Note: Descriptions are shown in the official language in which they were submitted.
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TREATMENT OF NSCLC PATIENTS WITH TUMOR INFILTRATING LYMPHOCYTE
THERAPIES
CROSS-REFERENCE TO RELATED APPLICATIONS
100011 This application claims priority to international Patent Application
No. PCT/US21/53841,
filed October 6. 2021, which claims priority to U.S. Provisional Application
No. 63/088,282, filed
October 6, 2020; U.S. Provisional Application No. 63/127,027, filed December
17, 2020; and U.S.
Provisional Application No. 63/146,402, filed February 5, 2021, all of which
are herein incorporated
by reference in their entireties.
BACKGROUND OF THE INVENTION
100021 A significant unmet need exists for new treatment options for patients
with locally advanced
or metastatic non-small-cell lung cancer (NSCLC); these patients comprise
approximately 70% of
newly diagnosed patients with NSCLC (Molina JR, Yang P, Cassivi SD, Schild SE,
Adjei AA. Mayo
Clin Proc. 2008; 83(5):584-94). Lung cancer is the leading cause of cancer
deaths worldwide, with
approximately 1.7 million deaths reported in 2015. Of the lung cancer deaths,
> 80% were attributed
to non-small-cell lung cancer (NSCLC) (21). In 2020, there will be an
estimated 228,820 new cases
and 135,720 deaths attributed to lung and bronchus cancer in the United States
(Siegel RL, 2015. CA
Cancer J Clin. 2015; 65(1):5-29). Thus, despite the approval of checkpoint
inhibitors (CPIs), which
revolutionized NSCLC treatment and outcomes, there remains a significant unmet
medical need in
NSCLC.
100031 Overall, in men and women, the lifetime risk of developing lung cancer
regardless of smoking
status is approximately 1 in 14 and 1 in 17, respectively. However, the risk
of developing lung cancer
is much higher in smokers vs. nonsmokers, e.g., men who smoke are 23 times
more likely to develop
cancer; likewise, women who smoke are 13 times more likely to develop lung
cancer than those who
do not (American Lung Association - Lung cancer fact sheet 2017 [American Lung
Association -
Lung cancer fact sheet]; available from: the World Wide Web at lung.org/lung-
health-and-
diseases/lung-disease-lookup/lung-cancer/resource-library/lung-cancer-fact-
sheet.html).
100041 Prior to checkpoint inhibitors, platinum doublet chemotherapy was
utilized in the initial
treatment of patients with incurable NSCLC (Schiller J. H. et al. 2002 NEngl
./Med 346 (2):92-8.),
and it produced an objective response rate (ORR) of 20% to 30% with limited
durability.
Pembrolizumab, alone or in combination with cytotoxic therapy, revolutionized
the treatment,
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producing higher response rates that have proven more durable as well (Gandhi
Lcena et al. 2018 N
Eng! J Med 378 (22):2078-2092; Paz-Ares L. et al. 2018 N Engl J Med 379
(21):2040-2051; Viteri S.
et al. 2020 Trans/Lung Cancer Res 9 (3):828-832; Pacheco J. M. 2020 Trans/Lung
Cancer Res 9
(1):148-153; Mok T. S. K. et al. 2019 Lancet 393 (10183):1819-1830; Nosaki K.
et al. 2019 Lung
Cancer 135:188-195; Morgensztern D. 2019.1 Thorac Dis 11 (Suppl 15): S 1963-S
1965 ; Reck M. et
al. 2016 N Engl J Med 375 (19):1823-1833). Other CPIs have demonstrated
improved ORR and
durability in combination with chemotherapy in the first-line setting for
NSCLC (Socinski MA et al
2018 New Engl J Med 378:2288-301). Despite these impressive results, there are
few complete
responses; almost all patients either fail to respond or progress, thus
necessitating subsequent therapy.
[0005] For NSCLC patients with identified driver mutations, the preferred
option is treatment with
targeted TKIs directed against the relevant mutation (e.g., osimertinib for
epidermal growth factor
receptor [EGFR] mutations, ceritinib for ALK mutations or crizotinib for ROS-1
mutations). For
previously untreated patients with NSCLC whose tumors express PD-Li, the
available treatment
options include pembrolizumab monotherapy (commonly used only for patients
with tumor
proportion score (TPS) for PD-L1 expression of at least 50%) or pembrolizumab
in combination with
chemotherapy. For patients with NSCLC and PD-L1 expression < 50%, the
preferred option is the
combination of pemetrexed, carboplatin or cisplatin, and pembrolizumab.
Patients with TPS for PD-
Li < 1% and no actionable mutations have no viable treatment options and are
not candidates for PD-
1 or PD-L1 CPIs. The combination of platinum-based doublet chemotherapy,
bevacizumab, and
atezolizumab is another potential therapeutic alternative in patients with
NSCLC, as is a combination
of nivolumab, ipilimumab and cytotoxic therapy (Hellmann et al. 2019 New Engl
J Med 381:2020-
31) .
[0006] Once a patient has disease progression on checkpoint inhibitor therapy
plus chemotherapy,
treatment options are limited and suffer from low efficacy, with objective
response rate ¨ 10% and
short progression-free survival (PFS), as well as high toxicity. The most
commonly used agent is
docetaxol, though other single agent cytotoxics or cytotoxics combined with
VEGF inhibitors are
sometimes employed, but all have similar, poor, outcomes. Thus, there is an
urgent need for better
therapeutic options in the second line treatment following checkpoint
inhibitor therapy plus
chemotherapy.
[0007] Furthermore, current TIL manufacturing and treatment processes are
limited by length, cost,
sterility concerns, and other factors described herein such that the potential
to treat patients which are
refractory to anti-PD-1 and/or anit-PD-L1 and/or VEGF inhibitor treatments and
as such have been
severly limited. There is an urgent need to provide TIL manufacturing
processes and therapies based
on such processes that are appropriate for use in treating patients for whom
very few or no viable
treatment options remain. The present invention meets this need by providing a
shortened
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manufacturing process for use in generating TILs which can then be employed in
the treatment of
non-small cell lung carcinoma (NSCLC) patients whom are refractory to anti-PD-
1, anti-PD-L1,
and/or VEGF inhibitor (including VEGF-A inhibitor) treatment.
BRIEF SUMMARY OF THE INVENTION
100081 The present invention provides improved and/or shortened methods for
expanding TILs and
producing therapeutic populations of TILs for use in treatment of non-small
cell lung carcinoma
(NSCLC) patients whom are refractory to anti-PD-1 treatment.
100091 In some embodiments, the present invention provides a method of
treating non-small cell lung
carcinoma (NSCLC) by administering a population of tumor infiltrating
lymphocytes (T1Ls) to a
subject or patient in need thereof, wherein obtaining the population of the
TILs comprises
multilesional sampling, wherein the subject or patient has at least one of:
i. a predetermined tumor proportion score (TPS) of PD-L1 of < 1%,
ii. a TPS score of PD-Li of 1%-49%, or
iii. a predetermined absence of one or more driver mutations.
100101 In some embodiments, the present invention provides a method of
treating non-small cell lung
carcinoma (NSCLC) by administering a population of tumor infiltrating
lymphocytes (TILs) to a
subject or patient in need thereof, wherein obtaining the population of the
TILs comprises
multilesional sampling, wherein the subject or patient has at least one of:
i. a predetermined tumor proportion score (TPS) of PD-Li of < 1%,
ii. a TPS score of PD-Li of 1%-49%, or
iii. a predetermined absence of one or more driver mutations;
wherein the method comprises:
(a) obtaining and/or receiving a first population of TILs from a tumor
resected from the
subject or patient by processing a tumor sample obtained from the subject into
multiple
tumor fragments;
(b) adding the first population of TILs into a closed system;
(c) performing a first expansion by culturing the first population of TILs in
a cell culture
medium comprising IL-2 to produce a second population of TILs, wherein the
first
expansion is performed in a closed container providing a first gas-permeable
surface area,
wherein the first expansion is performed for about 3-14 days to obtain the
second
population of TILs, and wherein the transition from step (b) to step (c)
occurs without
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opening the system;
(d) performing a second expansion by supplementing the cell culture medium of
the second
population of TILs with additional 1L-2, OKT-3, and antigen presenting cells
(APCs), to
produce a third population of TILs, wherein the second expansion is performed
for about
7-14 days to obtain the third population of TILs, wherein the third population
of TILs is a
therapeutic population of IlLs, wherein the second expansion is performed in a
closed
container providing a second gas-permeable surface area, and wherein the
transition from
step (c) to step (d) occurs without opening the system;
(e) harvesting therapeutic population of TILs obtained from step (d), wherein
the transition
from step (d) to step (e) occurs without opening the system; and
(f) transferring the harvested TIL population from step (e) to an infusion
bag, wherein the
transfer from step (e) to (f) occurs without opening the system;
(g) cryopreserving the infusion bag comprising the harvested TIL population
from step (f)
using a cryopreservation process; and
(h) administering a therapeutically effective dosage of the third population
of TILs from the
infusion bag in step (g) to the subject or patient.
100111 In some embodiments, the present invention provides a method of
treating non-small cell lung
carcinoma (NSCLC), wherein the NSCLC is refractory or resistant to treatment
with an anti-PD-1
and/or anti-PD-Ll antibody, by administering a population of tumor
infiltrating lymphocytes (TILs)
to a subject or patient in need thereof, wherein obtaining the population of
the TILs comprises
multilesional sampling, wherein the subject or patient has at least one of:
1. a predetermined tumor proportion score (TPS) of PD-L1 of <
1%,
ii. a TPS score of PD-L1 of 1%-49%, or
iii. a predetermined absence of one or more driver mutations;
wherein the method comprises:
(a) obtaining a first population of TILs from a tumor resected from a subject
by processing a
tumor sample obtained from the subject into multiple tumor fragments;
(b) adding the tumor fragments into a closed system;
(c) performing a first expansion by culturing the first population of TILs in
a cell culture
medium comprising IL-2 to produce a second population of TILs, wherein the
first
expansion is performed in a closed container providing a first gas-permeable
surface area,
wherein the first expansion is performed for about 3-11 days to obtain the
second
population of TILs, and wherein the transition from step (b) to step (c)
occurs without
opening the system;
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(d) performing a second expansion by supplementing the cell culture medium of
the second
population of TILs with additional IL-2, OKT-3, and antigen presenting cells
(APCs), to
produce a third population of TILs, wherein the second expansion is performed
for about
7-11 days to obtain the third population of TILs, wherein the second expansion
is
performed in a closed container providing a second gas-permeable surface area,
and
wherein the transition from step (c) to step (d) occurs without opening the
system;
(e) harvesting the third population of TILs obtained from step (d), wherein
the transition
from step (d) to step (e) occurs without opening the system;
(f) transferring the harvested third TIL population from step (e) to an
infusion bag, wherein
the transfer from step (e) to (f) occurs without opening the system;
(g) cryopreserving the infusion bag comprising the harvested TIL population
from step (f)
using a cryopreservation process; and
(h) administering a therapeutically effective dosage of the third population
of TILs from the
infusion bag in step (g) to the subject.
100121 In some embodiments, the present invention provides a method of
treating non-small cell lung
carcinoma (NSCLC) by administering a population of tumor infiltrating
lymphocytes (TILs) to a
subject or patient in need thereof, wherein obtaining the population of the
TILs comprises
multilesional sampling, wherein the subject or patient has at least one of:
i. a predetermined tumor proportion score (TPS) of PD-Ll of < 1%,
ii. a TPS score of PD-Li of 1%-49%, or
iii. a predetermined absence of one or more driver mutations;
wherein the method comprises:
(a) obtaining and/or receiving a first population of TILs from surgical
resection, needle
biopsy, core biopsy, small biopsy, or other means for obtaining a sample that
contains a
mixture of tumor and TIL cells from a NSCLC tumor in the subject or patient,
(b) adding the first population of TILs into a closed system;
(c) performing a first expansion by culturing the first population of TILs in
a cell culture
medium comprising IL-2 to produce a second population of TILs, wherein the
first
expansion is performed in a closed container providing a first gas-permeable
surface area,
wherein the first expansion is performed for about 3-11 days to obtain the
second
population of TILs, and wherein the transition from step (b) to step (c)
occurs without
opening the system;
(d) performing a second expansion by supplementing the cell culture medium of
the second
population of TILs with additional IL-2, OKT-3, and antigen presenting cells
(APCs), to
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produce a third population of TILs, wherein the second expansion is performed
for about
7-11 days to obtain the third population of TILs, wherein the second expansion
is
performed in a closed container providing a second gas-permeable surface area,
and
wherein the transition from step (c) to step (d) occurs without opening the
system;
(e) harvesting the third population of TILs obtained from step (d), wherein
the transition
from step (d) to step (e) occurs without opening the system;
(f) transferring the harvested third TIL population from step (e) to an
infusion bag, wherein
the transfer from step (e) to (0 occurs without opening the system;
(g) cryopreserving the infusion bag comprising the harvested TIL population
from step (0
using a cryopreservation process; and
(h) administering a therapeutically effective dosage of the third population
of TILs from the
infusion bag in step (g) to the subject or patient.
100131 In some embodiments, the present invention provides a method of
treating non-small cell lung
carcinoma (NSCLC) by administering a population of tumor infiltrating
lymphocytes (TILs) to a
subject or patient in need thereof, wherein obtaining the population of the
TILs comprises
multilesional sampling, wherein the subject or patient has at least one of:
i. a predetermined tumor proportion score (IPS) of PD-Li of < 1%,
ii. a TPS score of PD-L1 of 1%-49%, or
iii. a predetermined absence of one or more driver mutations;
wherein the method comprises:
(a) resecting a NSCLC tumor from the subject or patient, the tumor comprising
a first
population of TILs, optionally from surgical resection, needle biopsy, core
biopsy, small
biopsy, or other means for obtaining a sample that contains a mixture of tumor
and TIL
cells from a NSCLC tumor;
(b) adding the tumor fragments into a closed system;
(c) performing a first expansion by culturing the first population of TILs in
a cell culture
medium comprising IL-2 to produce a second population of TILs, wherein the
first
expansion is performed in a closed container providing a first gas-permeable
surface area;
wherein the first expansion is performed for about 3-11 days to obtain the
second
population of TILs, and wherein the transition from step (b) to step (c)
occurs without
opening the system;
(d) performing a second expansion by supplementing the cell culture medium of
the second
population of TILs with additional IL-2, OKT-3, and antigen presenting cells
(APCs), to
produce a third population of TILs, wherein the second expansion is performed
for about
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7-11 days to obtain the third population of TILs, wherein the second expansion
is
performed in a closed container providing a second gas-permeable surface area,
and
wherein the transition from step (c) to step (d) occurs without opening the
system;
(e) harvesting the third population of TILs obtained from step (d), wherein
the transition
from step (d) to step (e) occurs without opening the system;
(t) transferring the harvested third T1L population from step (c) to an
infusion bag, wherein
the transfer from step (e) to (f) occurs without opening the system;
(g) cryopreserving the infusion bag comprising the harvested TIL population
from step (f)
using a cryopreservation process; and
(h) administering a therapeutically effective dosage of the third population
of TILs from the
infusion bag in step (g) to the subject.
100141 In some embodiments, the second population of TILs is at least 50-fold
greater in number
than the first population of TILs.
[0015] In some embodiments, the present invention provides a method of
treating non-small cell lung
carcinoma (NSCLC) by administering a population of tumor infiltrating
lymphocytes (TILs) to a
subject or patient in need thereof, wherein obtaining the population of the
TILs comprises
multilesional sampling, wherein the subject or patient has at least one of:
i. a predetermined tumor proportion score (TPS) of PD-Li of < 1%,
ii. a TPS score of PD-Li of 1%-49%, or
iii. a predetermined absence of one or more driver mutations;
wherein the method comprises:
(a) obtaining and/or receiving a first population of TILs from surgical
resection, needle
biopsy, core biopsy, small biopsy, or other means for obtaining a sample that
contains a
mixture of tumor and T1L cells from the subject or patient;
(c) contacting the first population of TILs with a first cell culture medium;
(d) performing an initial expansion (or priming first expansion) of the first
population of TILs
in the first cell culture medium to obtain a second population of TILs,
wherein the second
population of TILs is at least 5-fold greater in number than the first
population of TILs,
wherein the first cell culture medium comprises IL-2, optionally, where the
priming first
expansion occurs for a period of 1 to 8 days;
(e) performing a rapid expansion of the second population of TILs in a second
cell culture
medium to obtain a third population of TILs, wherein the second cell culture
medium
comprises IL-2, OKT-3 (anti-CD3 antibody), and optionally irradiated
allogeneic
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peripheral blood mononuclear cells (PBMCs); and wherein the rapid expansion is
performed over a period of 14 days or less, optionally the rapid expansion can
proceed for
1 day, 2 days, 3 days, 4, days, 5 days, 6 days, 7 days, 8 days, 9 days or 10
days after
initiation of the rapid expansion;
(f) harvesting the third population of TILs; and
(g) administering a therapeutically effective portion of the third population
of TILs to the
subject or patient with the NSCLC.
100161 In some embodiments, the present invention provides a method of
treating non-small cell lung
carcinoma (NSCLC) by administering a population of tumor infiltrating
lymphocytes (TILs) to a
subject or patient in need thereof, wherein obtaining the population of the
TILs comprises
multilesional sampling, wherein the subject or patient has at least one of:
i. a predetermined tumor proportion score (TPS) of PD-Ll of < 1%,
ii. a TPS score of PD-Li of 1%-49%, or
iii. a predetermined absence of one or more driver mutations;
wherein the method comprises:
(a) resecting a NSCLC tumor from the subject or patient, the tumor comprising
a first
population of TILs, optionally from surgical resection, needle biopsy, core
biopsy, small
biopsy, or other means for obtaining a sample that contains a mixture of tumor
and TIL
cells from a NSCLC tumor;
(b) fragmenting the tumor into tumor fragments;
(c) contacting the tumor fragments with a first cell culture medium;
(d) performing an initial expansion (or priming first expansion) of the first
population of TILs
in the first cell culture medium to obtain a second population of TILs,
wherein the second
population of TILs is at least 5-fold greater in number than the first
population of TILs,
wherein the first cell culture medium comprises 1L-2, optionally, where the
priming first
expansion occurs for a period of 1 to 8 days;
(e) performing a rapid expansion of the second population of TTLs in a second
cell culture
medium to obtain a third population of TILs, wherein the second cell culture
medium
comprises IL-2, OKT-3 (anti-CD3 antibody), and optionally irradiated
allogeneic
peripheral blood mononuclear cells (PBMCs); and wherein the rapid expansion is
performed over a period of 14 days or less, optionally the rapid expansion can
proceed for
1 day, 2 days, 3 days, 4, days, 5 days, 6 days, 7 days, 8 days, 9 days or 10
days after
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initiation of the rapid expansion;
(f) harvesting the third population of TILs; and
(g) administering a therapeutically effective portion of the third population
of TILs to the
subject or patient with the NSCLC.
[0017] A method of treating non-small cell lung carcinoma (N SCLC), wherein
the NSCLC is
refractory or resistant to treatment with an anti-PD-1 and/or anti-PD-Li
antibody, by administering a
population of tumor infiltrating lymphocytes (TILs) to a subject or patient in
need thereof, wherein
obtaining the population of the TILs comprises multilesional sampling, wherein
the subject or patient
has at least one of:
i. a predetermined tumor proportion score (TPS) of PD-Ll of < 1%,
ii. a TPS score of PD-Li of 1%-49%, or
iii. a predetermined absence of one or more driver mutations;
wherein the method comprises:
(a) obtaining and/or receiving a first population of TILs from surgical
resection, needle
biopsy, core biopsy, small biopsy, or other means for obtaining a sample that
contains a
mixture of tumor and TIL cells from the subject or patient;
(c) contacting the first population of TILs with a first cell culture medium;
(d) performing an initial expansion (or priming first expansion) of the first
population of TILs
in the first cell culture medium to obtain a second population of TILs,
wherein the second
population of TILs is at least 5-fold greater in number than the first
population of TILs,
wherein the first cell culture medium comprises IL-2, optionally, where the
priming first
expansion occurs for a period of 1 to 8 days;
(e) performing a rapid expansion of the second population of TILs in a second
cell culture
medium to obtain a third population of TILs; wherein the second cell culture
medium
comprises 1L-2, OKT-3 (anti-CD3 antibody), and optionally irradiated
allogeneic
peripheral blood mononuclear cells (PBMCs); and wherein the rapid expansion is
performed over a period of 14 days or less, optionally the rapid expansion can
proceed for
1 day, 2 days, 3 days, 4, days, 5 days, 6 days, 7 days, 8 days, 9 days or 10
days after
initiation of the rapid expansion;
(f) harvesting the third population of TILs; and
(g) administering a therapeutically effective portion of the third population
of TILs to the
subject or patient with the NSCLC.
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100181 In some embodiments, the third population of TILs is at least 50-fold
greater in number than
the second population of TILs after 7-8 days from the start of the rapid
expansion.
100191 In some embodiments, the patient or subject has a TPS of PD-Li of < 1%.
100201 In some embodiments, the patient or subject has a TPS of PD-Li of 1%-
49%.
100211 In some embodiments, the patient or subject has a NSCLC that is not
indicated for treatment
by an EGFR inhibitor, a BRAF inhibitor, an ALK inhibitor, a c-Ros inhibitor, a
RET inhibitor, an
ERBB2 inhibitor, BRCA inhibitor, a MAP2K1 inhibitor, PIK3CA inhibitor, CDKN2A
inhibitor, a
PTEN inhibitor, an UMD inhibitor, an NRAS inhibitor, a KRAS inhibitor, an NF1
inhibitor, MET
inhibitor a TP53 inhibitor, a CREBBP inhibitor, a KMT2C inhibitor, a KMT2D
mutation, an
AR1D1A mutation, a RB1 inhibitor, an ATM inhibitor, a SETD2 inhibitor, a FLT3
inhibitor, a
PTPN11 inhibitor, a FGER1 inhibitor, an EP300 inhibitor, a MYC inhibitor, an
EZH2 inhibitor, a
JAK2 inhibitor, a FBXW7 inhibitor, a CCND3 inhibitor, and a GNAll inhibitor.
100221 In some embodiments, the patient or subject has a predetermined absence
of one or more
driver mutations.
100231 In some embodiments, the patient or subject has a TPS of PD-Li of < 1%
and has a
predetermined absence of one or more driver mutations.
100241 In some embodiments, the one or more driver is selected from the group
consisting of an
EGFR mutation, an EGFR insertion, EGFR exon20, a KRAS mutation, a BRAF-
mutation, a BRAF
V600 mutation, an ALK-mutation, a c-ROS-mutation (ROS1-mutation), a ROS1
fusion, a RET
mutation, a RET fusion, an ERBB2 mutation, an ERBB2 amplification, a BRCA
mutation, a
MAP2K1 mutation, PIK3CA, CDKN2A, a PTEN mutation, an UMD mutation, an NRAS
mutation, a
KRAS mutation, an NF1 mutation, a MET mutation, a MET splice and/or altered
MET signaling, a
TP53 mutation, a CREBBP mutation, a KMT2C mutation, a KMT2D mutation, an ARID
1A
mutation, a RB 1 mutation, an ATM mutation, a SETD2 mutation, a FLT3 mutation,
a PTPN11
mutation, a FGFR1 mutation, an EP3 00 mutation, a MYC mutation, an EZH2
mutation, a JAK2
mutation, a FBXW7 mutation, a CCND3 mutation, and a GNAll mutation.
100251 In some embodiments, the patient or subject has a TPS of < 1% and has a
NSCLC that is not
indicated for treatment by an EGFR inhibitor, a BRAF inhibitor, an ALK
inhibitor, a c-Ros inhibitor,
a RET inhibitor, an ERBB2 inhibitor, BRCA inhibitor, a MAP2K1 inhibitor,
PIK3CA inhibitor,
CDKN2A inhibitor, a PTEN inhibitor, an UMD inhibitor, an NRAS inhibitor, a
KRAS inhibitor, an
NF1 inhibitor, MET inhibitor a TP53 inhibitor, a CREBBP inhibitor, a KMT2C
inhibitor, a KMT2D
mutation, an ARID lA mutation, a RB1 inhibitor, an ATM inhibitor, a SETD2
inhibitor, a FLT3
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inhibitor, a PTPN11 inhibitor, a FGFR1 inhibitor, an EP300 inhibitor, a MYC
inhibitor, an EZH2
inhibitor, a JAK2 inhibitor, a FBXW7 inhibitor, a CCND3 inhibitor, and a GNAll
inhibitor.
100261 In some embodiments, the NSCLC has low or no expression of PD-Li.
[0027] In some embodiments, the NSCLC is refractory or resistant to treatment
with a
chemotherapeutic agent.
100281 In some embodiments, the NSCLC is refractory or resistant to treatment
with a VEGF-A
inhibitor.
100291 In some embodiments, the NSCLC has been treated with a chemotherapeutic
agent but is not
being currently treated with a chemotherapeutic agent.
[0030] In some embodiments, the NSCLC has been treated with a chemotherapeutic
agent but is not
being currently treated with a chemotherapeutic agent and has a TPS of< 1%.
100311 In some embodiments, the NSCLC has been treated with a VEGF-A inhibitor
but is not being
currently treated with a VEGF-A inhibitor.
100321 In some embodiments, the NSCLC has been treated with a VEGF-A inhibitor
but is not being
currently treated with a VEGF-A inhibitor and has a TPS of < 1%.
[0033] In some embodiments, the NSCLC has been treated with a chemotherapeutic
agent and/or a
VEGF-A inhibitor, but is not being currently treated with a chemotherapeutic
agent and/or a VEGF-A
inhibitor.
100341 In some embodiments, the NSCLC has been treated with a chemotherapeutic
agent and/or a
VEGF-A inhibitor but is not being currently treated with a chemotherapeutic
agent and/or a VEGF-A
inhibitor and has a TPS of < 1%.
100351 In some embodiments, the NSCLC has not been previously treated with an
anti-PD-1 and/or
anti-PD-Li antibody.
100361 In some embodiments, the NSCLC has not been previously treated with an
anti-PD-1 and/or
anti-PD-Li antibody and has been previously treated a chemotherapeutic agent
and/or a VEGF-A
inhibitor.
[0037] In some embodiments, the NSCLC has not been previously treated with an
anti-PD-1 and/or
anti-PD-Li antibody and has been previously treated a chemotherapeutic agent
and/or a VEGF-A
inhibitor but is not being currently treated with a chemotherapeutic agent
and/or a VEGF-A inhibitor.
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100381 In some embodiments, the NSCLC has not been previously treated with an
anti-PD-1 and/or
anti-PD-L1 antibody and has been previously treated a VEGF-A inhibitor but is
not being currently
treated with a VEGF-A inhibitor.
100391 In some embodiments, the NSCLC has not been previously treated with an
anti-PD-1 and/or
anti-PD-L1 antibody and has been previously treated a chemotherapeutic agent
and/or a VEGF-A
inhibitor but is not being currently treated with a chemotherapeutic agent
and/or a VEGF-A inhibitor.
100401 In some embodiments, the NSCLC has not been previously treated with an
anti-PD-1 and/or
anti-PD-Li antibody and has low or no expression of PD-Li.
100411 In some embodiments, the NSCLC has not been previously treated with an
anti-PD-1 and/or
anti-PD-Li antibody and has been previously treated a chemotherapeutic agent
and/or a VEGF-A
inhibitor but is not being currently treated with a chemotherapeutic agent
and/or a VEGF-A inhibitor
and has a TPS of < 1%.
100421 In some embodiments, the NSCLC has been previously treated with an anti-
PD-1 and/or anti-
PD-Li and/or anti-PD-L2 antibody.
100431 In some embodiments, the NSCLC has been previously treated with an anti-
PD-1 and/or anti-
PD-Li antibody and has been previously treated a chemotherapeutic agent and/or
a VEGF-A
inhibitor.
100441 In some embodiments, the NSCLC is refractory or resistant to treatment
with an anti-PD-1
and/or anti-PD-Li antibody.
100451 In some embodiments, the NSCLC has been previously treated with an anti-
PD-1 and/or anti-
PD-Li antibody and the tumor proportion score was determined prior to the anti-
PD-1 and/or anti-
PD-Li antibody treatment.
100461 In some embodiments, the NSCLC has been previously treated with an anti-
PD-Li antibody
and the tumor proportion score was determined prior to the anti-PD-LI antibody
treatment, or the
NSCLC has been previously treated with an anti-PD-1 antibody and the tumor
proportion score was
determined prior to the anti-PD-1 antibody treatment.
100471 In some embodiments, the NSCLC has been treated with a chemotherapeutic
agent and/or a
VEGF-A inhibitor.
100481 In some embodiments, the NSCLC has not been previously treated with an
anti-PD-1 and/or
anti-PD-L1 antibody and has bulky disease at baseline.
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100491 In some embodiments, the NSCLC has been previously treated with an anti-
PD-1 and/or anti-
PD-L I antibody and has bulky disease at baseline.
100501 In some embodiments, the NSCLC has been treated with a chemotherapeutic
agent and has
bulky disease at baseline.
100511 In some embodiments, the NSCLC has been treated with a chemotherapeutic
agent and/or
VEGF-A inhibitor but is not being currently treated with a chemotherapeutic
agent and/or VEGF-A
inhibitor and has bulky disease at baseline.
100521 In some embodiments, bulky disease is indicated where the maximal tumor
diameter is
greater than 7 cm measured in either the transverse or coronal plane or
swollen lymph nodes with a
short-axis diameter of 20 mm or greater.
100531 In some embodiments, the NSCLC is refractory or resistant to at least
two prior systemic
treatment courses, not including neo-adjuvant or adjuvant therapies.
100541 In some embodiments, the NSCLC is refractory or resistant to an anti-PD-
1 or an anti-PD-L1
antibody selected from the group consisting of nivolumab, pembrolizumab,
JS001, TSR-042,
pidilizum BGB-A317, SHR-1210, REGN2810, MDX-1106, PDR001, anti-PD-1 from
clone:
RMP1-14, anti-PD-1 antibodies disclosed in U.S. Patent No. 8,008,449,
durvalumab, atezolizumab,
avelumab, and fragments, derivatives, variants, as well as biosimilars
thereof.
100551 In some embodiments, the NSCLC is refractory or resistant to
pembrolizumab or a biosimilar
thereof.
100561 In some embodiments, the NSCLC is refractory or resistant to nivolumab
or a biosimilar
thereof
100571 In some embodiments, the NSCLC is refractory or resistant to an anti-
CTLA-4 antibody.
100581 In some embodiments, the NSCLC is refractory or resistant to an anti-
CTLA-4 antibody and
pembrolizumab or a biosimilar thereof.
100591 In some embodiments, the NSCLC is refractory or resistant to an anti-
CTLA-4 antibody, and
nivolumab or a biosimilar thereof
100601 In some embodiments, the anti- CTLA-4 antibody is ipilimumab or a
biosimilar thereof
100611 In some embodiments, the NSCLC is refractory or resistant to durvalumab
or a biosimilar
thereof
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[0062] In some embodiments, the NSCLC is refractory or resistant to
atezolizumab or a biosimilar
thereof.
[0063] In some embodiments, the NSCLC is refractory or resistant to avelumab
or a biosimilar
thereof.
[0064] In some embodiments, the chemotherapeutic agent is a platinum doublet
chemotherapeutic
agent(s).
[0065] In some embodiments, the platinum doublet chemotherapeutic agent
therapy comprises:
i) a first chemotherapeutic agent selected from the group consisting of
cisplatin and
carboplatin,
ii) and a second chemotherapeutic agent selected from the group consisting of
vinorelbine,
gemcitabine and a taxane (including for example, paclitaxel, docetaxel or nab-
paclitaxel).
[0066] In some embodiments, the chemotherapeutic agent, including the first
and/or second
chemotherapeutic agent, is in combination with pemetrexed.
[0067] In some embodiments, the NSCLC is refractory or resistant to a
combination therapy
comprising carboplatin, paclitaxel, pemetrexed, and cisplatin.
[0068] In some embodiments, the NSCLC is refractory or resistant to a
combination therapy
comprising carboplatin, paclitaxel, pemetrexed, cisplatin, nivolumab, and
ipilimumab.
[0069] In some embodiments, the NSCLC is refractory or resistant to a VEGF-A
inhibitor.
[0070] In some embodiments, the NSCLC is refractory or resistant to a VEGF-A
inhibitor selected
from the group consisting of bevacizumab, ranibizumab, and icrucumab.
[0071] In some embodiments, the NSCLC is refractory or resistant to
bevacizumab.
[0072] In some embodiments, the NSCLC has been analyzed for the absence or
presence of one or
more driver mutations.
[0073] In some embodiments, one or more driver mutations are not present.
[0074] In some embodiments, the NSCLC treatment is independent of the presence
or absence of one
or more driver mutations.
[0075] In some embodiments, the one or more driver mutations is selected from
the group consisting
of an EGFR mutation, an EGFR insertion, a KRAS mutation, a BRAF-mutation, an
ALK-mutation, a
c-ROS-mutation a c-ROS-mutation, EML4-ALK, and MET mutation.
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[0076] In some embodiments, the EGFR mutation results in tumor transformation
from NSCLC to
small cell lung cancer (SCLC).
[0077] In some embodiments, the NSCLC treatment is independent of the presence
or absence of
high-tumor mutational burden (high-TMB) and/or microsatellite instability-high
(MSI-high) status.
[0078] In some embodiments, the NSCLC exhibits high-IMB and/or MS1-high
status.
[0079] In some embodiments, the IL-2 is present at an initial concentration of
between 1000 IU/mL
and 6000 IU/mT, in the first cell culture medium, when present.
[0080] In some embodiments, the 1L-2 is present at an initial concentration of
between 1000 IU/mL
and 6000 IU/mL and the OKT-3 antibody is present at an initial concentration
of about 30 ng/mL in
the second cell culture medium, when present.
[0081] In some embodiments, the initial expansion, when present, is performed
using a gas
permeable container.
[0082] In some embodiments, the rapid expansion, when present, is performed
using a gas permeable
container.
[0083] In some embodiments, the first cell culture medium, when present,
further comprises a
cytokine selected from the group consisting of IL-4, IL-7, IL-15, IL-21, and
combinations thereof.
[0084] In some embodiments, the second cell culture medium, when present,
further comprises a
cytokine selected from the group consisting of IL-4, IL-7, IL-15, IL-21, and
combinations thereof.
[0085] In some embodiments, the method further comprises the step of treating
the patient with a
non-myeloablative lymphodepletion regimen prior to administering the third
population of TILs to the
patient.
[0086] In some embodiments, the non-myeloablative lymphodepletion regimen
comprises the steps
of administration of cyclophosphamide at a dose of 60 mg/m2/day for two days
followed by
administration of fludarabine at a dose of 25 mg/m2/day for five days.
[0087] In some embodiments, the non-myeloablative lymphodepletion regimen
comprises the steps
of administration of cyclophosphamide at a dose of 60 mg/m2/day and
fludarabine at a dose of 25
mg/m)/day for two days followed by administration of fludarabine at a dose of
25 mg/m7day for three
days.
[0088] In some embodiments, the cyclophosphamide is administered with mesna.
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[0089] In some embodiments, the method further comprises the step of treating
the patient with an
IL-2 regimen starting on the day after administration of the third population
of TILs to the patient.
[0090] In some embodiments, the method further comprises the step of treating
the patient with an
IL-2 regimen starting on the same day as administration of the third
population of TILs to the patient.
100911 In some embodiments, the IL-2 regimen is a high-dose IL-2 regimen
comprising 600,000 or
720,000 IU/kg of aldesleukin, or a biosimilar or variant thereof, administered
as a 15-minute bolus
intravenous infusion every eight hours until tolerance.
[0092] In some embodiments, a therapeutically effective population of TILs is
administered and
comprises from about 2.3 x 101 to about 13.7x 1010 TILs.
[0093] In some embodiments, the initial expansion is performed over a period
of 21 days or less.
[0094] In some embodiments, the initial expansion is performed over a period
of 7 days or less.
100951 In some embodiments, the rapid expansion is performed over a period of
7 days or less.
[0096] In some embodiments, the first expansion in step (c) and the second
expansion in step (d) are
each individually performed within a period of 11 days.
100971 In some embodiments, the steps (a) through (f) are performed in about
10 days to about 24
days.
[0098] In some embodiments, the steps (a) through (f) are performed in about
10 days to about 22
days.
BRIEF DESCRIPTION OF THE DRAWINGS
[0099] Figure I: Exemplary Gen 2 (Process 2A) type chart providing an overview
of Steps A
through F.
[00100] Figure 2: Exemplary process flow chart of Gen 2 (Process 2A) type
process.
[00101] Figure 3: Shows a diagram of an embodiment of a cryopreserved TIL
exemplary
manufacturing process (-22 days).
[00102] Figure 4: Shows a diagram of an embodiment of Gen 2, a 22-day process
for TIL
manufacturing.
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[00103] Figure 5: Comparison table of Steps A through F from exemplary
embodiments of process
1C and Gen 2.
[00104] Figure 6: Detailed comparison of an embodiment of process 1C and an
embodiment of Gen
2.
1001051 Figure 7: Exemplary Gen 3 type process for NSCLC tumors.
[00106] Figure 8A-8D: A) Shows a comparison between the 2A process
(approximately 22-day
process) and an embodiment of the Gen 3 process for TIT, manufacturing
(approximately 14-days to
16-days process). B) Exemplary Process Gen 3 chart providing an overview of
Steps A through F
(approximately 14-days to 16-days process). C) Chart providing three exemplary
Gen 3 processes
with an overview of Steps A through F (approximately 14-days to 16-days
process) for each of the
three process variations. D) Exemplary Modified Gen 2-like process providing
an overview of Steps
A through F (approximately 22-days process).
1001071 Figure 9: Provides an experimental flow chart for comparability
between Gen 2 (Gen 2)
versus Gen 3.
[00108] Figure 10: Shows a comparison between various Gen 2 (2A process) and
the Gen 3.1
process embodiment.
[00109] Figure 11: Table describing various features of embodiments of the Gen
2, Gen 2.1 and
Gen 3.0 process.
[00110] Figure 12: Overview of the media conditions for an embodiment of the
Gen 3 process,
referred to as Gen 3.1.
[00111] Figure 13: Table describing various features of embodiments of the Gen
2, Gen 2.1 and
Gen 3.0 process.
[00112] Figure 14: Table comparing various features of embodiments of the Gen
2 and Gen 3.0
processes.
[00113] Figure 15: Table providing media uses in the various embodiments of
the described
expansion processes.
[00114] Figure 16: Schematic of an exemplary embodiment of the Gen 3 process
(a 16-day
process).
[00115] Figure 17: Schematic of an exemplary embodiment of a method for
expanding T cells from
hematopoietic malignancies using Gen 3 expansion platform.
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[00116] Figure 18: Provides the structures I-A and I-B, the cylinders refer to
individual polypeptide
binding domains. Structures I-A and I-B comprise three linearly-linked TNFRSF
binding domains
derived from e.g., 4-1BBL or an antibody that binds 4-1BB, which fold to form
a trivalent protein,
which is then linked to a second trivalent protein through IgGl-Fc (including
CH3 and CH2 domains)
is then used to link two of the trivalent proteins together through disulfide
bonds (small elongated
ovals), stabilizing the structure and providing an agonists capable of
bringing together the intracellular
signaling domains of the six receptors and signaling proteins to form a
signaling complex. The
TNFRSF binding domains denoted as cylinders may be scFv domains comprising,
e.g., a VH and a
VL chain connected by a linker that may comprise hydrophilic residues and Gly
and Ser sequences
for flexibility, as well as Glu and Lys for solubility.
[00117] Figure 19: Schematic of an exemplary embodiment of the Gen 3 process
(a 16-day
process).
[00118] Figure 20: Provides a process overview for an exemplary embodiment of
the Gen 3.1
process (a 16 day process).
[00119] Figure 21: Schematic of an exemplary embodiment of the Gen 3.1 Test
process (a 16-17
day process).
[00120] Figure 22: Schematic of an exemplary embodiment of the Gen 3 process
(a 16-day
process).
[00121] Figure 23A-23B: Comparison table for exemplary Gen 2 and exemplary Gen
3 processes.
[00122] Figure 24: Schematic of an exemplary embodiment of the Gen 3 process
(a 16/17 day
process) preparation timeline.
[00123] Figure 25: Schematic of an exemplary embodiment of the Gen 3 process
(a 14-16 day
process).
[00124] Figure 26A-26B: Schematic of an exemplary embodiment of the Gen 3
process (a 16 day
process).
[00125] Figure 27: Schematic of an exemplary embodiment of the Gen 3 process
(a 16 day
process).
[00126] Figure 28: Comparison of Gen 2, Gen 2.1 and an embodiment of the Gen 3
process (a 16
day process).
[00127] Figure 29: Comparison of Gen 2, Gen 2.1 and an embodiment of the Gen 3
process (a 16
day process).
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[00128] Figure 30: Gen 3 embodiment components.
[00129] Figure 31: Gen 3 embodiment flow chart comparison (Gen 3.0, Gen 3.1
control, Gen 3.1
Test).
[00130] Figure 32: Shown are the components of an exemplary embodiment of the
Gen 3 process
(a 16-17 day process).
[00131] Figure 33: Acceptance criteria table.
[00132] Figure 34: Diagram of Study Design related to study described in
Example 19.
[00133] Figure 35: Schematic of TIL-based immunotherapy banufacturing process
related to the
study described in Example 19. Abbreviations: CMO = contract manufacturing
organization; GMP =
Good Manufacturing Practices; IL-2 = interleukin-2; OKT3 = monoclonal antibody
to CD3; TIL ¨
tumor infiltrating lymphocytes.
[00134] Figure 36: Study Flowchart (all four cohorts).
[00135] Figure 37: Patient journey and central Gen 2 GMP manufacturing.
[00136] Figure 38: Cohort 3B patient treatment schema.
[00137] Figure 39: Patient disposition.
[00138] Figure 40: Adverse events over time (FAS).
[00139] Figure 41: Best percentage change from baseline in target lesion sum
of diameters
(efficacy-evaluable set).
[00140] Figure 42: Time to first response, duration of response, and time on
efficacy assessment for
confirmed responders who achieved PR or better.
[00141] Figure 43: Percentage change from baseline in target lesion sum of
diameters (FAS).
BRIEF DESCRIPTION OF THE SEQUENCE LISTING
[00142] SEQ ID NO:1 is the amino acid sequence of the heavy chain of
muromonab.
[00143] SEQ ID NO:2 is the amino acid sequence of the light chain of
muromonab.
[00144] SEQ ID NO:3 is the amino acid sequence of a recombinant human IL-2
protein.
[00145] SEQ ID NO:4 is the amino acid sequence of aldesleukin.
[00146] SEQ ID NO:5 is an IL-2 form.
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[00147] SEQ ID NO:6 is the amino acid sequence of nemvaleukin alfa.
[00148] SEQ ID NO:7 is an IL-2 form.
[00149] SEQ ID NO:8 is a mucin domain polypeptide.
[00150] SEQ ID NO:9 is the amino acid sequence of a recombinant human IL-4
protein.
[00151] SEQ ID NO: 10 is the amino acid sequence of a recombinant human 1L-7
protein.
[00152] SEQ ID NO:11 is the amino acid sequence of a recombinant human IL-15
protein.
[00153] SEQ ID NO:12 is the amino acid sequence of a recombinant human IL-21
protein.
[00154] SEQ ID NO:13 is an IL-2 sequence.
[00155] SEQ ID NO:14 is an IL-2 mutein sequence.
[00156] SEQ ID NO:15 is an 1L-2 mutcin sequence.
[00157] SEQ ID NO:16 is the HCDR1 IL-2 for IgG.IL2R67A.H1.
[00158] SEQ ID NO:17 is the HCDR2 for IgG.IL2R67A.H1.
[00159] SEQ ID NO:18 is the HCDR3 for IgG.IL2R67A.H1.
[00160] SEQ ID NO:19 is the HCDR1 IL-2 kabat for IgG.IL2R67A.H1.
[00161] SEQ ID NO:20 is the HCDR2 kabat for IgG.IL2R67A.H1.
[00162] SEQ ID NO:21 is the HCDR3 kabat for IgG.IL2R67A.H1.
[00163] SEQ ID NO:22 is the HCDR1 IL-2 clothia for IgG.IL2R67A.H1.
[00164] SEQ ID NO:23 is the HCDR2 clothia for IgG.IL2R67A.H1.
[00165] SEQ ID NO:24 is the HCDR3 clothia for IgG.IL2R67A.H1.
[00166] SEQ ID NO:25 is the HCDR1 IL-2 IMGT for IgG.IL2R67A.H1.
[00167] SEQ ID NO:26 is the HCDR2 IMGT for IgG.IL2R67A.H1.
[00168] SEQ ID NO:27 is the HCDR3 IMGT for IgG.IL2R67A.H1.
[00169] SEQ ID NO:28 is the VH chain for IgG.IL2R67A.H1.
[00170] SEQ ID NO:29 is the heavy chain for IgG.IL2R67A.H1.
[00171] SEQ ID NO:30 is the LCDR1 kabat for IgG.IL2R67A.H1.
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[00172] SEQ ID NO:31 is the LCDR2 kabat for IgG.IL2R67A.H1.
[00173] SEQ ID NO:32 is the LCDR3 kabat for IgG.IL2R67A.H1.
[00174] SEQ ID NO:33 is the LCDR1 chothia for IgG.IL2R67A.H1.
[00175] SEQ ID NO:34 is the LCDR2 chothia for IgG.IL2R67A.H1.
[00176] SEQ ID NO:35 is the LCDR3 chothia for IgGIL2R67A.H I .
[00177] SEQ ID NO:36 is a VL chain.
[00178] SEQ ID NO:37 is a light chain.
[00179] SEQ ID NO:38 is a light chain.
[00180] SEQ ID NO:39 is a light chain.
[00181] SEQ ID NO:40 is the amino acid sequence of human 4-1BB.
[00182] SEQ ID NO:41 is the amino acid sequence of murine 4-1BB.
[00183] SEQ ID NO:42 is the heavy chain for the 4-1BB agonist monoclonal
antibody utomilumab
(PF-05082566).
[00184] SEQ ID NO:43 is the light chain for the 4-1BB agonist monoclonal
antibody utomilumab
(PF-05082566).
[00185] SEQ ID NO:44 is the heavy chain variable region (VH) for the 4-1BB
agonist monoclonal
antibody utomilumab (PF-05082566).
[00186] SEQ ID NO:45 is the light chain variable region (VI) for the 4-1BB
agonist monoclonal
antibody utomilumab (PF-05082566).
[00187] SEQ ID NO:46 is the heavy chain CDR1 for the 4-1BB agonist monoclonal
antibody
utomilumab (PF-05082566).
[00188] SEQ ID NO:47 is the heavy chain CDR2 for the 4-1BB agonist monoclonal
antibody
utomilumab (PF-05082566).
[00189] SEQ ID NO:48 is the heavy chain CDR3 for the 4-1BB agonist monoclonal
antibody
utomilumab (PF-05082566).
[00190] SEQ ID NO:49 is the light chain CDR1 for the 4-1BB agonist monoclonal
antibody
utomilumab (PF-05082566).
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[00191] SEQ ID NO:50 is the light chain CDR2 for the 4-1BB agonist monoclonal
antibody
utomilumab (PF-05082566).
[00192] SEQ ID NO:51 is the light chain CDR3 for the 4-1BB agonist monoclonal
antibody
utomilumab (PF-05082566).
[00193] SEQ ID NO:52 is the heavy chain for the 4-1BB agonist monoclonal
antibody urelumab
(BMS-663513).
[00194] SF() ID NO:53 is the light chain for the 4-1BB agonist monoclonal
antibody urelumab
(BMS-663513).
[00195] SEQ ID NO:54 is the heavy chain variable region (VH) for the 4-1BB
agonist monoclonal
antibody urelumab (BMS-663513).
[00196] SEQ ID NO:55 is the light chain variable region (VL) for the 4-1BB
agonist monoclonal
antibody urelumab (BMS-663513).
[00197] SEQ ID NO:56 is the heavy chain CDR1 for the 4-1BB agonist monoclonal
antibody
urelumab (BMS-663513).
[00198] SEQ ID NO:57 is the heavy chain CDR2 for the 4-1BB agonist monoclonal
antibody
urelumab (BMS-663513).
[00199] SEQ ID NO:58 is the heavy chain CDR3 for the 4-1BB agonist monoclonal
antibody
urelumab (BMS-663513).
[00200] SEQ ID NO:59 is the light chain CDR1 for the 4-1BB agonist monoclonal
antibody
urelumab (BMS-663513).
[00201] SEQ ID NO:60 is the light chain CDR2 for the 4-1BB agonist monoclonal
antibody
urelumab (BMS-663513).
[00202] SEQ ID NO:61 is the light chain CDR3 for the 4-1BB agonist monoclonal
antibody
ureltimab (BMS-663513),
[00203] SEQ ID NO:62 is an Fc domain for a TNFRSF agonist fusion protein.
[00204] SEQ ID NO:63 is a linker for a TNFRSF agonist fusion protein.
[00205] SEQ ID NO:64 is a linker for a TNFRSF agonist fusion protein.
[00206] SEQ ID NO:65 is a linker for a TNFRSF agonist fusion protein.
[00207] SEQ ID NO:66 is a linker for a TNFRSF agonist fusion protein.
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[00208] SEQ ID NO:67 is a linker for a TNFRSF agonist fusion protein.
[00209] SEQ ID NO:68 is a linker for a TNFRSF agonist fusion protein.
[00210] SEQ ID NO:69 is a linker for a TNFRSF agonist fusion protein.
[00211] SEQ ID NO:70 is a linker for a TNFRSF agonist fusion protein.
[00212] SEQ ID NO:71 is a linker for a TNFRSF agonist fusion protein.
[00213] SEQ ID NO:72 is a linker for a TNFRSF agonist fusion protein.
[00214] SEQ ID NO:73 is an Fc domain for a TNFRSF agonist fusion protein.
[00215] SEQ ID NO:74 is a linker for a TNFRSF agonist fusion protein.
[00216] SEQ ID NO:75 is a linker for a TNFRSF agonist fusion protein.
[00217] SEQ ID NO:76 is a linker for a TNFRSF agonist fusion protcin.
[00218] SEQ ID NO:77 is a 4-1BB ligand (4-1BBL) amino acid sequence.
[00219] SEQ ID NO:78 is a soluble portion of 4-1BBL polypeptide.
[00220] SEQ ID NO:79 is a heavy chain variable region (Vx) for the 4-1BB
agonist antibody 4B4-
1-1 version 1.
[00221] SEQ ID NO:80 is a light chain variable region (VI) for the 4-1BB
agonist antibody 4B4-1-1
version 1.
[00222] SEQ ID NO:81 is a heavy chain variable region (VH) for the 4-1BB
agonist antibody 4B4-
1-1 version 2.
[00223] SEQ ID NO:82 is a light chain variable region (VI) for the 4-1BB
agonist antibody 4B4-1-1
version 2.
[00224] SEQ ID NO:83 is a heavy chain variable region (YE) for the 4-1BB
agonist antibody
H39E3-2.
[00225] SEQ ID NO:84 is a light chain variable region (VI) for the 4-1BB
agonist antibody H39E3-
2.
[00226] SEQ ID NO:85 is the amino acid sequence of human 0X40.
[00227] SEQ ID NO:86 is the amino acid sequence of murine 0X40.
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[00228] SEQ ID NO:87 is the heavy chain for the 0X40 agonist monoclonal
antibody
tavolixizumab (MEDI-0562).
[00229] SEQ ID NO:88 is the light chain for the 0X40 agonist monoclonal
antibody tavolixizumab
(MEDI-0562).
[00230] SEQ ID NO:89 is the heavy chain variable region (VH) for the 0X40
agonist monoclonal
antibody tavolixizumab (MEDI-0562).
[00231] SEQ ID NO:90 is the light chain variable region (VI) for the 0X40
agonist monoclonal
antibody tavolixizumab (MEDI-0562).
[00232] SEQ ID NO:91 is the heavy chain CDR1 for the 0X40 agonist monoclonal
antibody
tavolixizumab (MEDI-0562).
[00233] SEQ ID NO:92 is the heavy chain CDR2 for the 0X40 agonist monoclonal
antibody
tavolixizumab (MEDI-0562).
[00234] SEQ ID NO:93 is the heavy chain CDR3 for the 0X40 agonist monoclonal
antibody
tavolixizumab (MEDI-0562).
[00235] SEQ ID NO:94 is the light chain CDR1 for the 0X40 agonist monoclonal
antibody
tavolixizumab (MEDI-0562).
[00236] SEQ ID NO:95 is the light chain CDR2 for the 0X40 agonist monoclonal
antibody
tavolixizumab (MEDI-0562).
[00237] SEQ ID NO:96 is the light chain CDR3 for the 0X40 agonist monoclonal
antibody
tavolixizumab (MEDI-0562).
[00238] SEQ ID NO:97 is the heavy chain for the 0X40 agonist monoclonal
antibody 11D4.
[00239] SEQ ID N0:98 is the light chain for the 0X40 agonist monoclonal
antibody 11D4.
[00240] SEQ ID NO:99 is the heavy chain variable region (VE) for the 0X40
agonist monoclonal
antibody 11D4.
[00241] SEQ ID NO:100 is the light chain variable region (VI) for the 0X40
agonist monoclonal
antibody 11D4.
[00242] SEQ ID NO:101 is the heavy chain CDR1 for the 0X40 agonist monoclonal
antibody
11D4.
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[00243] SEQ ID NO:102 is the heavy chain CDR2 for the 0X40 agonist monoclonal
antibody
11D4.
[00244] SEQ ID NO:103 is the heavy chain CDR3 for the 0X40 agonist monoclonal
antibody
11D4.
[00245] SEQ ID NO:104 is the light chain CDR1 for the 0X40 agonist monoclonal
antibody 11D4.
[00246] SEQ ID NO:105 is the light chain CDR2 for the OX40 agonist monoclonal
antibody 11D4.
[00247] SEQ ID NO:106 is the light chain CDR3 for the OX40 agonist monoclonal
antibody 11D4.
[00248] SEQ ID NO:107 is the heavy chain for the OX40 agonist monoclonal
antibody 18D8.
[00249] SEQ ID NO:108 is the light chain for the 0X40 agonist monoclonal
antibody 18D8.
[00250] SEQ ID NO:109 is the heavy chain variable region (VH) for the 0X40
agonist monoclonal
antibody 18D8.
[00251] SEQ ID NO:110 is the light chain variable region (VI) for the 0X40
agonist monoclonal
antibody 18D8.
[00252] SEQ ID NO:111 is the heavy chain CDR1 for the 0X40 agonist monoclonal
antibody
18D8.
[00253] SEQ ID NO:112 is the heavy chain CDR2 for the 0X40 agonist monoclonal
antibody
18D8.
[00254] SEQ ID NO:113 is the heavy chain CDR3 for the 0X40 agonist monoclonal
antibody
18D8.
[00255] SEQ ID NO:114 is the light chain CDR1 for the OX40 agonist monoclonal
antibody 18D8.
[00256] SEQ ID NO:115 is the light chain CDR2 for the 0X40 agonist monoclonal
antibody 18D8.
[00257] SEQ ID NO:116 is the light chain CDR3 for the OX40 agonist monoclonal
antibody 18D8.
[00258] SEQ ID NO:117 is the heavy chain variable region (VH) for the 0X40
agonist monoclonal
antibody Hu119-122.
[00259] SEQ ID NO:118 is the light chain variable region (VI) for the 0X40
agonist monoclonal
antibody Hu I 19-122.
[00260] SEQ ID NO:119 is the heavy chain CDR1 for the OX40 agonist monoclonal
antibody
Hu119-122.
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[00261] SEQ ID NO:120 is the heavy chain CDR2 for the 0X40 agonist monoclonal
antibody
Hu119-122.
[00262] SEQ ID NO:121 is the heavy chain CDR3 for the 0X40 agonist monoclonal
antibody
Hu119-122.
[00263] SEQ ID NO:122 is the light chain CDR1 for the 0X40 agonist monoclonal
antibody
Hu119-122.
[00264] SEQ ID NO:] 23 is the light chain CDR2 for the 0X40 agonist monoclonal
antibody
Hu119-122.
[00265] SEQ ID NO:124 is the light chain CDR3 for the 0X40 agonist monoclonal
antibody
Hu119-122.
[00266] SEQ ID NO:125 is the heavy chain variable region (VII) for the 0X40
agonist monoclonal
antibody Hu106-222.
[00267] SEQ ID NO: 126 is the light chain variable region (VI) for the 0X40
agonist monoclonal
antibody Hu106-222.
[00268] SEQ ID NO:127 is the heavy chain CDR1 for the 0X40 agonist monoclonal
antibody
Hu106-222.
[00269] SEQ ID NO:128 is the heavy chain CDR2 for the 0X40 agonist monoclonal
antibody
Hu106-222.
[00270] SEQ ID NO:129 is the heavy chain CDR3 for the 0X40 agonist monoclonal
antibody
Hu106-222.
[00271] SEQ ID NO:130 is the light chain CDR1 for the OX40 agonist monoclonal
antibody
Hu106-222.
[00272] SEQ ID NO:131 is the light chain CDR2 for the OX40 agonist monoclonal
antibody
Hu106-222.
[00273] SEQ ID NO:132 is the light chain CDR3 for the 0X40 agonist monoclonal
antibody
Hu106-222.
[00274] SEQ ID NO:133 is an 0X40 ligand (OX4OL) amino acid sequence.
[00275] SEQ ID NO:134 is a soluble portion of OX4OL polypeptide.
[00276] SEQ ID NO:135 is an alternative soluble portion of OX4OL polypeptide.
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[00277] SEQ ID NO:136 is the heavy chain variable region (VH) for the 0X40
agonist monoclonal
antibody 008.
[00278] SEQ ID NO:137 is the light chain variable region (VI) for the 0X40
agonist monoclonal
antibody 008.
[00279] SEQ ID NO:138 is the heavy chain variable region (VH) for the 0X40
agonist monoclonal
antibody 011.
[00280] SEQ ID NO:139 is the light chain variable region (VI) for the 0X40
agonist monoclonal
antibody 011.
[00281] SEQ ID NO:140 is the heavy chain variable region (VH) for the 0X40
agonist monoclonal
antibody 021.
[00282] SEQ ID NO:141 is the light chain variable region (VI) for the 0X40
agonist monoclonal
antibody 021.
[00283] SEQ ID NO: 142 is the heavy chain variable region (VH) for the 0X40
agonist monoclonal
antibody 023.
[00284] SEQ ID NO:143 is the light chain variable region (VI) for the 0X40
agonist monoclonal
antibody 023.
[00285] SEQ ID NO:144 is the heavy chain variable region (VH) for an 0X40
agonist monoclonal
antibody.
[00286] SEQ ID NO:145 is the light chain variable region (VI) for an OX40
agonist monoclonal
antibody.
[00287] SEQ ID NO:146 is the heavy chain variable region (VH) for an 0X40
agonist monoclonal
antibody.
[00288] SEQ ID NO:147 is the light chain variable region NO for an OX40
agonist monoclonal
antibody.
[00289] SEQ ID NO:148 is the heavy chain variable region (VH) for a humanized
0X40 agonist
monoclonal antibody.
[00290] SEQ ID NO:149 is the heavy chain variable region (VH) for a humanized
OX40 agonist
monoclonal antibody.
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[00291] SEQ ID NO:150 is the light chain variable region (VI) for a humanized
0X40 agonist
monoclonal antibody.
[00292] SEQ ID NO:151 is the light chain variable region (VI) for a humanized
OX40 agonist
monoclonal antibody.
[00293] SEQ ID NO:152 is the heavy chain variable region (VH) for a humanized
0X40 agonist
monoclonal antibody.
[00294] SEQ ID NO:] 53 is the heavy chain variable region (VII) for a
humanized 0X40 agonist
monoclonal antibody.
[00295] SEQ ID NO:154 is the light chain variable region (VI) for a humanized
0X40 agonist
monoclonal antibody.
[00296] SEQ ID NO:155 is the light chain variable region (VI) for a humanized
OX40 agonist
monoclonal antibody.
[00297] SEQ ID NO: 156 is the heavy chain variable region (VI) for an 0X40
agonist monoclonal
antibody.
[00298] SEQ ID NO:157 is the light chain variable region (VI) for an OX40
agonist monoclonal
antibody.
[00299] SEQ ID NO:158 is the heavy chain amino acid sequence of the PD-1
inhibitor nivolumab.
[00300] SEQ ID NO:159 is the light chain amino acid sequence of the PD-1
inhibitor nivolumab.
[00301] SEQ ID NO:160 is the heavy chain variable region (Vu) amino acid
sequence of the PD-1
inhibitor nivolumab.
[00302] SEQ ID NO:161 is the light chain variable region (VI) amino acid
sequence of the PD-1
inhibitor nivolumab.
[00303] SEQ ID NO:162 is the heavy chain CDR1 amino acid sequence of the PD-1
inhibitor
nivolumab.
[00304] SEQ ID NO:163 is the heavy chain CDR2 amino acid sequence of the PD-1
inhibitor
nivolumab.
[00305] SEQ ID NO:164 is the heavy chain CDR3 amino acid sequence of the PD-1
inhibitor
nivolumab.
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[00306] SEQ ID NO:165 is the light chain CDR1 amino acid sequence of the PD-1
inhibitor
nivolumab.
[00307] SEQ ID NO: i66 is the light chain CDR2 amino acid sequence of the PD-1
inhibitor
nivolumab.
[00308] SEQ ID NO:167 is the light chain CDR3 amino acid sequence of the PD-1
inhibitor
nivolumab.
[00309] SEQ ID NO:168 is the heavy chain amino acid sequence ofthe PD-1
inhibitor
pembrolizumab.
[00310] SEQ ID NO: i69 is the light chain amino acid sequence of the PD-1
inhibitor
pembrolizumab.
[00311] SEQ ID NO: i70 is the heavy chain variable region (VII) amino acid
sequence of the PD-1
inhibitor pembrolizumab.
[00312] SEQ ID NO:171 is the light chain variable region (VI) amino acid
sequence of the PD-1
inhibitor pembrolizumab.
[00313] SEQ ID NO: i72 is the heavy chain CDR1 amino acid sequence of the PD-1
inhibitor
pembrolizumab.
[00314] SEQ ID NO: i73 is the heavy chain CDR2 amino acid sequence of the PD-1
inhibitor
pembrolizumab.
[00315] SEQ ID NO:174 is the heavy chain CDR3 amino acid sequence of the PD-1
inhibitor
pembrolizumab.
[00316] SEQ ID NO:175 is the light chain CDR1 amino acid sequence of the PD-1
inhibitor
pembrolizumab.
[00317] SEQ ID NO: i76 is the light chain CDR2 amino acid sequence of the PD-1
inhibitor
pembrolizumab.
[00318] SEQ ID NO: i77 is the light chain CDR3 amino acid sequence of the PD-1
inhibitor
pembrolizumab.
[00319] SEQ ID NO:178 is the heavy chain amino acid sequence of the PD-Li
inhibitor
durvalumab.
[00320] SEQ ID NO: i79 is the light chain amino acid sequence of the PD-Li
inhibitor durvalumab.
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[00321] SEQ ID NO:180 is the heavy chain variable region (VH) amino acid
sequence of the PD-Li
inhibitor durvalumab.
[00322] SEQ ID NO:181 is the light chain variable region (VI) amino acid
sequence of the PD-Li
inhibitor durvalumab.
[00323] SEQ ID NO:182 is the heavy chain CDR1 amino acid sequence of the PD-L1
inhibitor
durvalumab.
[00324] SEQ ID NO:] 83 is the heavy chain CDR2 amino acid sequence of the PD-
I,1 inhibitor
durvalumab.
[00325] SEQ ID NO:184 is the heavy chain CDR3 amino acid sequence of the PD-Ll
inhibitor
durvalumab.
[00326] SEQ ID NO:185 is the light chain CDR1 amino acid sequence of the PD-L1
inhibitor
durvalumab.
[00327] SEQ ID NO: 186 is the light chain CDR2 amino acid sequence of the PD-L
I inhibitor
durvalumab.
[00328] SEQ ID NO:187 is the light chain CDR3 amino acid sequence of the PD-Li
inhibitor
durvalumab.
[00329] SEQ ID NO:188 is the heavy chain amino acid sequence of the PD-L1
inhibitor avelumab.
[00330] SEQ ID NO:189 is the light chain amino acid sequence of the PD-L1
inhibitor avelumab.
[00331] SEQ ID NO:190 is the heavy chain variable region (Vu) amino acid
sequence of the PD-Li
inhibitor avelumab.
[00332] SEQ ID NO:191 is the light chain variable region (VI) amino acid
sequence of the PD-Li
inhibitor avelumab.
[00333] SEQ ID NO:192 is the heavy chain CDR1 amino acid sequence of the PD-Ll
inhibitor
avelumab.
[00334] SEQ ID NO:193 is the heavy chain CDR2 amino acid sequence of the PD-Ll
inhibitor
avelumab.
[00335] SEQ ID NO:194 is the heavy chain CDR3 amino acid sequence of the PD-Ll
inhibitor
avelumab.
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[00336] SEQ ID NO:195 is the light chain CDR1 amino acid sequence of the PD-Li
inhibitor
avelumab.
[00337] SEQ ID NO: i96 is the light chain CDR2 amino acid sequence of the PD-
Li inhibitor
avelumab.
[00338] SEQ ID NO: i97 is the light chain CDR3 amino acid sequence of the PD-
L1 inhibitor
avelumab.
[00339] SEQ ID NO:198 is the heavy chain amino acid sequence ofthe PD-Li
inhibitor
atezolizumab.
[00340] SEQ ID NO:199 is the light chain amino acid sequence of the PD-Li
inhibitor
atezolizumab.
[00341] SEQ ID NO:200 is the heavy chain variable region (VII) amino acid
sequence of the PD-Li
inhibitor atezolizumab.
[00342] SEQ ID NO:201 is the light chain variable region (VI) amino acid
sequence of the PD-L I
inhibitor atezolizumab.
[00343] SEQ ID NO:202 is the heavy chain CDR1 amino acid sequence of the PD-L1
inhibitor
atezolizumab.
[00344] SEQ ID NO:203 is the heavy chain CDR2 amino acid sequence of the PD-Li
inhibitor
atezolizumab.
[00345] SEQ ID NO:204 is the heavy chain CDR3 amino acid sequence of the PD-L1
inhibitor
atezolizumab.
[00346] SEQ ID NO:205 is the light chain CDR1 amino acid sequence of the PD-Li
inhibitor
atezolizumab.
[00347] SEQ ID NO:206 is the light chain CDR2 amino acid sequence of the PD-Li
inhibitor
atezolizumab.
[00348] SEQ ID NO:207 is the light chain CDR3 amino acid sequence of the PD-Li
inhibitor
atezolizumab.
[00349] SEQ ID NO:208 is the heavy chain amino acid sequence of the CTLA-4
inhibitor
ipilimumab.
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[00350] SEQ ID NO:209 is the light chain amino acid sequence of the CTLA-4
inhibitor
ipilimumab.
[00351] SEQ ID NO:210 is the heavy chain variable region (VII) amino acid
sequence of the CTLA-
4 inhibitor ipilimumab.
[00352] SEQ ID NO:211 is the light chain variable region (VL) amino acid
sequence of the CTLA-4
inhibitor ipilimumab.
[00353] SEQ ID NO:212 is the heavy chain CDR] amino acid sequence of the CTLA-
4 inhibitor
ipilimumab.
[00354] SEQ ID NO:213 is the heavy chain CDR2 amino acid sequence of the CTLA-
4 inhibitor
ipilimumab.
[00355] SEQ ID NO:214 is the heavy chain CDR3 amino acid sequence of the CTLA-
4 inhibitor
ipilimumab.
[00356] SEQ ID NO:215 is the light chain CDR1 amino acid sequence of the CTLA-
4 inhibitor
ipilimumab.
[00357] SEQ ID NO:216 is the light chain CDR2 amino acid sequence of the CTLA-
4 inhibitor
ipilimumab.
[00358] SEQ ID NO:217 is the light chain CDR3 amino acid sequence of the CTLA-
4 inhibitor
ipilimumab.
[00359] SEQ ID NO:218 is the heavy chain amino acid sequence of the CTLA-4
inhibitor
tremelimumab.
[00360] SEQ ID NO:219 is the light chain amino acid sequence of the CTLA-4
inhibitor
tremelimumab.
[00361] SEQ ID NO:220 is the heavy chain variable region (VII) amino acid
sequence of the CTLA-
4 inhibitor tremelimumab.
[00362] SEQ ID NO:221 is the light chain variable region (VI) amino acid
sequence of the CTLA-4
inhibitor tremelimumab.
[00363] SEQ ID NO:222 is the heavy chain CDR1 amino acid sequence of the CTLA-
4 inhibitor
tremelimumab.
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[00364] SEQ ID NO:223 is the heavy chain CDR2 amino acid sequence of the CTLA-
4 inhibitor
tremelimumab.
[00365] SEQ ID NO:224 is the heavy chain CDR3 amino acid sequence of the CTLA-
4 inhibitor
tremelimumab.
[00366] SEQ ID NO:225 is the light chain CDR1 amino acid sequence of the CTLA-
4 inhibitor
tremelimumab.
[00367] SEQ ID NO:226 is the light chain CDR2 amino acid sequence ofthe CTT,A-
4 inhibitor
tremelimumab.
[00368] SEQ ID NO:227 is the light chain CDR3 amino acid sequence of the CTLA-
4 inhibitor
tremelimumab.
[00369] SEQ ID NO:228 is the heavy chain amino acid sequence of the CTLA-4
inhibitor
zalifrelimab.
[00370] SEQ ID NO:229 is the light chain amino acid sequence of the CTLA-4
inhibitor
zalifrelimab.
[00371] SEQ ID NO:230 is the heavy chain variable region (Vii) amino acid
sequence of the CTLA-
4 inhibitor zalifrelimab.
[00372] SEQ ID NO:231 is the light chain variable region (VI) amino acid
sequence of the CTLA-4
inhibitor zalifrelimab.
[00373] SEQ ID NO:232 is the heavy chain CDR1 amino acid sequence of the CTLA-
4 inhibitor
zalifrelimab.
[00374] SEQ ID NO:233 is the heavy chain CDR2 amino acid sequence of the CTLA-
4 inhibitor
zalifrelimab.
[00375] SEQ ID NO:234 is the heavy chain CDR3 amino acid sequence of the CTLA-
4 inhibitor
zalifrelimab.
[00376] SEQ ID NO:235 is the light chain CDR1 amino acid sequence of the CTLA-
4 inhibitor
zalifrelimab.
[00377] SEQ ID NO:236 is the light chain CDR2 amino acid sequence of the CTLA-
4 inhibitor
zalifrclimab.
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[00378] SEQ ID NO:237 is the light chain CDR3 amino acid sequence of the CTLA-
4 inhibitor
zalifrelimab.
DETAILED DESCRIPTION OF THE INVENTION
I. Introduction
[00379] Adoptive cell therapy utilizing TILs cultured ex vivo by the Rapid
Expansion Protocol
(REP) has produced successful adoptive cell therapy following host
immunosuppression in patients
with cancer such as melanoma. Current infusion acceptance parameters rely on
readouts of the
composition of TILs (e.g., CD28, CD8, or CD4 positivity) and on the numerical
folds of expansion
and viability of the REP product.
[00380] Current REP protocols give little insight into the health of the TIL
that will be infused into
the patient. T cells undergo a profound metabolic shift during the course of
their maturation from
naive to effector T cells (see Chang, et at., Nat. Immunol. 2016, 17 364,
hereby expressly
incorporated in its entirety, and in particular for the discussion and markers
of anaerobic and aerobic
metabolism). For example, naïve T cells rely on mitochondrial respiration to
produce ATP, while
mature, healthy effector T cells such as TIL are highly glycolytic, relying on
aerobic glycolysis to
provide the bioenergetics substrates they require for proliferation,
migration, activation, and anti-
tumor efficacy.
[00381] Current TIL manufacturing and treatment processes are limited by
length, cost, sterility
concerns, and other factors described herein such that the potential to treat
patients which are
refractory to anti-PD1 and as such have been severly limited. There is an
urgent need to provide TIL
manufacturing processes and therapies based on such processes that are
appropriate for use in treating
patients for whom very few or no viable treatment options remain. The present
invention meets this
need by providing a shortened manufacturing process for use in generating TILs
which can then be
employed in the treatment of non-small cell lung carcinoma (NSCLC) patients
whom are refractory to
anti-PD-1 treatment.
Definitions
[00382] Unless defined otherwise, all technical and scientific terms used
herein have the same
meaning as is commonly understood by one of skill in the art to which this
invention belongs. All
patents and publications referred to herein are incorporated by reference in
their entireties.
[00383] The terms "co-administration," "co-administering,"
"administered in combination
with, "administering in combination with," "simultaneous," and "concurrent,"
as used herein,
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encompass administration of two or more active pharmaceutical ingredients (in
a preferred
embodiment of the present invention, for example, a plurality of TILs) to a
subject so that both active
pharmaceutical ingredients and/or their metabolites are present in the subject
at the same time. Co-
administration includes simultaneous administration in separate compositions,
administration at
different times in separate compositions, or administration in a composition
in which two or more
active pharmaceutical ingredients arc present. Simultaneous administration in
separate compositions
and administration in a composition in which both agents are present are
preferred.
[00384] The term "in viva" refers to an event that takes place in a subject's
body.
[00385] The term "in vitro" refers to an event that takes places outside of a
subject's body. In vitro
assays encompass cell-based assays in which cells alive or dead are employed
and may also
encompass a cell-free assay in which no intact cells are employed.
[00386] The term -ex vivo" refers to an event which involves treating or
performing a procedure on
a cell, tissue and/or organ which has been removed from a subject's body.
Aptly, the cell, tissue
and/or organ may be returned to the subject's body in a method of surgery or
treatment.
[00387] The term "rapid expansion" means an increase in the
number of antigen-specific TILs
of at least about 3-fold (or 4-, 5-, 6-, 7-, 8-, or 9-fold) over a period of a
week, more preferably at least
about 10-fold (or 20-, 30-, 40-, 50-, 60-, 70-, 80-, or 90-fold) over a period
of a week, or most
preferably at least about 100-fold over a period of a week. A number of rapid
expansion protocols are
described herein.
[00388] By "tumor infiltrating lymphocytes" or -TILs" herein is meant a
population of cells
originally obtained as white blood cells that have left the bloodstream of a
subject and migrated into a
tumor. TILs include, but are not limited to, CD8 cytotoxic T cells
(lymphocytes), Thl and Th17
CD4 T cells, natural killer cells, dendritic cells and M1 macrophages. TILs
include both primary and
secondary TILs. "Primary TILs" are those that are obtained from patient tissue
samples as outlined
herein (sometimes referred to as "freshly harvested"), and "secondary TILs"
are any TIL cell
populations that have been expanded or proliferated as discussed herein,
including, but not limited to
bulk TILs and expanded TILs (REP TILs" or -post-REP TILs"). TIL cell
populations can include
genetically modified TILs.
[00389] By "population of cells" (including TILs) herein is meant a number of
cells that share
common traits. In general, populations generally range from 1 X 106 to 1 X
1010 in number, with
different TIL populations comprising different numbers. For example, initial
growth of primary TILs
in the presence of IL-2 results in a population of bulk TILs of roughly 1 ><
108 cells. REP expansion is
generally done to provide populations of 1.5 x 109 to 1.5 x 101 cells for
infusion.
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[00390] By "cryopreserved TILs" herein is meant that TILs, either primary,
bulk, or expanded (REP
TILs), are treated and stored in the range of about -150 C to -60 C. General
methods for
cryopreservation are also described elsewhere herein, including in the
Examples. For clarity,
cryopreserved TILs" are distinguishable from frozen tissue samples which may
be used as a source
of primary TILs.
[00391] By "thawed cryopreserved TILs" herein is meant a population of TILs
that was previously
cryopreserved and then treated to return to room temperature or higher,
including but not limited to
cell culture temperatures or temperatures wherein TILs may be administered to
a patient.
[00392] TILs can generally be defined either biochemically, using cell surface
markers, or
functionally, by their ability to infiltrate tumors and effect treatment. TILs
can be generally
categorized by expressing one or more of the following biomarkers: CD4, CD8,
TCR aI3, CD27,
CD28, CD56, CCR7, CD45Ra, CD95, PD-1, and CD25. Additionally and
alternatively, TILs can be
functionally defined by their ability to infiltrate solid tumors upon
reintroduction into a patient.
[00393] The term "cryopreservation media" or "cryopreservation medium" refers
to any medium
that can be used for cryopreservation of cells. Such media can include media
comprising 7% to 10%
DMSO. Exemplary media include CryoStor CS10, Hyperthermasol, as well as
combinations thereof.
The term "CS10" refers to a cryopreservation medium which is obtained from
Stemcell Technologies
or from Biolifc Solutions. The CS10 medium may be referred to by the trade
name "CryoStor0
CS10". The CS10 medium is a serum-free, animal component-free medium which
comprises DMSO.
[00394] The term -central memory T refers to a subset of T cells that
in the human arc
CD45R0+ and constitutively express CCR7 (CCR7111) and CD62L (CD62111). The
surface phenotype of
central memory T cells also includes TCR, CD3, CD127 (IL-7R), and IL-15R.
Transcription factors
for central memory T cells include BCL-6, BCL-6B, MBD2, and BMIl. Central
memory T cells
primarily secret IL-2 and CD4OL as effector molecules after TCR triggering.
Central memory T cells
are predominant in the CD4 compartment in blood, and in the human are
proportionally enriched in
lymph nodes and tonsils.
[00395] The term "effector memory T cell" refers to a subset of human or
mammalian T cells that,
like central memory T cells, are CD45R0+, but have lost the constitutive
expression of CCR7
(CCR71 ) and are heterogeneous or low for CD62L expression (CD62L1 ). The
surface phenotype of
central memory '1 cells also includes "'CR, CD3, CD127 (1L-7R), and 1L-15R.
Transcription factors
for central memory T cells include BLIMP 1. Effector memory T cells rapidly
secret high levels of
inflammatory cytokines following antigenic stimulation, including interferon-
y, IL-4, and IL-5.
Effector memory T cells are predominant in the CD8 compartment in blood, and
in the human are
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proportionally enriched in the lung, liver, and gut. CD8+ effector memory T
cells carry large amounts
of perforM.
[00396] The term "closed system" refers to a system that is closed to the
outside environment. Any
closed system appropriate for cell culture methods can be employed with the
methods of the present
invention. Closed systems include, for example, but are not limited to closed
G-containers. Once a
tumor segment is added to the closed system, the system is no opened to the
outside environment until
the TILs are ready to be administered to the patient.
[00397] The terms "fragmenting," "fragment," and "fragmented," as used herein
to describe
processes for disrupting a tumor, includes mechanical fragmentation methods
such as crushing,
slicing, dividing, and morcellating tumor tissue as well as any other method
for disrupting the
physical structure of tumor tissue.
[00398] The terms -peripheral blood mononuclear cells" and -PBMCs" refers to a
peripheral blood
cell having a round nucleus, including lymphocytes (T cells, B cells, NK
cells) and monocytes. When
used as an antigen presenting cell (PBMCs are a type of antigen-presenting
cell), the peripheral blood
mononuclear cells are preferably irradiated allogeneic peripheral blood
mononuclear cells.
[00399] The terms "peripheral blood lymphocytes" and "PBLs" refer to T cells
expanded from
peripheral blood. In some embodiments, PBLs arc separated from whole blood or
aphcrcsis product
from a donor. In some embodiments, PBLs are separated from whole blood or
apheresis product from
a donor by positive or negative selection of a T cell phenotype, such as the T
cell phenotype of CD3+
CD45+.
[00400] The term "anti-CD3 antibody" refers to an antibody or variant thereof,
e.g., a monoclonal
antibody and including human, humanized, chimeric or murine antibodies which
are directed against
the CD3 receptor in the T cell antigen receptor of mature T cells. Anti-CD3
antibodies include OKT-
3, also known as muromonab. Anti-CD3 antibodies also include the UHCT1 clone,
also known as T3
and CD3e. Other anti-CD3 antibodies include, for example, otelixizumab,
teplizumab, and
visilizumab.
[00401] The term "OKT-3" (also referred to herein as "OKT3") refers to a
monoclonal antibody or
biosimilar or variant thereof, including human, humanized, chimeric, or murine
antibodies, directed
against the CD3 receptor in the T cell antigen receptor of mature T cells, and
includes commercially-
available forms such as OKT-3 (30 ng/mL, MACS GMP CD3 pure, Miltenyi Biotech,
Inc., San
Diego, CA, USA) and muromonab or variants, conservative amino acid
substitutions, glycoforms, or
biosimilars thereof The amino acid sequences of the heavy and light chains of
muromonab are given
in Table 1 (SEQ ID NO:1 and SEQ ID NO:2). A hybridoma capable of producing OKT-
3 is deposited
37
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with the American Type Culture Collection and assigned the ATCC accession
number CRL 8001. A
hybridoma capable of producing OKT-3 is also deposited with European
Collection of Authenticated
Cell Cultures (ECACC) and assigned Catalogue No. 86022706.
TABLE 1. Amino acid sequences of muromonab (exemplary OKT-3 antibody).
Identifier Sequence (One-Letter Amino Acid
Symbols)
SEQ ED NO:1 QVQLQQSGAE LARPGASVKM SCKASGYTYT RYTMHWVKQR
PGQGLEWIGY INPSRGYTNY 60
muromonab heavy NQKFKDKATL TTDHSSOTAY MQLSSLTSED SAVYYCARYY
DDNYCLDYWG Q3TTLTVSSA 120
chain KTTAPSVYPL APVCGGTTGS SVTLGCLVKG YFPEPVTLTW
NSGSLSSGVH TEPAVLQSDL 180
YTLSSSVTVT SSTWPSQSIT CNVAHPASST KVDKKIEPRP KSCDKTNTCP PCPAPELLGG
240
PSVFLFPPKP KDTLMISRTP EVTCVVVDVS HEDPEVKFNW YVDGVEVHNA KTKPREEQYN
300
STYRVVSVLT VLHQDWLNGK EYKCKVSNKA LPAPIEKTIS KAKGQPREPQ VYTLPPSRDE
360
LTENQVSLTC LVKGFYPSDI AAIEWESNGQP ENNYKTTPPV LDSDGSFFLY SKLTVDKSRW
420
QQGNVFSCSV MHEALHNHYT QKSLSLSPGK
450
SEQ ED NO:2 QIVLTQSPAI MSASPGEKVT MTCSASSSVS YMNWYQQHSG
TSPKRWIYDT SHLASGVPAN 60
muromonab light FRGSGSGTSY SLTISGMEAE DAATYYCQQW 5SNPFTEGSG
THLEINRADT APTVSIFFP5 120
chain SEQLTSGGAS VVCFLNNFYP KDINVKWKID GSERQNGVLN
SWTDQDSKDS TYSMSSTLTL 180
TKDEYERNNS YTCLATNKTS TSPIVKS.LNR NEC
213
[00402] The term -IL-2" (also referred to herein as -IL2") refers to the T
cell growth factor known
as interleukin-2, and includes all forms of IL-2 including human and mammalian
forms, conservative
amino acid substitutions, glycoforms, biosimilars, and variants thereof. IL-2
is described, e.g., in
Nelson, I Immunol. 2004, 172, 3983-88 and Malek, Annit. Rev. Immunol. 2008,
26, 453-79, the
disclosures of which are incorporated by reference herein. The amino acid
sequence of recombinant
human IL-2 suitable for use in the invention is given in Table 2 (SEQ ID
NO:3). For example, the
term IL-2 encompasses human, recombinant forms of IL-2 such as aldesleukin
(PROLEUKIN,
available commercially from multiple suppliers in 22 million 1U per single use
vials), as well as the
form of recombinant IL-2 commercially supplied by CellGenix, Inc., Portsmouth,
NH, USA
(CELLGRO GMP) or ProSpec-Tany TechnoGene Ltd., East Brunswick, NJ, USA (Cat.
No. CYT-
209-b) and other commercial equivalents from other vendors. Aldesleukin (des-
alanyl-1, serine-125
human IL-2) is a nonglycosylated human recombinant form of 1L-2 with a
molecular weight of
approximately 15 kDa. The amino acid sequence of aldesleukin suitable for use
in the invention is
given in Table 2 (SEQ ID NO:4). The term IL-2 also encompasses pegylated forms
of IL-2, as
described herein, including the pegylated 1L2 prodrug bempegaldesleukin (NKTR-
214, pegylated
human recombinant IL-2 as in SEQ ID NO:4 in which an average of 6 lysine
residues are N6
substituted with [(2,7-his{ rmethylpoly(oxyethylene)lcarbamoyl -9H-fluoren-9-
yl)methoxylcarbonyl),
which is available from Nektar Therapeutics, South San Francisco, CA, USA, or
which may be
prepared by methods known in the art, such as the methods described in Example
19 of International
Patent Application Publication No. WO 2018/132496 Al or the method described
in Example 1 of
U.S. Patent Application Publication No. US 2019/0275133 Al, the disclosures of
which are
incorporated by reference herein. Bempegaldesleukin (NKTR-214) and other
pcgylated IL-2
molecules suitable for use in the invention is described in U.S. Patent
Application Publication No. US
38
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2014/0328791 Al and International Patent Application Publication No. WO
2012/065086 Al, the
disclosures of which are incorporated by reference herein. Alternative forms
of conjugated IL-2
suitable for use in the invention are described in U.S. Patent Nos. 4,766,106,
5,206,344, 5,089,261
and 4,902,502, the disclosures of which are incorporated by reference herein.
Formulations of IL-2
suitable for use in the invention are described in U.S. Patent No. 6,706,289,
the disclosure of which is
incorporated by reference herein.
[00403] In some embodiments, an 1L-2 form suitable for use in the
present invention is
THOR-707. available from Synthorx, Inc. The preparation and properties of THOR-
707 and
additional alternative forms of IL-2 suitable for use in the invention are
described in U.S. Patent
Application Publication Nos. US 2020/0181220 Al and US 2020/0330601 Al, the
disclosures of
which are incorporated by reference herein. In some embodiments, and IL-2 form
suitable for use in
the invention is an interleukin 2 (IL-2) conjugate comprising: an isolated and
purified IL-2
polypeptide; and a conjugating moiety that binds to the isolated and purified
IL-2 polypeptide at an
amino acid position selected from K35, T37, R38, T41, F42, K43, F44, Y45, E61,
E62, E68, K64,
P65. V69, L72, and Y107, wherein the numbering of the amino acid residues
corresponds to SEQ ID
NO:5. In some embodiments, the amino acid position is selected from T37, R38,
141, F42, F44, Y45,
E61, E62, E68, K64, P65, V69, L72, and Y107. In some embodiments, the amino
acid position is
selected from T37, R38, T41, F42, F44, Y45, E61, E62, E68, P65, V69, L72, and
Y107. In some
embodiments, the amino acid position is selected from T37, T41, F42, F44, Y45,
P65, V69, L72, and
Y107. In some embodiments, the amino acid position is selected from R38 and
K64. In some
embodiments, the amino acid position is selected from E61, E62, and E68. In
some embodiments, the
amino acid position is at E62. In some embodiments, the amino acid residue
selected from K35, T37,
R38, T41, F42, K43, F44, Y45, E61, E62, E68, K64, P65, V69, L72, and Y107 is
further mutated to
lysine, cysteine, or histidine. In some embodiments, the amino acid residue is
mutated to cysteine. In
some embodiments, the amino acid residue is mutated to lysine. In some
embodiments, the amino
acid residue selected from K35, T37, R38, T41, F42, K43, F44, Y45, E61, E62,
E68, K64, P65, V69,
L72, and Y107 is further mutated to an unnatural amino acid. In some
embodiments, the unnatural
amino acid comprises N6-azidoethoxy-L-lysine (AzK), N6-propargylethoxy-L-
lysine (PraK), BCN-L-
lysine, norbornene lysine, TCO-lysinc, methyltetrazine lysine,
allyloxycarbonyllysine, 2-amino-8-
oxononanoic acid, 2-amino-8-oxooctanoic acid, p-acetyl-L-phenylalanine, p-
azidomethyl-L-
phenylalanine (pAMF), p-iodo-L-phenylalanine, m-acetylphenylalanine, 2-amino-8-
oxononanoic
acid, p-propargyloxyphenylalanine, p-propargyl-phenylalanine, 3-methyl-
phenylalanine, L-Dopa,
fluorinated phenylalanine, isopropyl-L-phenylalanine, p-azido-L-phenylalanine,
p-acyl-L-
phenylalanine, p-benzoyl-L-phenylalanine, p-bromophenylalanine, p-amino-L-
phenylalanine,
isopropyl-L-phenylalanine, 0-allyltyrosine, 0-methyl-L-tyrosine, 0-4-allyl-L-
tyrosine, 4-propyl-L-
tyrosine, phosphonotyrosine, tri-O-acetyl-G1cNAcp-serine, L-phosphoserine,
phosphonoserine, L-3-
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(2-naph-thypalanine, 2-amino-3-((2-((3-(benzyloxy)-3-
oxopropyl)amino)ethyl)selanyl)propanoic acid,
2-amino-3-(phenvlselanyl)propanoic, or selenocysteine. In some embodiments,
the IL-2 conjugate has
a decreased affinity to TL-2 receptor a (IL-2Ra) subunit relative to a wild-
type 1L-2 polypeptide. In
some embodiments, the decreased affinity is about 10%, 20%, 30%, 40%, 50%,
60%, 70%, 80%,
90%, 95%, 99%, or greater than 99% decrease in binding affinity to IL-2Ra
relative to a wild-type IL-
2 polypeptide. In some embodiments, the decreased affinity is about 1-fold, 2-
fold, 3-fold, 4-fold, 5-
fold, 6-fold, 7-fold, 8-fold, 9-fold, 10-fold, 30-fold, 50-fold, 100-fold, 200-
fold, 300-fold, 500-fold,
1000-fold, or more relative to a wild-type IL-2 polypeptide. In some
embodiments, the conjugating
moiety impairs or blocks the binding of IL-2 with IL-2Ra. In some embodiments,
the conjugating
moiety comprises a water-soluble polymer. In some embodiments, the additional
conjugating moiety
comprises a water-soluble polymer. In some embodiments, each of the water-
soluble polymers
independently comprises polyethylene glycol (PEG), poly(propylene glycol)
(PPG), copolymers of
ethylene glycol and propylene glycol, poly(oxyethylatcd polyol), poly(olcfinic
alcohol),
poly(vinylpyrrolidone), poly(hydroxyalkylmethacrylamide),
poly(hydroxyalkylmethacrylate),
poly(saccharides), poly(a-hydroxy acid), poly(vinyl alcohol), polyphosphazene,
polyoxazolines
(POZ), poly(N-acryloylmorpholine), or a combination thereof. In some
embodiments, each of the
water-soluble polymers independently comprises PEG. In some embodiments, the
PEG is a linear
PEG or a branched PEG. In some embodiments, each of the water-soluble polymers
independently
comprises a polysaccharide. In some embodiments, the polysaccharide comprises
dextran, polysialic
acid (PSA), hyaluronic acid (HA), amylose, heparin, heparan sulfate (HS),
dextrin, or hydroxyethyl-
starch (HES). In some embodiments, each of the water-soluble polymers
independently comprises a
glycan. In some embodiments, each of the water-soluble polymers independently
comprises
polyamine. In some embodiments, the conjugating moiety comprises a protein. In
some embodiments,
the additional conjugating moiety comprises a protein. In some embodiments,
each of the proteins
independently comprises an albumin, a transferrin, or a transthyrctin. In some
embodiments, each of
the proteins independently comprises an Fe portion. In some embodiments, each
of the proteins
independently comprises an Fe portion of IgG. In some embodiments, the
conjugating moiety
comprises a polypeptide. In some embodiments, the additional conjugating
moiety comprises a
polypeptide. In some embodiments, each of the polypeptides independently
comprises a XTEN
peptide, a glycine-rich homoamino acid polymer (HAP), a PAS polypeptide, an
elastin-like
polypeptide (ELP), a CTP peptide, or a gelatin-like protein (GLK) polymer. In
some embodiments,
the isolated and purified IL-2 polypeptide is modified by glutamylation. In
some embodiments, the
conjugating moiety is directly bound to the isolated and purified IL-2
polypeptide. In some
embodiments, the conjugating moiety is indirectly bound to the isolated and
purified IL-2 poly-peptide
through a linker. In some embodiments, the linker comprises a homobifunctional
linker. In some
embodiments, the homobifunctional linker comprises Lomant's reagent dithiobis
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(succinimidylpropionate) DSP, 3'3'-dithiobis(sulfosuccinimidyl proprionate)
(DTSSP), disuccinimidyl
suberate (DSS), bis(sulfosuccinimidyl)suberate (BS), disuccinimidyl tartrate
(DST),
di sulfosuccinimidyl tartrate (sulfo DST), ethylene glycobi s(succinim idyl
succinate) (EGS),
disuccinimidyl glutarate (DSG), N,N'-disuccinimidyl carbonate (DSC), dimethyl
adipimidate (DMA),
dimethyl pimelimidate (DMP), dimethyl suberimidate (DMS), dimethyl-3,3'-
dithiobispropionimidate
(DTBP), 1,4-di-(3'-(2'-pyridyldithio)propionamido)butane (DPDPB),
bismaleimidohcxane (BMH),
aryl halide-containing compound (DFDNB), such as e.g. 1,5-difluoro-2,4-
dinitrobenzene or 1,3-
difluoro-4,6-dinitrobenzene, 4,4'-difluoro-3,3'-dinitrophenylsulfone (DFDNPS),
bis-[13-(4-
azidosalicylamido)ethyll disulfide (BASED), formaldehyde, glutaraldehyde, 1,4-
butanediol diglycidyl
ether, adipie acid dihydrazide, carbohydrazide, o-toluidine, 3,3'-
dimethylbenzidine, benzidine, a,ce-p-
diaminodiphenyl, diiodo-p-xylene sulfonic acid, N,N'-ethylene-
bis(iodoacetamide), or N,N'-
hexamethylene-bis(iodoacetamide). In some embodiments, the linker comprises a
heterobifunctional
linker. In some embodiments, the hctcrobifunctional linker comprises N-
succinimidyl 3-(2-
pyridyldithio)propionate (sPDP), long-chain N-succinimidyl 3-(2-
pyridyldithio)propionate (LC-
sPDP), water-soluble-long-chain N-succinimidyl 3-(2-pyridyldithio) propionate
(sulfo-LC-sPDP),
succinimidyloxycarbonyl-a-methyl-a-(2-pyridyldithio)toluene (sMPT),
sulfosuccinimidy1-64a-
methyl-a-(2-pyridyldithio)toluamido]hexanoate (sulfo-LC-sMPT), succinimidy1-4-
(N-
maleimidomethyl)cyclohexane-1-carboxylate (sMCC), sulfosuccinimidy1-4-(N-
maleimidomethyl)cyclohexane-1-carboxylate (sulfo-sMCC), m-maleimidobenzoyl-N-
hydroxysuccinimide ester (MBs), m-maleimidobenzoyl-N-hydroxysulfosuccinimide
ester (sulfo-
MBs), N-succinimidy1(4-iodoactevl)aminobenzoate (sIAB), sulfosuccinimidy1(4-
iodoacteyl)aminobenzoate (sulfo-sIAB), succinimidyl-4-(p-
maleimidophenyl)butyrate (sMPB),
sulfosuccinimidy1-4-(p-maleimidophenyl)butyrate (sulfo-sMPB), N-(y-
maleimidobutyryloxy)succinimide ester (GMBs), N-(y-maleimidobutyryloxy)
sulfosuccinimide ester
(sulfo-GMBs), succinimidyl 6-((iodoacetyl)amino)hexanoate (sIAX), succinimidyl
(((iodoacetyl)amino)hexanoyl)aminolhexanoate (slAXX), succinimidyl 4-
(((iodoacetyl)amino)methyl)cyclohexane-1-carboxylate (sIAC), succinimidyl
644((4-
iodoacetyl)amino)methyl)cyclohexane-1-carbonyl)amino) hexanoate (sIACX), p-
nitrophenyl
iodoacetate (NPIA), carbonyl-reactive and sulfhydryl-reactive cross-linkers
such as 4-(4-N-
maleimidophenyl)butyric acid hydrazide (MPBH), 4-(N-
maleimidomethyl)cyclohexane-1-carboxyl-
hydrazide-8 (M2C2H), 3-(2-pyridyldithio)propionyl hydrazide (PDPH), N-
hydroxysuccinimidy1-4-
azidosalicylic acid (NHs-AsA), N-hydroxysulfosuccinimidy1-4-azidosalicylic
acid (sulfo-NHs-AsA),
sulfosuccinimidy1-(4-azidosalicylamido)hexanoate (sulfo-NHs-LC-AsA),
sulfosuccinimidy1-2-(p-
azidosalicylamido)ethy1-1,3'-dithiopropionate (sAsD), N-hydroxysuccinimidy1-4-
azidobenzoate
(HsAB), N-hydroxysulfosuccinimidy1-4-azidobenzoate (sulfo-HsAB), N-
succinimidyl 6 (4' azido-2'-
nitrophenyl amino)hexanoate (sANPAH), sulfosuccinimidy1-6-(4'-azido-2'-
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nitrophenylamino)hexanoate (sulfo-sANPAH), N-5-azido-2-
nitrobenzoyloxysuccinimide (ANB-
N0s), sulfosuccinimidyl-2-(m-azido-o-nitrobenzamido)-ethy1-1,3'-
dithiopropionate (sAND), N-
succinimidy1-4(4-azidopheny1)1,3'-dithiopropionate (sADP), N-
sulfosuccinimidy1(4-azidopheny1)-
1,3'-dithiopropionate (sulfo-sADP), sulfosuccinimidyl 4-(p-
azidophenyl)butyrate (sulfo-sAPB),
sulfosuccinimidyl 2-(7-azido-4-methylcoumarin-3-acetamide)ethy1-1,3'-
dithiopropionate (sAED),
sulfosuccinimidyl 7-azido-4-methylcoumain-3-acetate (sulfo-sAMCA), p-
nitrophcnyl diazopyruvatc
(pNPDP), p-nitropheny1-2-diazo-3,3,3-trifluoropropionate (PNP-DTP), 1-(p-
azidosalicylamido)-4-
(iodoacetamido)butane (AsIB), N44-(p-azidosalicylamido)buty11-3'-(2'-
pyridyldithio) propionamide
(APDP), benzophenone-4-iodoacetamide, p-azidobenzoyl hydrazide (ABH), 4-(p-
azidosalicylamido)butylamine (AsBA), or p-azidophenyl glyoxal (APG). In some
embodiments, the
linker comprises a cleavable linker, optionally comprising a dipeptide linker.
In some embodiments,
the dipeptide linker comprises Val-Cit, Phe-Lys, Val-Ala, or Val-Lys. In some
embodiments, the
linker comprises a non-cleavable linker. In some embodiments, the linker
comprises a malcimide
group, optionally comprising maleimidocaproyl (mc), succinimidy1-4-(N-
maleimidomethyl)cyclohexane-1-carboxylate (sMCC), or sulfosuccinimidyl -4-(N-
maleimidomethyl)cyclohexa.ne- 1-carboxylate (sulfo-sMCC). In some embodiments,
the linker further
comprises a spacer. In some embodiments, the spacer comprises p-aminobenzyl
alcohol (PAB), p-
aminobenzyoxycarbonyl (PABC), a derivative, or an analog thereof. In some
embodiments, the
conjugating moiety is capable of extending the serum half-life of the IL-2
conjugate. In some
embodiments, the additional conjugating moiety is capable of extending the
serum half-life of the IL-
2 conjugate. In some embodiments, the IL-2 form suitable for use in the
invention is a fragment of
any of the IL-2 forms described herein. In some embodiments, the IL-2 form
suitable for use in the
invention is pegylated as disclosed in U.S. Patent Application Publication No.
US 2020/0181220 Al
and U.S. Patent Application Publication No. US 2020/0330601 Al. In some
embodiments, the IL-2
form suitable for use in the invention is an 1L-2 conjugate comprising: an 1L-
2 polypeptide
comprising an N6-azidoethoxy-L-lysine (AzK) covalently attached to a
conjugating moiety
comprising a polyethylene glycol (PEG), wherein: the IL-2 polypeptide
comprises an amino acid
sequence having at least 80% sequence identity to SEQ ID NO:5; and the AzK
substitutes for an
amino acid at position K35, F42, F44, K43, E62, P65, R38, T41, E68, Y45, V69,
or L72 in reference
to the amino acid positions within SEQ ID NO:5. In some embodiments, the IL-2
polypeptide
comprises an N-terminal deletion of one residue relative to SEQ ID NO:5. In
some embodiments, the
IL-2 form suitable for use in the invention lacks IL-2R alpha chain engagement
but retains normal
binding to the intermediate affinity IL-2R beta-gamma signaling complex. In
some embodiments, the
IL-2 form suitable for use in the invention is an IL-2 conjugate comprising:
an IL-2 polypeptide
comprising an N6-azidoethoxy-L-lysine (AzK) covalently attached to a
conjugating moiety
comprising a polyethylene glycol (PEG), wherein: the IL-2 polypeptide
comprises an amino acid
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sequence having at least 90% sequence identity to SEQ ID NO:5; and the AzK
substitutes for an
amino acid at position K35, F42, F44, K43, E62, P65, R38, T41, E68, Y45, V69,
or L72 in reference
to the amino acid positions within SEQ ID NO:5. In some embodiments, the 1L-2
form suitable for
use in the invention is an IL-2 conjugate comprising: an IL-2 polypeptide
comprising an N6-
azidoethoxy-L-lysine (AzK) covalently attached to a conjugating moiety
comprising a polyethylene
glycol (PEG), wherein: the 1L-2 polypeptide comprises an amino acid sequence
having at least 95%
sequence identity to SEQ ID NO:5; and the AzK substitutes for an amino acid at
position K35, F42,
F44, 1(43, E62, P65, R38, T41, E68, Y45, V69, or L72 in reference to the amino
acid positions within
SEQ ID NO:5. In some embodiments, the IL-2 form suitable for use in the
invention is an IL-2
conjugate comprising: an IL-2 polypeptide comprising an N6-azidoethoxy-L-
lysine (AzK) covalently
attached to a conjugating moiety comprising a polyethylene glycol (PEG),
wherein: the IL-2
polypeptide comprises an amino acid sequence having at least 98% sequence
identity to SEQ ID
NO:5; and the AzK substitutes for an amino acid at position K35, F42, F44,
K43, E62, P65, R38, T41,
E68, Y45, V69, or L72 in reference to the amino acid positions within SEQ ID
NO:5.
[00404] In some embodiments, an IL-2 form suitable for use in the
invention is nemvaleukin
alfa, also known as ALKS-4230 (SEQ ID NO:6), which is available from Alkermes,
Inc.
Nemvaleukin alfa is also known as human interleukin 2 fragment (1-59), variant
(Cys'>Ser51), fused
via peptidyl linker (60GG61) to human interleukin 2 fragment (62-132), fused
via peptidyl linker
(133GSGGGS138) to human interleukin 2 receptor a-chain fragment (139-303),
produced in Chinese
hamster ovary (CHO) cells, glycosylated, human interleukin 2 (IL-2) (75-133)-
peptide
[Cys'(51)>Serl-mutant (1-59), fused via a G2 peptide linker (60-61) to human
interleukin 2 (IL-2)
(4-74)-peptide (62-132) and via a GSG3S peptide linker (133-138) to human
interleukin 2 receptor a-
chain (1L2R subunit alpha, IL2Ra, 1L2RA) (1-165)-peptide (139-303), produced
in Chinese hamster
ovary (CHO) cells, glycoform alfa. The amino acid sequence of nemvaleukin alfa
is given in SEQ ID
NO:6. In some embodiments, nemvaleukin alfa exhibits the following post-
translational
modifications: disulfide bridges at positions: 31-116, 141-285, 184-242, 269-
301, 166-197 or 166-
199, 168-199 or 168-197 (using the numbering in SEQ ID NO:6), and
glycosylation sites at positions:
N187, N206, T212 using the numbering in SEQ ID NO:6. The preparation and
properties of
nemvaleukin alfa, as well as additional alternative forms of IL-2 suitable for
use in the invention, is
described in U.S. Patent Application Publication No. US 2021/0038684 Al and
U.S. Patent No.
10,183,979, the disclosures of which are incorporated by reference herein. In
some embodiments, an
1L-2 form suitable for use in the invention is a protein having at least 80%,
at least 90%, at least 95%,
or at least 90% sequence identity to SEQ ID NO:6. In some embodiments, an IL-2
form suitable for
use in the invention has the amino acid sequence given in SEQ ID NO:6 or
conservative amino acid
substitutions thereof. In some embodiments, an 1L-2 form suitable for use in
the invention is a fusion
protein comprising amino acids 24-452 of SEQ ID NO:7, or variants, fragments,
or derivatives
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thereof. In some embodiments, an IL-2 form suitable for use in the invention
is a fusion protein
comprising an amino acid sequence having at least 80%, at least 90%, at least
95%, or at least 90%
sequence identity to amino acids 24-452 of SEQ TD NO:7, or variants,
fragments, or derivatives
thereof Other IL-2 forms suitable for use in the present invention are
described in U.S. Patent No.
10,183,979, the disclosures of which are incorporated by reference herein.
Optionally, in some
embodiments, an 1L-2 form suitable for use in the invention is a fusion
protein comprising a first
fusion partner that is linked to a second fusion partner by a mucin domain
polypeptide linker, wherein
the first fusion partner is IL-1Ra or a protein having at least 98% amino acid
sequence identity to IL-
1Ra and having the receptor antagonist activity of IL-Ra, and wherein the
second fusion partner
comprises all or a portion of an immunoglobulin comprising an Fe region,
wherein the mucin domain
polypeptide linker comprises SEQ ID NO:8 or an amino acid sequence having at
least 90% sequence
identity to SEQ ID NO:8 and wherein the half-life of the fusion protein is
improved as compared to a
fusion of the first fusion partner to the second fusion partner in the absence
of the mucin domain
polypeptide linker.
TABLE 2. Amino acid sequences of interleukins.
Identifier Sequence (One-Letter Amino Acid
Symbols)
SEQ ID NO:3 MAPTSSSTKK TQLQLEHLLL DLQMILNGIN NYKNPILLTRM
LTEKEYMPKK ATELKHLQCL 60
recombinant EEELKPLEEV LNLAQSKNFH LRPRELISNI NVIVLELKGS
ETTFMCEYAD ETATIVEFLN 120
human IL 2 RWITECQSTE STLT
134
(LhIL-2)
SEQ ID NO:4 PTSSSTKETQ LQLEHLLLDL QMILNGINNY HNPHLTRMLT
FKEYMPKKAT ELHHLQCLEE 60
Aldesleukin ELKPLEEVLN LAQSKNFHLR PRDLISNINV IVLELKGSET
TFMCEYADET ATIVEFLNRW 120
ITESQSIIST LT
132
SEQ ID NO:5 APTSSSTEKT QLQLEHLLLD LQMILNGINN YKNPKLTRML
TEKEYMPKKA TELKHLQCLE 60
IL-2 form EELKPLEEVL NLAQSKNEHL RPRDLISNIN VIVLELKGSE
TTFMCEYADE TATIVEFLNR 120
WITECQSITS IL?
133
SEQ ID NO:6 SKNEHLRPRD LISNINVIVL ELKGSETTEM CEYADETATI
VEFLNRWITF SQSIISTLTG 60
Nemvaleukin rife GSSSTKKTQL QLEHLLEDILQ MILNGINNYK NYKLTRMITY 15-
EME3KA2E LEHLQCLEEE 120
LKPLEEVLNL AQGSGGGSEL CDDDPPEIPH ATFKAMAYKE GTMLNCECKR GFRRIKSGSL
180
YMLCTGNSSH SSWDNQCQCT SSATRNTTICQ VTPQPEEQHE RHTTEMQSPM QPVDQASLPG
240
HCREPPPWEN EATERIYHEV VGQMVYYQCV QGYRALHRGP AESVCKMTHG KTRWTQPQLI
300
CTG
303
SEQ ID NO:7 MDAMKRGLCC VLLLCGAVEV SARRPSGRKS SKMQAFRIWD
VNGKTFYLRN NQLVAGYLGG 60
IL-2 form PNMNLEEKID VVPIEPNALF LGIHGgKMCL SCVKSGDETR
LQLEAVNITD LSENRKQDKR 120
FAFIRSDSGP TTSFESAACP GWFLCTAMEA DQPVSLTNMP DEGVMVTKEY FQEDESGSGG
180
ASSESSASSD GPHID/DPESR ASSESSASSD GPHDVITESR EPASSDKTHT CPRCPAs'ELL
240
GGPSVFLFPP KPKDTLMISR TPEVTCVVVD VSHEDPEVKF NWYVDGVEVH NANTKPREEQ
300
YNETYRVVSV LTVLHQDWLN GKEYRCKVSN KALPAPIEKT ISKAKGQFRE P2VYTL2PER
360
EEMTKNQVSL TCLVKGFYPS 2IAVEWESNG QPENNYKTTP PVLDS2GSFY LYSKLTV2KS
420
RWQQGNVFSC SVMHEALHNH YTQKSLSLSP GK
452
SEQ ID NO:8 SESSASSDGP HPVITP
16
mucin domain
polypeptide
SEQ ID NO:9 MHXCDITLQE IIKTLNSLTE QKTLCTELTV TDIFAASKNT
TEKETFCRAA TVLPQFYSHH 60
recombinant EKDTRCLGAT AQQEHRHHQL IRELKRLDRN LWGLAGLNSC
FVHEANQSTL ENELERLKTI 120
human IL-4 MREKYSKCSS
130
(rhIL-4)
SEQ ID NO:10 MDCDIEGEDG KQYESVLMVS IDQLLDSMKE IGSNCLNNEF
NFFKRHICDA NKEGMFLFRA 60
recombinant ARKLRQFLKM NSTGDFDLHL LKVSEGTTIL LNCTGQVKGR
KPAALGEAQP TKSLEENKSL 120
human IL-7 KEQKKLNDLC FLKRLLQEIK TCWNHILMGT HEH
153
(rhIL-7)
SEQ ID NO:11 MNWVNVISDL KNIEDLIQSM HIDATLYTES DVHPSCKVTA
MIKCELLELQV ISLESGDASI 60
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recombinant HDTVENLIIL ANNSLSSNGN VTESGCKECE ELEEKNIKEF
LQSFVHIVQM FINTS 115
human IL-15
(rhIL-15)
SEQ =D NO:12 MQDRHMIRMR QLIDIVDQLK NYVNELVREF LPAPEDVETN
CEWSAFSCFQ KAQLKSANTG -- 60
recombinant NNER=INVSI KHLHRKPFST NAGRRQKHRL TCPSODSYEK
KETKEELERF HSLLQHMIHQ -- 120
human 1L-21 HL.5SRTHGSE DS
132
(rhII-21)
[00405] In some embodiments, an IL-2 form suitable for use in the
invention includes a
antibody cytokine engrafted protein comprises a heavy chain variable region
(VII), comprising
complementarity determining regions HCDR1, HCDR2, HCDR3; a light chain
variable region (VL).
comprising LCDR1, LCDR2, LCDR3; and an IL-2 molecule or a fragment thereof
engrafted into a
CDR of the VII or the VL, wherein the antibody cytokine engrafted protein
preferentially expands T
effector cells over regulatory T cells. Insome embodiments, the antibody
cytokine engrafted protein
comprises a heavy chain variable region (VH), comprising complementarity
determining regions
HCDR1, HCDR2, HCDR3; a light chain variable region (VL), comprising LCDR1,
LCDR2, LCDR3;
and an IL-2 molecule or a fragment thereof engrafted into a CDR of the Vu or
the VL, wherein the IL-
2 molecule is a mutein, and wherein the antibody cytokine engrafted protein
preferentially expands T
effector cells over regulatory T cells. In some embodiments, the IL-2 regimen
comprises
administration of an antibody described in U.S. Patent Application Publication
No. US 2020/0270334
Al, the disclosures of which are incorporated by reference herein. In some
embodiments, the antibody
cytokine engrafted protein comprises a heavy chain variable region (VH),
comprising
complementarity determining regions HCDR1, HCDR2, HCDR3; a light chain
variable region (VL),
comprising LCDR1, LCDR2, LCDR3; and an IL-2 molecule or a fragment thereof
engrafted into a
CDR of the VI{ or the VL, wherein the 1L-2 molecule is a mutcin, wherein the
antibody cytokine
engrafted protein preferentially expands T effector cells over regulatory T
cells, and wherein the
antibody further comprises an IgG class heavy chain and an IgG class light
chain selected from the
group consisting of: a IgG class light chain comprising SEQ ID NO:39 and a IgG
class heavy chain
comprising SEQ ID NO:38; a IgG class light chain comprising SEQ ID NO:37 and a
IgG class heavy
chain comprising SEQ ID NO:29; a IgG class light chain comprising SEQ ID NO:39
and a IgG class
heavy chain comprising SEQ ID NO:29; and a IgG class light chain comprising
SEQ ID NO:37 and a
IgG class heavy chain comprising SEQ ID NO:38.
[00406] In some embodiments, an IL-2 molecule or a fragment
thereof is engrafted into
HCDR1 of the VH, wherein the 1L-2 molecule is a mutein. In some embodiments,
an 1L-2 molecule or
a fragment thereof is engrafted into HCDR2 of the VH, wherein the IL-2
molecule is a mutein. In
some embodiments, an IL-2 molecule or a fragment thereof is engrafted into
HCDR3 of the VH,
wherein the IL-2 molecule is a mutein. In some embodiments, an IL-2 molecule
or a fragment thereof
is engrafted into LCDR1 of the VL, wherein the IL-2 molecule is a mutein. In
some embodiments, an
1L-2 molecule or a fragment thereof is engrafted into LCDR2 of the VL, wherein
the IL-2 molecule is
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a mutein. In some embodiments, an IL-2 molecule or a fragment thereof is
engrafted into LCDR3 of
the VL, wherein the IL-2 molecule is a mutein.
[00407] The insertion of the IL-2 molecule can be at or near the
N-terminal region of the
CDR, in the middle region of the CDR or at or near the C-terminal region of
the CDR. In some
embodiments, the antibody cytokine engrafted protein comprises an IL-2
molecule incorporated into a
CDR, wherein the IL2 sequence does not frameshift the CDR sequence. In some
embodiments, the
antibody cytokine engrafted protein comprises an IL-2 molecule incorporated
into a CDR, wherein
the IL-2 sequence replaces all or part of a CDR sequence. The replacement by
the IL-2 molecule can
be the N-terminal region of the CDR, in the middle region of the CDR or at or
near the C-terminal
region the CDR. A replacement by the IL-2 molecule can be as few as one or two
amino acids of a
CDR sequence, or the entire CDR sequences.
[00408] In some embodiments, an IL-2 molecule is engrafted
directly into a CDR without a
peptide linker, with no additional amino acids between the CDR sequence and
the IL-2 sequence. In
some embodiments, an IL-2 molecule is engrafted indirectly into a CDR with a
peptide linker, with
one or more additional amino acids between the CDR sequence and the IL-2
sequence.
[00409] In some embodiments, the TL-2 molecule described herein
is an 1L-2 mutein. In some
instances, the IL-2 mutein comprising an R67A substitution. In some
embodiments, the IL-2 mutein
comprises the amino acid sequence SEQ ID NO:14 or SEQ ID NO:15. In some
embodiments, the IL-
2 mutcin comprises an amino acid sequence in Table 1 in U.S. Patent
Application Publication No. US
2020/0270334 Al, the disclosure of which is incorporated by reference herein.
[00410] In some embodiments, the antibody cytokine engrafted
protein comprises an HCDR1
selected from the group consisting of SEQ ID NO:16, SEQ ID NO:19, SEQ ID NO:22
and SEQ ID
NO:25. In some embodiments, the antibody cytokine engrafted protein comprises
an HCDR1 selected
from the group consisting of SEQ ID NO:7, SEQ ID NO: 10, SEQ ID NO:13 and SEQ
ID NO:16. In
some embodiments, the antibody cytokine engrafted protein comprises an HCDR1
selected from the
group consisting of HCDR2 selected from the group consisting of SEQ ID NO:17,
SEQ ID NO:20,
SEQ ID NO:23, and SEQ ID NO:26. In some embodiments, the antibody cytokine
engrafted protein
comprises an HCDR3 selected from the group consisting of SEQ ID NO:18, SEQ ID
NO :21, SEQ ID
NO:24, and SEQ ID NO:27. In some embodiments, the antibody cytokine engrafted
protein comprises
a VH region comprising the amino acid sequence of SEQ ID NO:28. In some
embodiments, the
antibody cytokine engrafted protein comprises a heavy chain comprising the
amino acid sequence of
SEQ ID NO:29. In some embodiments, the antibody cytokine engrafted protein
comprises a VL region
comprising the amino acid sequence of SEQ ID NO:36. In some embodiments, the
antibody cytokine
engrafted protein comprises a light chain comprising the amino acid sequence
of SEQ ID NO:37. In
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some embodiments, the antibody cytokine engrafted protein comprises a VII
region comprising the
amino acid sequence of SEQ ID NO:28 and a VL region comprising the amino acid
sequence of SEQ
ID NO:36. In sonic embodiments, the antibody cytokine engrafted protein
comprises a heavy chain
region comprising the amino acid sequence of SEQ ID NO:29 and a light chain
region comprising the
amino acid sequence of SEQ ID NO:37. In some embodiments, the antibody
cytokine engrafted
protein comprises a heavy chain region comprising the amino acid sequence of
SEQ ID NO:29 and a
light chain region comprising the amino acid sequence of SEQ ID NO:39. In some
embodiments, the
antibody cytokine engrafted protein comprises a heavy chain region comprising
the amino acid
sequence of SEQ ID NO:38 and a light chain region comprising the amino acid
sequence of SEQ ID
NO:37. In some embodiments, the antibody cytokine engrafted protein comprises
a heavy chain
region comprising the amino acid sequence of SEQ ID NO:38 and a light chain
region comprising the
amino acid sequence of SEQ ID NO:39. In some embodiments, the antibody
cytokine engrafted
protein comprises IgG.IL2F71A.H1 or IgGIL2R67A.H1 of U.S. Patent Application
Publication No.
2020/0270334 Al, or variants, derivatives, or fragments thereof, or
conservative amino acid
substitutions thereof, or proteins with at least 80%, at least 90%, at least
95%, or at least 98%
sequence identity thereto. In some embodiments, the antibody components of the
antibody cytokine
engrafted protein described herein comprise immunoglobulin sequences,
framework sequences, or
CDR sequences of palivizumab. In some embodiments, the antibody cytokine
engrafted protein
described herein has a longer serum half-life that a wild-type IL-2 molecule
such as, but not limited
to, aldesleukin or a comparable molecule. In some embodiments, the antibody
cytokine engrafted
protein described herein has a sequence as set forth in Table 3.
TABLE 3: Sequences of exemplary palivizumab antibody-IL-2 engrafted proteins
Identifier Sequence (One-Letter Amino Acid
Symbols)
SEQ ID NO:13 MYRMQLLSCI ALSLALVTNS APTSSSTKRT QLQLEHLLLD LQMILNGINN
YKN2KLIRML 60
IL-2 TEKEYMPKKA TELKHLQCLE EELKPLEEVL NLAQSKNFHL RPRDLISNIN
VIVLELKGSE 120
TTFMCEYADE TATIVEFLNR WITFCQSIIS TLT 153
SEQ ID NO:14 APTSSSTKKT QLQLEHLLLD LQMILNGINN YKNPKLTAML TFHFYMPKKA
TELKHLQCLE .. 60
IL-2 mutein EELKPLEEVL NLAQSKNFHL RPRDLISNIN VIVLELKGSE TTFMCEYADE
TATIVEFLNR 120
WiTFCQSIIS TLT 133
SEQ ID NO:15 APTSSSTKKT QLQLEHLLLD LQMILNGINN YKNPKLTRML TAKFYMPKKA
TELKHLQCLE 60
IL 2 mutein EELKPLEEVL NLAQSKNFHL RPRDLISNIN VIVLELKGSE TTFMCEYADE
TATIVEFLNR 120
WITFCQSIIS TLT 133
SEQ ID NO:16 GFSLAPTSSS TKKTQLQLEH LLLDLQMILN GINNYKNPKL TAMLTEKEYM
PKKATELKHL 60
HCDR1_IL-2 QCLEEELKFL EEVLNLAQSK NEHLR2RDLI SNINVIVLEL KGSETTFMCE
YADETATIVE 120
FLNRWITECQ SIISTLTSTS GMSVG 145
SEQ ID NO:17 D=WWDDKKDY NPSLKS 16
HCDR2
SEQ ID NO:18 SMITNWYFDV 10
HCDR3
SEQ ID NO:19 APTSSSTKKT QLQLEHLLLD LcMILNGINN YKNPKLTAML TEKEYMPKKA
TELKHLQCLE 60
HCDR1_IL-2 EELKPLEEVL NLAQSKNFHL RPRDLISNIN VIVLELKGSE TTFMCEYADE
TATIVEFLNR 120
kabat WITFCQSITS TLTSTSGMSV G 141
SEQ ID NO:20 DIWWDDKKDY NPSLKS 16
tiC1).R2 kabat
SEQ ID NO:21 SMITNWYFDV 10
HCDR3 kabat
SEQ ID NO:22 GFSLAPTSSS TKKTQLQLEH LLLDLQMILN GINNYKNPKL TAMLTFKFYM
PKKATELKHL 60
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HCDR1_IL-2 QCLEEELKPL EEVLNLAQSK NEHLRPRDLI SNINVIVLEL KGSETTFMCE
YADETATIVE 120
c1othia FLNRWITFCQ SIISTLTSTS GM 142
SEQ ID NO:23 WWDDK
HCDR2 c1othia
SEQ ID NO:24 SMITNWYFDV 10
HCDR3 c1othia
SEQ ID NO:25 GFSLAPTSSS TKKTQLQLEH LLLDLQMILN GINNYKNPKL TAMLTEKEYM
PKKATELKHL 60
HCDR1_IL-2 QCLEEELKPL EEVLNLAQSK NENLRPRDLI SNINVIVLEL KGSETTFMCE
YADETATIVE 120
IMGT FLNRWITFCQ SIISTLTSTS GMS 143
SEQ ID NO:26 IWWDDKK
7
HCDR2 IMGT
SEQ ID NO:27 ARSMITNWYF DV 12
HCOR3 iMGT
SEQ ID NO:28 QVTLRESGPA LVKFTQTLTL TCTFSGFSLA FTSSSTKKTQ LQLEHLLLDL
QMILNGINNY 60
Vu KNPKLTAMLT EKFYMPKKAT ELKHLQCLEE ELKPLEEVLN LAQSKNEHLR
PRDLISNINV 120
IVLELKGSET TFMCEYADET ATIVEYLNRW ITFCQSIIST LTSTSGMSVG WIRQPPGKAL
180
EWLADIWWDD EKDYNPSLKS RLTISKDTSK NQVVLKVTNM DPADTATYYC ARSMITNWYF
240
DVWGAGTTVT VSS 253
SEQ ID NO:29 QMILNGINNY KNPHLTAMLT FKFYM2KKAT ELKHLQCLEE ELKPLEEVLN
LAQSKNEHLR 60
Heavy chain PRDLISNINV IVLELKGSET TEMCEYADET ATIVEFLNRW ITECOSIIST
LTSTSGMSVG 120
WERQPPGKAL EWLADIWWDD KKDYN2SLYE RLTISKDTSK NQVVLKVTNM DPADTATYYC 180
ARSMITNWYF DVWGAGTTVT VSSASTKGPS VTPLAPSSKS TSGGTAALGC LNTHDYPPEPV
240
TVSWNSGALT SGVIMFFAVL QSSGLYSLSS VVTVPSSSLG TQTYICNVNH KPSNTKVDKR
300
VEPKSCDKTH TCPPCPAPEL LGGPSVFLFP PKPKDTLMIS RTPEVTCVVV AVSHEDPEVY
360
PNWYVDGVEV IINAKTKPREE QYNSTYRVVS VLTVLHQ2WL DIGEYHCKVS NRALAAPIEK 420
TISKAKGQPR EPQVYTLPPS REEMTKNQVS LTCLVKGFYP SDIAVEWESN GQPENNYKTT
480
PPVLDSDGSF FLYSKLTVDK SRWQQGNVFS CSVMHEALHN HETQKSLSLS PGK
533
SEQ ID NO:30 KAQLSVGYMH 10
LCDR1 kabat
SEQ ID NO:31 D=KLAS
7
LCOR2 kabat
SEQ ID NO:32 FQGSGYPFT 9
LCDR3 kabat
SEQ ID NO:33 QLSVGY
6
LCDR1 choLhia
SEQ ID N0:34 DTS
3
LCDR2 chothia
sn ID ND:35 GSGYPY
6
LCDR3 chothia
SEQ ID NO:36 DEQMTQSFST LSASVGDRVT ITCKAQLSVG YMHWYQQHFG KAPHLLIYDT
SKLASGVPSR 60
Vu FSGSGSGTEF TLTISSLQPD DFATYYCFQG SGYPFTFGGG TKLEIK 106
SEQ ID NO:37 D1QMTQSPST LSASVGDRVT ITCKAQLSVG YMHWYQQKPG KAPKLLIYDT
SKLASGVPSR 60
Light chain FSGSGSGTEF TLTISSLQPD DFATYYCFQG SGYPFTEGGG TKLEIKRTVA
APSVFIFPPS 120
DEQLKSGTAS VVCLLNNFYP REAKVQWKVD NALQSGNSQE SVTEQDSKDS TYSLSSTLTL
180
SKADEEKHKV EACEVTHQGL SSPVTKSFNR GEC
213
SEQ ID NO:38 QVTLRESGPA LVKPTQTLTL TCTFSGFSLA PTSSSTKKTQ LQLEHLLLDL
QMILNGINNY 60
Light chain KNPKLTRMLT AKFYMPKKAT ELKHLQCLEE FLKPLEEVLN LAQSKNEHLR
PRDLISNINV 120
IVLELKGSET IFMCEYAPTP AIIVEJ'LNRW iTrcQsiisT LTSTSGMSVG WiRQPPGKAL
180
EWLADIWWDD ZKDYNPSLKS RLTISKDTSK NQVVIKVTNM DPADTATYYC ARSMITNWYF
240
DVWGAGTTVT VSSASTKGPS VFPLAPSSES TSGGTAALGC LVHDYFPEPV TVSWNSGALT
300
SGVHTFPAVL QSSGLESLSS VVTVPSSSLG TQTYICNVNH KPSNTHVDKR VEPKSCDKTH
360
TCPPCPAPEL LGGPSVFLFP PKPKDTLMIS RTPEVTCVVV AVSHEDPEVK FNWYVDGVEV
420
HNAKTKPREE GENSTERVVS VLTVLHQDWL NGKEYKCKVS NKALAAPIEK TISKAKGQPR
480
EPQVYTLPPS REEMTKNQVS LTCLVKGFYP SDIAMEWESN GQPENNYKTT PPVLDSDGSF
540
FLYSKLTVDH SRWQQGNVFS CSVMHEALHN HYTQHSLSLS PGH
583
SEQ ID ND:39 D_QMTUSPST LSASVGDRVT ITCKAQLSVG YMHWYQQAPG KAPALL1YDT
SKLASGVPSR 60
Light chain FSGSGSGTEF TLTISSLQPD DFATYYCFQG SGYPFTEGGG TKLEIKRTVA
A2SVFIFPPS 120
DEQLKSGTAS VVCLLNNEYP REAKVQWKVD NALQSGNSQE SVTEQDSKDS TYSLSSTLTL
180
SKADYEKHKV YACEVTHQGL SSPVTKSFNR DEC
213
[00411] The term "IL-4" (also referred to herein as "IL4") refers to the
cytokine known as
interleukin 4, which is produced by Th2 T cells and by eosinophils, basophils,
and mast cells. 1L-4
regulates the differentiation of naïve helper T cells (Th0 cells) to Th2 T
cells. Steinke and Borish,
Respir. Res. 2001, 2, 66-70. Upon activation by IL-4, Th2 T cells subsequently
produce additional IL-
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4 in a positive feedback loop. IL-4 also stimulates B cell proliferation and
class II MHC expression,
and induces class switching to IgE and IgGI expression from B cells.
Recombinant human IL-4
suitable for use in the invention is commercially available from multiple
suppliers, including ProSpec-
Tany TechnoGene Ltd., East Brunswick, NJ, USA (Cat. No. CYT-211) and
ThermoFisher Scientific,
Inc., Waltham, MA, USA (human IL-15 recombinant protein, Cat. No. Gibco
CTP0043). The amino
acid sequence of recombinant human 1L-4 suitable for use in the invention is
given in Table 2 (SEQ
ID NO:9).
[00412] The term "IL-7" (also referred to herein as "IL7") refers to a
glycosylated tissue-derived
cytokine known as interleukin 7, which may be obtained from stromal and
epithelial cells, as well as
from dendritic cells. Fry and Mackall, Blood 2002, 99, 3892-904. 1L-7 can
stimulate the development
of T cells. IL-7 binds to the IL-7 receptor, a heterodimer consisting of IL-7
receptor alpha and
common gamma chain receptor, which in a series of signals important for T cell
development within
the thymus and survival within the periphery. Recombinant human IL-7 suitable
for use in the
invention is commercially available from multiple suppliers, including ProSpec-
Tany TechnoGene
Ltd., East Brunswick, NJ, USA (Cat. No. CYT-254) and ThermoFisher Scientific,
Inc.. Waltham,
MA, USA (human IL-15 recombinant protein, Cat. No. Gibco PHC0071). The amino
acid sequence
of recombinant human IL-7 suitable for use in the invention is given in Table
2 (SEQ ID NO: 10).
[00413] The term "1L-15" (also referred to herein as "1L15") refers to the T
cell growth factor
known as interleukin-15, and includes all forms of IL-2 including human and
mammalian forms,
conservative amino acid substitutions, glycoforms, biosimilars, and variants
thereof. IL-15 is
described, e.g., in Fehniger and Caligiuri, Blood 2001, 97, 14-32, the
disclosure of which is
incorporated by reference herein. IL-15 shares 13 and y signaling receptor
subunits with IL-2.
Recombinant human IL-15 is a single, non-glycosylated polypeptide chain
containing 114 amino
acids (and an N-terminal methionine) with a molecular mass of 12.8 kDa.
Recombinant human IL-15
is commercially available from multiple suppliers, including ProSpec-Tany
TechnoGene Ltd., East
Brunswick, NJ, USA (Cat. No. CYT-230-b) and ThermoFisher Scientific, Inc.,
Waltham, MA, USA
(human IL-15 recombinant protein, Cat. No. 34-8159-82). The amino acid
sequence of recombinant
human IL-15 suitable for use in the invention is given in Table 2 (SEQ ID
NO:11).
[00414] The term "IL-21" (also referred to herein as "IL21") refers to the
pleiotropic cytokine
protein known as interleukin-21, and includes all forms of IL-21 including
human and mammalian
forms, conservative amino acid substitutions, glycoforms, biosimilars, and
variants thereof. IL-21 is
described, e.g., in Spolski and Leonard, Nat. Rev. Drug. Disc. 2014, /3, 379-
95, the disclosure of
which is incorporated by reference herein. IL-21 is primarily produced by
natural killer T cells and
activated human CD4 T cells. Recombinant human IL-21 is a single, non-
glycosylated polypeptide
chain containing 132 amino acids with a molecular mass of 15.4 kDa.
Recombinant human IL-21 is
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commercially available from multiple suppliers, including ProSpec-Tany
TechnoGene Ltd., East
Brunswick, NJ, USA (Cat. No. CYT-408-b) and ThermoFisher Scientific, Inc.,
Waltham, MA, USA
(human 1L-21 recombinant protein, Cat. No. 14-8219-80). The amino acid
sequence of recombinant
human IL-21 suitable for use in the invention is given in Table 2 (SEQ ID
NO:12).
[00415] When "an anti-tumor effective amount", "a tumor-inhibiting effective
amount", or
"therapeutic amount- is indicated, the precise amount of the compositions of
the present invention to
be administered can be determined by a physician with consideration of
individual differences in age,
weight, tumor size, extent of infection or metastasis, and condition of the
patient (subject). It can
generally be stated that a pharmaceutical composition comprising the tumor
infiltrating lymphocytes
(e.g., secondary TILs or genetically modified cytotoxic lymphocytes) described
herein may be
administered at a dosage of 104 to 1011 cells/kg body weight (e.g., 105 to
106, 105 to 1010, 105 to 1011,
106 to 1019, 106to 1011,107to 1011, 107 ot 010,
108 to 1011,
108 to 1 n 10,
1 09 tO 1011, or 109 to 1010
cells/kg body weight), including all integer values within those ranges. TILs
(including in some cases,
genetically modified cytotoxic lymphocytes) compositions may also be
administered multiple times at
these dosages. The TILs (inlcuding in some cases, genetically) can be
administered by using infusion
techniques that are commonly known in immunotherapy (see, e.g., Rosenberg et
al., New Eng. J of
Med. 1988, 319: 1676). The optimal dosage and treatment regime for a
particular patient can readily
be determined by one skilled in the art of medicine by monitoring the patient
for signs of disease and
adjusting the treatment accordingly.
[00416] The term "hematological malignancy", "hematologic malignancy" or terms
of correlative
meaning refer to mammalian cancers and tumors of the hematopoietic and
lymphoid tissues, including
but not limited to tissues of the blood, bone marrow, lymph nodes, and
lymphatic system.
Hematological malignancies are also referred to as "liquid tumors."
Hematological malignancies
include, but are not limited to, acute lymphoblastic leukemia (ALL), chronic
lymphocytic lymphoma
(CLL), small lymphocytic lymphoma (SLL), acute myelogenous leukemia (AML),
chronic
myelogenous leukemia (CML), multiple myeloma, acute monocytic leukemia (AMoL),
Hodgkin's
lymphoma, and non-Hodgkin's lymphomas. The term "B cell hematological
malignancy- refers to
hematological malignancies that affect B cells.
[00417] The term "liquid tumor" refers to an abnormal mass of cells that is
fluid in nature. Liquid
tumor cancers include, but are not limited to, leukemias, myelomas, and
lymphomas, as well as other
hematological malignancies. TILs obtained from liquid tumors may also be
referred to herein as
marrow infiltrating lymphocytes (MILs). TILs obtained from liquid tumors,
including liquid tumors
circulating in peripheral blood, may also be referred to herein as PBLs. The
terms MIL, TIL, and PBL
are used interchangeably herein and differ only based on the tissue type from
which the cells are
derived.
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[00418] The term "microenvironment," as used herein, may refer to the solid or
hematological
tumor microenvironment as a whole or to an individual subset of cells within
the microenvironment.
The tumor microenvironment, as used herein, refers to a complex mixture of
"cells, soluble factors,
signaling molecules, extracellular matrices, and mechanical cues that promote
neoplastic
transformation, support tumor growth and invasion, protect the tumor from host
immunity, foster
therapeutic resistance, and provide niches for dominant metastases to thrive,"
as described in Swartz,
et al., Cancer Res., 2012, 72, 2473. Although tumors express antigens that
should be recognized by T
cells, tumor clearance by the immune system is rare because of immune
suppression by the
microenvironment.
[00419] In some embodiments, the invention includes a method of treating a
cancer with a
population of TILs, wherein a patient is pre-treated with non-myeloablative
chemotherapy prior to an
infusion of TILs according to the invention. In some embodiments, the
population of TILs may be
provided wherein a patient is pre-treated with nonmyeloablative chemotherapy
prior to an infusion of
TILs according to the present invention. In some embodiments, the non-
myeloablative chemotherapy
is cyclophosphamide 60 mg/kg/d for 2 days (days 27 and 26 prior to TIL
infusion) and fludarabine 25
mg/m2/d for 5 days (days 27 to 23 prior to TIL infusion). In some embodiments,
after non-
myeloablative chemotherapy and TIL infusion (at day 0) according to the
invention, the patient
receives an intravenous infusion of IL-2 intravenously at 720,000 IU/kg every
8 hours to physiologic
tolerance.
[00420] Experimental findings indicate that lymphodepletion prior to adoptive
transfer of tumor-
specific T lymphocytes plays a key role in enhancing treatment efficacy by
eliminating regulatory T
cells and competing elements of the immune system ("cytokine sinks").
Accordingly, some
embodiments of the invention utilize a lymphodepletion step (sometimes also
referred to as
"immunosuppressive conditioning") on the patient prior to the introduction of
the TILs of the
invention.
[00421] The term -effective amount" or -therapeutically effective amount"
refers to that amount of
a compound or combination of compounds as described herein that is sufficient
to effect the intended
application including, but not limited to, disease treatment. A
therapeutically effective amount may
vary depending upon the intended application (in vitro or in vivo), or the
subject and disease condition
being treated (e.g., the weight, age and gender of the subject), the severity
of the disease condition, or
the manner of administration. The term also applies to a dose that will induce
a particular response in
target cells (e.g., the reduction of platelet adhesion and/or cell migration).
The specific dose will vary
depending on the particular compounds chosen, the dosing regimen to be
followed, whether the
compound is administered in combination with other compounds, timing of
administration, the tissue
to which it is administered, and the physical delivery system in which the
compound is carried.
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[00422] The terms "treatment'', "treating", "treat", and the like, refer to
obtaining a desired
pharmacologic and/or physiologic effect. The effect may be prophylactic in
terms of completely or
partially preventing a disease or symptom thereof and/or may be therapeutic in
terms of a partial or
complete cure for a disease and/or adverse effect attributable to the disease.
"Treatment", as used
herein, covers any treatment of a disease in a mammal, particularly in a
human, and includes: (a)
preventing the disease from occurring in a subject which may be predisposed to
the disease but has
not yet been diagnosed as having it; (b) inhibiting the disease, i.e.,
arresting its development or
progression; and (c) relieving the disease, i.e., causing regression of the
disease and/or relieving one
or more disease symptoms. -Treatment" is also meant to encompass delivery of
an agent in order to
provide for a pharmacologic effect, even in the absence of a disease or
condition. For example,
"treatment" encompasses delivery of a composition that can elicit an immune
response or confer
immunity in the absence of a disease condition, e.g., in the case of a
vaccine.
[00423] The term -heterologous" when used with reference to portions of a
nucleic acid or protein
indicates that the nucleic acid or protein comprises two or more subsequences
that are not found in the
same relationship to each other in nature. For instance, the nucleic acid is
typically recombinantly
produced, having two or more sequences from unrelated genes arranged to make a
new functional
nucleic acid, e.g., a promoter from one source and a coding region from
another source, or coding
regions from different sources. Similarly, a heterologous protein indicates
that the protein comprises
two or more subsequences that are not found in the same relationship to each
other in nature (e.g., a
fusion protein).
[00424] The terms "sequence identity," "percent identity," and "sequence
percent identity" (or
synonyms thereof, e.g., 99% identical") in the context of two or more nucleic
acids or polypeptides,
refer to two or more sequences or subsequences that are the same or have a
specified percentage of
nucleotides or amino acid residues that arc the same, when compared and
aligned (introducing gaps, if
necessary) for maximum correspondence, not considering any conservative amino
acid substitutions
as part of the sequence identity. The percent identity can be measured using
sequence comparison
software or algorithms or by visual inspection. Various algorithms and
software are known in the art
that can be used to obtain alignments of amino acid or nucleotide sequences.
Suitable programs to
determine percent sequence identity include for example the BLAST suite of
programs available from
the U.S. Government's National Center for Biotechnology Information BLAST web
site.
Comparisons between two sequences can be carried using either the BLASTN or
BLASTP algorithm.
BLASTN is used to compare nucleic acid sequences, while BLASTP is used to
compare amino acid
sequences. ALIGN, ALIGN-2 (Genentech, South San Francisco, California) or
MegAlign, available
from DNASTAR, are additional publicly available software programs that can be
used to align
sequences. One skilled in the art can determine appropriate parameters for
maximal alignment by
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particular alignment software. In certain embodiments, the default parameters
of the alignment
software are used.
[00425] As used herein, the term -variant" encompasses but is not limited to
antibodies or fusion
proteins which comprise an amino acid sequence which differs from the amino
acid sequence of a
reference antibody by way of one or more substitutions, deletions and/or
additions at certain positions
within or adjacent to the amino acid sequence of the reference antibody. The
variant may comprise
one or more conservative substitutions in its amino acid sequence as compared
to the amino acid
sequence of a reference antibody. Conservative substitutions may involve,
e.g., the substitution of
similarly charged or uncharged amino acids. The variant retains the ability to
specifically bind to the
antigen of the reference antibody. The term variant also includes pegylated
antibodies or proteins.
[00426] By "tumor infiltrating lymphocytes" or "TiLs" herein is meant a
population of cells
originally obtained as white blood cells that have left the bloodstream of a
subject and migrated into a
tumor. TILs include, but are not limited to, CD8 cytotoxic T cells
(lymphocytes), Thl and Th17
CD4+ T cells, natural killer cells, dendritic cells and M1 macrophages. TILs
include both primary and
secondary TILs. "Primary TILs" are those that are obtained from patient tissue
samples as outlined
herein (sometimes referred to as "freshly harvested"), and "secondary TILs"
are any TIL cell
populations that have been expanded or proliferated as discussed herein,
including, but not limited to
bulk TILs, expanded TILs (¶REP TILs") as well as "reREP TILs" as discussed
herein. reREP TILs
can include for example second expansion TILs or second additional expansion
TILs (such as, for
example, those described in Step D of Figure 8, including TILs referred to as
reREP TILs).
[00427] TILs can generally be defined either biochemically, using cell surface
markers, or
functionally, by their ability to infiltrate tumors and effect treatment. TILs
can be generally
categorized by expressing one or more of the following biomarkers: CD4. CD8,
TCR a13, CD27,
CD28, CD56, CCR7, CD45Ra, CD95, PD-1, and CD25. Additionally, and
alternatively, TILs can be
functionally defined by their ability to infiltrate solid tumors upon
reintroduction into a patient. TILs
may further be characterized by potency - for example, TILs may be considered
potent if, for
example, interferon (IFN) release is greater than about 50 pg/mL, greater than
about 100 pg/mL,
greater than about 150 pg/mL, or greater than about 200 pg/mL. TILs may be
considered potent if, for
example, interferon (IFN7) release is greater than about 50 pg/mL, greater
than about 100 pg/mL,
greater than about 150 pg/mL, or greater than about 200 pg/mL, greater than
about 300 pg/mL, greater
than about 400 pg/mL, greater than about 500 pg/mL, greater than about 600
pg/mL, greater than
about 700 pg/mL, greater than about 800 pg/mL, greater than about 900 pg/mL,
greater than about
1000 pg/mL.
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[00428] The term "deoxyribonucleotide" encompasses natural and synthetic,
unmodified and
modified deoxyribonucleotides. Modifications include changes to the sugar
moiety, to the base moiety
and/or to the linkages between deoxyribonucleotide in the oligonucleotide.
[00429] The term "RNA" defines a molecule comprising at least one
ribonucleotide residue. The
term "ribonucleotide" defines a nucleotide with a hydroxyl group at the 2'
position of a b-D-
ribofuranose moiety. The term RNA includes double-stranded RNA, single-
stranded RNA, isolated
RNA such as partially purified RNA, essentially pure RNA, synthetic RNA,
recombinantly produced
RNA, as well as altered RNA that differs from naturally occurring RNA by the
addition, deletion,
substitution and/or alteration of one or more nucleotides. Nucleotides of the
RNA molecules
described herein may also comprise non-standard nucleotides, such as non-
naturally occurring
nucleotides or chemically synthesized nucleotides or deoxynucleotides. These
altered RNAs can be
referred to as analogs or analogs of naturally-occurring RNA.
[00430] The terms "pharmaceutically acceptable carrier" or "pharmaceutically
acceptable excipient"
are intended to include any and all solvents, dispersion media, coatings,
antibacterial and antifungal
agents, isotonic and absorption delaying agents, and inert ingredients. The
use of such
pharmaceutically acceptable carriers or pharmaceutically acceptable excipients
for active
pharmaceutical ingredients is well known in the art. Except insofar as any
conventional
pharmaceutically acceptable carrier or pharmaceutically acceptable excipient
is incompatible with the
active pharmaceutical ingredient, its use in therapeutic compositions of the
invention is contemplated.
Additional active pharmaceutical ingredients, such as other drugs, can also be
incorporated into the
described compositions and methods.
[00431] The terms "about" and "approximately" mean within a statistically
meaningful range of a
value. Such a range can be within an order of magnitude, preferably within
50%, more preferably
within 20%, more preferably still within 10%, and even more preferably within
5% of a given value
or range. The allowable variation encompassed by the terms "about" or
"approximately" depends on
the particular system under study, and can be readily appreciated by one of
ordinary skill in the art.
Moreover, as used herein, the terms "about" and -approximately" mean that
dimensions, sizes,
formulations, parameters, shapes and other quantities and characteristics arc
not and need not be
exact, but may be approximate and/or larger or smaller, as desired, reflecting
tolerances, conversion
factors, rounding off, measurement error and the like, and other factors known
to those of skill in the
art. In general, a dimension, size, formulation, parameter, shape or other
quantity or characteristic is
"about" or "approximate" whether or not expressly stated to be such. It is
noted that embodiments of
very different sizes, shapes and dimensions may employ the described
arrangements.
[00432] The transitional terms "comprising," "consisting essentially of," and
"consisting of," when
used in the appended claims, in original and amended form, define the claim
scope with respect to
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what unrecited additional claim elements or steps, if any, are excluded from
the scope of the claim(s).
The term "comprising' is intended to be inclusive or open-ended and does not
exclude any additional,
unrecited element, method, step or material. The term "consisting of" excludes
any element, step or
material other than those specified in the claim and, in the latter instance,
impurities ordinary
associated with the specified material(s). The term "consisting essentially of-
limits the scope of a
claim to the specified elements, steps or material(s) and those that do not
materially affect the basic
and novel characteristic(s) of the claimed invention. All compositions,
methods, and kits described
herein that embody the present invention can, in alternate embodiments, be
more specifically defined
by any of the transitional terms "comprising," "consisting essentially o" and
"consisting of."
[00433] The terms "antibody" and its plural form -antibodies" refer to whole
immunoglobulins and
any antigen-binding fragment ("antigen-binding portion") or single chains
thereof. An "antibody-
further refers to a glycoprotein comprising at least two heavy (H) chains and
two light (L) chains
inter-connected by disulfide bonds, or an antigen-binding portion thereof.
Each heavy chain is
comprised of a heavy chain variable region (abbreviated herein as VII) and a
heavy chain constant
region. The heavy chain constant region is comprised of three domains, CHI,
CH2 and CH3. Each
light chain is comprised of a light chain variable region (abbreviated herein
as VL) and a light chain
constant region. The light chain constant region is comprised of one domain,
CL. The VH and VI
regions of an antibody may be further subdivided into regions of
hypervariability, which are referred
to as complemental* determining regions (CDR) or hypervariable regions (HVR),
and which can be
interspersed with regions that are more conserved, termed framework regions
(FR). Each VH and VL is
composed of three CDRs and four FRs, arranged from amino-terminus to carboxy-
terminus in the
following order: FR1, CDR1, FR2, CDR2, FR3, CDR3, FR4. The variable regions of
the heavy and
light chains contain a binding domain that interacts with an antigen epitope
or epitopes. The constant
regions of the antibodies may mediate the binding of the immunoglobulin to
host tissues or factors,
including various cells of the immune system (e. g. , effector cells) and the
first component (Clq) of the
classical complement system.
[00434] The term "antigen" refers to a substance that induces an immune
response. In some
embodiments, an antigen is a molecule capable of being bound by an antibody or
a TCR if presented
by major histocompatibility complex (MHC) molecules. The term "antigen", as
used herein, also
encompasses T cell epitopes. An antigen is additionally capable of being
recognized by the immune
system. In some embodiments, an antigen is capable of inducing a humoral
immune response or a
cellular immune response leading to the activation of B lymphocytes and/or T
lymphocytes. In some
cases, this may require that the antigen contains or is linked to a Th cell
epitope. An antigen can also
have one or more epitopes (e.g., B- and T-epitopes). In some embodiments, an
antigen will preferably
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react, typically in a highly specific and selective manner, with its
corresponding antibody or TCR and
not with the multitude of other antibodies or TCRs which may be induced by
other antigens.
[00435] The terms "monoclonal antibody," "mAb," "monoclonal antibody
composition," or their
plural forms refer to a preparation of antibody molecules of single molecular
composition. A
monoclonal antibody composition displays a single binding specificity and
affinity for a particular
epitope. Monoclonal antibodies specific to certain receptors can be made using
knowledge and skill in
the art of injecting test subjects with suitable antigen and then isolating
hybridomas expressing
antibodies having the desired sequence or functional characteristics. DNA
encoding the monoclonal
antibodies is readily isolated and sequenced using conventional procedures
(e.g., by using
oligonucleotide probes that are capable of binding specifically to genes
encoding the heavy and light
chains of the monoclonal antibodies). The hybridoma cells serve as a preferred
source of such DNA.
Once isolated, the DNA may be placed into expression vectors, which are then
transfected into host
cells such as E. colt cells, simian COS cells, Chinese hamster ovary (CHO)
cells, or myeloma cells
that do not otherwise produce immunoglobulin protein, to obtain the synthesis
of monoclonal
antibodies in the recombinant host cells. Recombinant production of antibodies
will be described in
more detail below.
[00436] The terms "antigen-binding portion" or "antigen-binding fragment" of
an antibody (or
simply "antibody portion" or "fragment"), as used herein, refers to one or
more fragments of an
antibody that retain the ability to specifically bind to an antigen. It has
been shown that the antigen-
binding function of an antibody can be performed by fragments of a full-length
antibody. Examples of
binding fragments encompassed within the term -antigen-binding portion- of an
antibody include (i) a
Fab fragment, a monovalent fragment consisting of the VL, VH, CL and CHI
domains; (ii) a F(ab')2
fragment, a bivalent fragment comprising two Fab fragments linked by a
disulfide bridge at the hinge
region; (iii) a Fd fragment consisting of the VH and CH1 domains; (iv) a FA/
fragment consisting of the
VL and VE domains of a single arm of an antibody, (v) a domain antibody (dAb)
fragment (Ward, et
at., Nature, 1989, 341, 544-546), which may consist of a VH or a VL domain;
and (vi) an isolated
complementarily determining region (CDR). Furthermore, although the two
domains of the Fv
fragment, VL and VII, are coded for by separate genes, they can be joined,
using recombinant methods,
by a synthetic linker that enables them to be made as a single protein chain
in which the VL and VH
regions pair to form monovalent molecules known as single chain Fv (scFv);
see, e.g., Bird, et al.,
Science 1988, 242, 423-426; and Huston, et al., Proc. Natl. Acad. Set. USA
1988, 85, 5879-5883).
Such scFAT antibodies are also intended to be encompassed within the terms
"antigen-binding portion"
or -antigen-binding fragment" of an antibody. These antibody fragments arc
obtained using
conventional techniques known to those with skill in the art, and the
fragments are screened for utility
in the same manner as are intact antibodies. In some embodiments, a scFy
protein domain comprises a
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VH portion and a VL portion. A scFv molecule is denoted as either VL-L-Vii if
the VL domain is the N-
terminal part of the scFv molecule, or as VL-L-VL if the VH domain is the N-
terminal part of the scFv
molecule. Methods for making say molecules and designing suitable peptide
linkers are described in
U.S. Pat. No. 4,704,692, U.S. Pat. No. 4,946,778, R. Raag and M. Whitlow,
"Single Chain Fvs."
FASEB Vol 9:73-80 (1995) and R. E. Bird and B. W. Walker, Single Chain
Antibody Variable
Regions, 11BTECH, Vol 9: 132-137 (1991), the disclosures of which are
incorporated by reference
herein.
[00437] The term "human antibody," as used herein, is intended to include
antibodies having
variable regions in which both the framework and CDR regions are derived from
human germline
immunoglobulin sequences. Furthermore, if the antibody contains a constant
region, the constant
region also is derived from human germline immunoglobulin sequences. The human
antibodies of the
invention may include amino acid residues not encoded by human germline
immunoglobulin
sequences (e.g., mutations introduced by random or site-specific mutagenesis
in vitro or by somatic
mutation in vivo). The term -human antibody", as used herein, is not intended
to include antibodies in
which CDR sequences derived from the germline of another mammalian species,
such as a mouse,
have been grafted onto human framework sequences.
[00438] The term "human monoclonal antibody" refers to antibodies displaying a
single binding
specificity which have variable regions in which both the framework and CDR
regions are derived
from human germline immunoglobulin sequences. In some embodiments, the human
monoclonal
antibodies are produced by a hybridoma which includes a B cell obtained from a
transgenic
nonhuman animal, e.g., a transgenic mouse, having a gcnomc comprising a human
heavy chain
transgene and a light chain transgene fused to an immortalized cell.
[00439] The term "recombinant human antibody", as used herein, includes all
human antibodies that
are prepared, expressed, created or isolated by recombinant means, such as (a)
antibodies isolated
from an animal (such as a mouse) that is transgenic or transchromosomal for
human immunoglobulin
genes or a hybridoma prepared therefrom (described further below), (b)
antibodies isolated from a
host cell transformed to express the human antibody, e.g., from a
transfectoma, (c) antibodies isolated
from a recombinant, combinatorial human antibody library, and (d) antibodies
prepared, expressed,
created or isolated by any other means that involve splicing of human
immunoglobulin gene
sequences to other DNA sequences. Such recombinant human antibodies have
variable regions in
which the framework and CDR regions are derived from human germline
immunoglobulin sequences.
In certain embodiments, however, such recombinant human antibodies can be
subjected to in vitro
mutagenesis (or, when an animal transgenic for human Ig sequences is used, in
vivo somatic
mutagenesis) and thus the amino acid sequences of the VH and VL regions of the
recombinant
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antibodies are sequences that, while derived from and related to human
germline VII and VL
sequences, may not naturally exist within the human antibody germline
repertoire in vivo.
[00440] As used herein, "isotype" refers to the antibody class (e.g., IgM or
IgG1) that is encoded by
the heavy chain constant region genes.
[00441] The phrases -an antibody recognizing an antigen" and "an antibody
specific for an antigen"
are used interchangeably herein with the term -an antibody which binds
specifically to an antigen."
[00442] The term "human antibody derivatives" refers to any modified form of
the human antibody,
including a conjugate of the antibody and another active pharmaceutical
ingredient or antibody. The
terms "conjugate," "antibody-drug conjugate", "ADC," or "immunoconjugate"
refers to an antibody,
or a fragment thereof, conjugated to another therapeutic moiety, which can be
conjugated to
antibodies described herein using methods available in the art.
[00443] The terms "humanized antibody," "humanized antibodies," and
"humanized" are intended
to refer to antibodies in which CDR sequences derived from the germline of
another mammalian
species, such as a mouse, have been grafted onto human framework sequences.
Additional framework
region modifications may be made within the human framework sequences.
Humanized forms of non-
human (for example, murine) antibodies are chimeric antibodies that contain
minimal sequence
derived from non-human immunoglobulin. For the most part, humanized antibodies
arc human
immunoglobulins (recipient antibody) in which residues from a hypervariable
region of the recipient
arc replaced by residues from a 15 hypervariable region of a non-human species
(donor antibody)
such as mouse, rat, rabbit or nonhuman primate having the desired specificity,
affinity, and capacity.
In some instances, Fv framework region (FR) residues of the human
immunoglobulin are replaced by
corresponding non-human residues. Furthermore, humanized antibodies may
comprise residues that
are not found in the recipient antibody or in the donor antibody. These
modifications are made to
further refine antibody performance. In general, the humanized antibody will
comprise substantially
all of at least one, and typically two, variable domains, in which all or
substantially all of the
hypervariable loops correspond to those of a non-human immunoglobulin and all
or substantially all
of the FR regions are those of a human immunoglobulin sequence. The humanized
antibody
optionally also will comprise at least a portion of an immunoglobulin constant
region (Fe), typically
that of a human immunoglobulin. For further details, see Jones, etal., Nature
1986, 321, 522-525;
Riechmann, et at., Nature 1988, 332, 323-329; and Presta, Curr. Op. Struct
Biol. 1992, 2, 593-596.
The antibodies described herein may also be modified to employ any Fe variant
which is known to
impart an improvement (e.g., reduction) in effector function and/or FcR
binding. The Fc variants may
include, for example, any one of the amino acid substitutions disclosed in
International Patent
Application Publication Nos. WO 1988/07089 Al, WO 1996/14339 Al, WO 1998/05787
Al, WO
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1998/23289 Al, WO 1999/51642 Al, WO 99/58572 Al, WO 2000/09560 A2, WO
2000/32767 Al,
WO 2000/42072 A2, WO 2002/44215 A2, WO 2002/060919 A2, WO 2003/074569 A2, WO
2004/016750 A2, WO 2004/029207 A2, WO 2004/035752 A2, WO 2004/063351 A2, WO
2004/074455 A2, WO 2004/099249 A2, WO 2005/040217 A2, WO 2005/070963 Al, WO
2005/077981 A2, WO 2005/092925 A2, WO 2005/123780 A2, WO 2006/019447 Al, WO
2006/047350 A2, and WO 2006/085967 A2; and U.S. Patent Nos. 5,648,260;
5,739,277; 5,834,250;
5,869,046; 6,096,871; 6,121,022; 6,194,551; 6,242,195; 6,277,375; 6,528,624;
6,538,124; 6,737,056;
6,821,505; 6,998,253; and 7,083,784; the disclosures of which are incorporated
by reference herein.
[00444] The term chimeric antibody" is intended to refer to antibodies in
which the variable region
sequences are derived from one species and the constant region sequences are
derived from another
species, such as an antibody in which the variable region sequences are
derived from a mouse
antibody and the constant region sequences are derived from a human antibody.
[00445] A "diabody" is a small antibody fragment with two antigen-binding
sites. The fragments
comprises a heavy chain variable domain (VII) connected to a light chain
variable domain (VL) in the
same polypeptide chain or Vi,-Vii). By using a linker that is too
short to allow pairing between
the two domains on the same chain, the domains are forced to pair with the
complementary domains
of another chain and create two antigen-binding sites. Diabodies are described
more fully in, e.g.,
European Patent No. EP 404,097, International Patent Publication No. WO
93/11161; and Bolliger, et
at., Proc. Natl. Acad. Sci. USA 1993, 90, 6444-6448.
[00446] The term "glycosylation" refers to a modified derivative of an
antibody. An aglycoslated
antibody lacks glycosylation. Glycosylation can be altered to, for example,
increase the affinity of the
antibody for antigen. Such carbohydrate modifications can be accomplished by,
for example, altering
one or more sites of glycosylation within the antibody sequence. For example,
one or more amino
acid substitutions can be made that result in elimination of one or more
variable region framework
glycosylation sites to thereby eliminate glycosylation at that site.
Aglycosylation may increase the
affinity of the antibody for antigen, as described in U.S. Patent Nos.
5,714,350 and 6,350,861.
Additionally or alternatively, an antibody can be made that has an altered
type of glycosylation, such
as a hypofucosylated antibody having reduced amounts of fucosyl residues or an
antibody having
increased bisecting GlcNac structures. Such altered glycosylation patterns
have been demonstrated to
increase the ability of antibodies. Such carbohydrate modifications can be
accomplished by, for
example, expressing the antibody in a host cell with altered glycosylation
machinery. Cells with
altered glycosylation machinery have been described in the art and can be used
as host cells in which
to express recombinant antibodies of the invention to thereby produce an
antibody with altered
glycosylation. For example, the cell lines Ms704, Ms705, and Ms709 lack the
fucosyltransferase
gene, FUT8 (alpha (1,6) fucosyltransferase), such that antibodies expressed in
the Ms704, Ms705, and
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Ms709 cell lines lack fucose on their carbohydrates. The Ms704, Ms705, and
Ms709 FUT8¨/¨ cell
lines were created by the targeted disruption of the FUT8 gene in CHO/DG44
cells using two
replacement vectors (see e.g. U.S. Patent Publication No. 2004/0110704 or
Yamane-Ohnuki, et al.,
Biotechnol. Bioeng., 2004,87, 614-622). As another example, European Patent
No. EP 1,176,195
describes a cell line with a functionally disrupted FUT8 gene, which encodes a
fucosyl transferase,
such that antibodies expressed in such a cell line exhibit hypofucosylation by
rcducing or eliminating
the alpha 1,6 bond-related enzyme, and also describes cell lines which have a
low enzyme activity for
adding fucose to the N-acetylglucosamine that binds to the Fc region of the
antibody or does not have
the enzyme activity, for example the rat myeloma cell line YB2/0 (ATCC CRL
1662). International
Patent Publication WO 03/035835 describes a variant CHO cell line, Lec 13
cells, with reduced
ability to attach fucose to Asn(297)-linked carbohydrates, also resulting in
hypofucosylation of
antibodies expressed in that host cell (see also Shields, et al., I Biol.
Chem. 2002, 277, 26733-26740.
International Patent Publication WO 99/54342 describes cell lines engineered
to express glycoprotein-
modifying glycosyl transferases (e.g., beta(1,4)-N-
acetylglucosaminyltransferase III (GnTIII)) such
that antibodies expressed in the engineered cell lines exhibit increased
bisecting GlcNac structures
which results in increased ADCC activity of the antibodies (see also Umana,
etal., Nat. Biotech.
1999, 17, 176-180). Alternatively, the fucose residues of the antibody may be
cleaved off using a
fucosidase enzyme. For example, the fucosidase alpha-L-fucosidase removes
fucosyl residues from
antibodies as described in Tarentino, etal., Biochem. 1975, 14, 5516-5523.
[00447] "Pegylation" refers to a modified antibody, or a fragment thereof,
that typically is reacted
with polyethylene glycol (PEG), such as a reactive ester or aldehyde
derivative of PEG, under
conditions in which one or more PEG groups become attached to the antibody or
antibody fragment.
Pcgylation may, for example, increase the biological (e.g., scrum) half life
of the antibody. Preferably,
the pegylation is carried out via an acylation reaction or an alkylation
reaction with a reactive PEG
molecule (or an analogous reactive water-soluble polymer). As used herein, the
term "polyethylene
glycol" is intended to encompass any of the forms of PEG that have been used
to derivatize other
proteins, such as mono (CI-Cio)alkoxy- or aryloxy-polyethylenc glycol or
polyethylene glycol-
maleimide. The antibody to be pegylated may be an aglycosylated antibody.
Methods for pegylation
are known in the art and can be applied to the antibodies of the invention, as
described for example in
European Patent Nos. EP 0154316 and EP 0401384 and U.S. Patent No. 5,824,778,
the disclosures of
each of which are incorporated by reference herein.
[00448] The term "biosimilar" means a biological product, including a
monoclonal antibody or
protein, that is highly similar to a U.S. licensed reference biological
product notwithstanding minor
differences in clinically inactive components, and for which there are no
clinically meaningful
differences between the biological product and the reference product in terms
of the safety, purity, and
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potency of the product. Furthermore, a similar biological or "biosimilar"
medicine is a biological
medicine that is similar to another biological medicine that has already been
authorized for use by the
European Medicines Agency. The term "biosimilar" is also used synonymously by
other national and
regional regulatory agencies. Biological products or biological medicines are
medicines that are made
by or derived from a biological source, such as a bacterium or yeast. They can
consist of relatively
small molecules such as human insulin or erythropoietin, or complex molecules
such as monoclonal
antibodies. For example, if the reference IL-2 protein is aldesleukin
(PROLEUKIN), a protein
approved by drug regulatory authorities with reference to aldesleukin is a
"biosimilar to" aldesleukin
or is a "biosimilar thereof' of aldesleukin. In Europe, a similar biological
or "biosimilar" medicine is
a biological medicine that is similar to another biological medicine that has
already been authorized
for use by the European Medicines Agency (EMA). The relevant legal basis for
similar biological
applications in Europe is Article 6 of Regulation (EC) No 726/2004 and Article
10(4) of Directive
2001/83/EC, as amended and therefore in Europe, the biosimilar may be
authorized, approved for
authorization or subject of an application for authorization under Article 6
of Regulation (EC) No
726/2004 and Article 10(4) of Directive 2001/83/EC. The already authorized
original biological
medicinal product may be referred to as a "reference medicinal product" in
Europe. Some of the
requirements for a product to be considered a biosimilar are outlined in the
CHMP Guideline on
Similar Biological Medicinal Products. In addition, product specific
guidelines, including guidelines
relating to monoclonal antibody biosimilars, are provided on a product-by-
product basis by the EMA
and published on its website. A biosimilar as described herein may be similar
to the reference
medicinal product by way of quality characteristics, biological activity,
mechanism of action, safety
profiles and/or efficacy. In addition, the biosimilar may be used or be
intended for use to treat the
same conditions as the reference medicinal product. Thus, a biosimilar as
described herein may be
deemed to have similar or highly similar quality characteristics to a
reference medicinal product.
Alternatively, or in addition, a biosimilar as described herein may be deemed
to have similar or highly
similar biological activity to a reference medicinal product. Alternatively,
or in addition, a biosimilar
as described herein may be deemed to have a similar or highly similar safety
profile to a reference
medicinal product. Alternatively, or in addition, a biosimilar as described
herein may be deemed to
have similar or highly similar efficacy to a reference medicinal product. As
described herein, a
biosimilar in Europe is compared to a reference medicinal product which has
been authorized by the
EMA. However, in some instances, the biosimilar may be compared to a
biological medicinal product
which has been authorized outside the European Economic Area (a non-EEA
authorized
"comparator") in certain studies. Such studies include for example certain
clinical and in vivo non-
clinical studies. As used herein, the term "biosimilar" also relates to a
biological medicinal product
which has been or may be compared to a non-EEA authorized comparator. Certain
biosimilars are
proteins such as antibodies, antibody fragments (for example, antigen binding
portions) and fusion
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proteins. A protein biosimilar may have an amino acid sequence that has minor
modifications in the
amino acid structure (including for example deletions, additions, and/or
substitutions of amino acids)
which do not significantly affect the function of the polypepti de. The
biosimilar may comprise an
amino acid sequence having a sequence identity of 97% or greater to the amino
acid sequence of its
reference medicinal product, e.g., 97%, 98%, 99% or 100%. The biosimilar may
comprise one or
more post-translational modifications, for example, although not limited to,
glycosylation, oxidation,
deamidation, and/or truncation which is/are different to the post-
translational modifications of the
reference medicinal product, provided that the differences do not result in a
change in safety and/or
efficacy of the medicinal product. The biosimilar may have an identical or
different glycosylation
pattern to the reference medicinal product. Particularly, although not
exclusively, the biosimilar may
have a different glycosylation pattern if the differences address or are
intended to address safety
concerns associated with the reference medicinal product. Additionally, the
biosimilar may deviate
from the reference medicinal product in for example its strength,
pharmaceutical form, formulation,
excipients and/or presentation, providing safety and efficacy of the medicinal
product is not
compromised. The biosimilar may comprise differences in for example
pliamiaeokinetic (PK) and/or
pharmacodynamic (PD) profiles as compared to the reference medicinal product
but is still deemed
sufficiently similar to the reference medicinal product as to be authorized or
considered suitable for
authorization. In certain circumstances, the biosimilar exhibits different
binding characteristics as
compared to the reference medicinal product, wherein the different binding
characteristics are
considered by a Regulatory Authority such as the EMA not to be a barrier for
authorization as a
similar biological product. The term "biosimilar- is also used synonymously by
other national and
regional regulatory agencies.
Gen 2 TIL Manufacturing Processes
[00449] An exemplary family of TIL processes known as Gen 2 (also known as
process 2A)
containing some of these features is depicted in Figures 1 and 2. An
embodiment of Gen 2 is shown in
Figure 2.
[00450] As discussed herein, the present invention can include a step relating
to the restimulation of
cryopre served TILs to increase their metabolic activity and thus relative
health prior to transplant into
a patient, and methods of testing said metabolic health. As generally outlined
herein, TILs are
generally taken from a patient sample and manipulated to expand their number
prior to transplant into
a patient. In some embodiments, the TILs may be optionally genetically
manipulated as discussed
below.
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[00451] In some embodiments, the TILs may be eryopreserved. Once thawed, they
may also be
restimulated to increase their metabolism prior to infusion into a patient.
[00452] In some embodiments, the first expansion (including processes referred
to as the preREP as
well as processes shown in Figure 1 as Step A) is shortened to 3 to 14 days
and the second expansion
(including processes referred to as the REP as well as processes shown in
Figure 1 as Step B) is
shorted to 7 to 14 days, as discussed in detail below as well as in the
examples and figures. In some
embodiments, the first expansion (for example, an expansion described as Step
B in Figure 1) is
shortened to 11 days and the second expansion (for example, an expansion as
described in Step D in
Figure 1) is shortened to 11 days. In some embodiments, the combination of the
first expansion and
second expansion (for example, expansions described as Step B and Step D in
Figure 1) is shortened
to 22 days, as discussed in detail below and in the examples and figures.
[00453] The Designations A, B, C, etc., below arc in reference to
Figure 1 and in reference to
certain embodiments described herein. The ordering of the Steps below and in
Figure 1 is exemplary
and any combination or order of steps, as well as additional steps, repetition
of steps, and/or omission
of steps is contemplated by the present application and the methods disclosed
herein.
A. STEP A: Obtain Patient Tumor Sample
[00454] In general, TILs are initially obtained from a patient tumor sample
and then expanded into a
larger population for further manipulation as described herein, optionally
cryopreserved, restimulated
as outlined herein and optionally evaluated for phenotype and metabolic
parameters as an indication
of TIL health.
[00455] A patient tumor sample may be obtained using methods known in the art,
generally via
surgical resection, needle biopsy, core biopsy, small biopsy, or other means
for obtaining a sample
that contains a mixture of tumor and TIL cells. In some embodiments,
multilesional sampling is used.
In some embodiments, surgical resection, needle biopsy, core biopsy, small
biopsy, or other means for
obtaining a sample that contains a mixture of tumor and TIL cells includes
multilesional sampling
(i.e., obtaining samples from one or more tumor sites and/or locations in the
patient, as well as one or
more tumors in the same location or in close proximity). In general, the tumor
sample may be from
any solid tumor, including primary tumors, invasive tumors or metastatic
tumors. The tumor sample
may also be a liquid tumor, such as a tumor obtained from a hematological
malignancy. The solid
tumor may be of lung tissue. In some embodiments, useful TILs are obtained
from non-small cell lung
carcinoma (NSCLC). The solid tumor may be of skin tissue. In some embodiments,
useful TILs are
obtained from a melanoma.
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[00456] Once obtained, the tumor sample is generally fragmented using sharp
dissection into small
pieces of between 1 to about 8 mml. with from about 2-3 mmi being particularly
useful. In some
embdoiments, the TILs are cultured from these fragments using enzymatic tumor
digests. Such tumor
digests may be produced by incubation in enzymatic media (e.g., Roswell Park
Memorial Institute
(RPMI) 1640 buffer, 2 mM glutamate, 10 mcg/mL gentamicine, 30 units/mL of
DNase and 1.0
mg/mL of collagenase) followed by mechanical dissociation (e.g., using a
tissue dissociator). Tumor
digests may be produced by placing the tumor in enzymatic media and
mechanically dissociating the
tumor for approximately 1 minute, followed by incubation for 30 minutes at 37
C in 5% CO2,
followed by repeated cycles of mechanical dissociation and incubation under
the foregoing conditions
until only small tissue pieces are present. At the end of this process, if the
cell suspension contains a
large number of red blood cells or dead cells, a density gradient separation
using FICOLL branched
hydrophilic polysaccharide may be performed to remove these cells. Alternative
methods known in
the art may be used, such as those described in U.S. Patent Application
Publication No, 2012/0244133
Al, the disclosure of which is incorporated by reference herein. Any of the
foregoing methods may be
used in any of the embodiments described herein for methods of expanding TILs
or methods treating
a cancer.
[00457] Tumor dissociating enzyme mixtures can include one or more
dissociating (digesting)
enzymes such as, but not limited to, collagenase (including any blend or type
of collagenase),
AccutaseTM, AccumaxTM, hyaluronidase, neutral protease (dispase),
chymotrypsin, chymopapain,
trypsin, caseinase, elastase, papain, protease type XIV (pronase),
deoxyribonuclease I (DNase),
trypsin inhibitor, any other dissociating or proteolytic enzyme, and any
combination thereof
[00458] In some embodiments, the dissociating enzymes are reconstituted from
lyophilized
enzymes. In some embodiments, lyophilized enzymes are reconstituted in an
amount of sterile buffer
such as HB SS.
[00459] In some instances, collagenase (such as animal free- type 1
collagenase) is reconstitued in
mL of sterile HBSS or another buffer. The lyophilized stock enzyme may be at a
concentration of
2892 PZ U/vial. In some embodiments, collagenase is reconstituted in 5 mL to
15 mL buffer. In some
embodiment, after reconstitution the collagenase stock ranges from about 100
PZ U/mL-about 400 PZ
U/mL, e.g., about 100 PZ U/mL-about 400 PZ U/mL, about 100 PZ U/mL-about 350
PZ U/mL, about
100 PZ U/mL-about 300 PZ U/mL, about 150 PZ U/mL-about 400 PZ U/mL, about 100
PZ U/mL,
about 150 PZ U/mL, about 200 PZ U/mL, about 210 PZ U/mL, about 220 PZ U/mL,
about 230 PZ
U/mL, about 240 PZ U/mL, about 250 PZ U/mL, about 260 PZ U/mL, about 270 PZ
U/mL, about 280
PZ U/mL, about 289.2 PZ U/mL, about 300 PZ U/mL, about 350 PZ U/mL, or about
400 PZ U/mL.
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[00460] In some embodiments, neutral protease is reconstituted in 1-ml of
sterile HESS or another
buffer. The lyophilized stock enzyme may be at a concentration of 175 DMC
U/vial. In some
embodiments, after reconstitution the neutral protease stock ranges from about
100 DMC/mL-about
400 DMC/mL, e.g., about 100 DMC/mL-about 400 DMC/mL, about 100 DMC/mL-about
350
DMC/mL, about 100 DMC/mL-about 300 DMC/mL, about 150 DMC/mL-about 400 DMC/mL,
about
100 DMC/mL, about 110 DMC/mL, about 120 DMC/mL, about 130 DMC/mL, about 140
DMC/mL,
about 150 DMC/mL, about 160 DMC/mL, about 170 DMC/mL, about 175 DMC/mL, about
180
DMC/mL, about 190 DMC/mL, about 200 DMC/mL, about 250 DMC/mL, about 300
DMC/mL,
about 350 DMC/mL, or about 400 DMC/mL.
[00461] In some embodiments, DNAse I is reconstituted in 1-ml of sterile HBSS
or another buffer.
The lyophilized stock enzyme was at a concentration of 4 KU/vial. In some
embodiments, after
reconstitution the DNase I stock ranges from about 1 KU/mL-10 KU/mL, e.g.,
about 1 KU/mL, about
2 KU/mL, about 3 KU/mL, about 4 KU/mL, about 5 KU/mL, about 6 KU/mL, about 7
KU/mL, about
8 KU/mL, about 9 KU/mL, or about 10 KU/mL.
[00462] In some embodiments, the stock of enzymes is variable and the
concentrations may need to
be determined. In some embodiments, the the concentration of the lyophilized
stock can be verified.
In some embodiments, the final amount of enzyme added to the digest cocktail
is adjusted based on
the determined stock concentration.
[00463] In some embodiment, the enzyme mixture includes about 10.2-ul of
neutral protease (0.36
DMC U/mL), 21.3-ul of collagenase (1.2 PZ/mL) and 250-ul of DNAse 1(200 U/mL)
in about 4.7-ml
of sterile HBSS.
[00464] As indicated above, in some embodiments, the TILs are derived from
solid tumors. In some
embodiments, the solid tumors are not fragmented. In some embodiments, the
solid tumors are not
fragmented and are subjected to enzymatic digestion as whole tumors. In some
embodiments, the
tumors are digested in in an enzyme mixture comprising collagenase, DNase, and
hyaluronidase. In
some embodiments, the tumors are digested in in an enzyme mixture comprising
collagenase, DNase,
and hyaluronidase for 1-2 hours. In some embodiments, the tumors are digested
in in an enzyme
mixture comprising collagenase, DNase, and hyaluronidase for 1-2 hours at 37
C, 5% CO2. In some
embodiments, the tumors are digested in in an enzyme mixture comprising
collagenase, DNase, and
hyaluronidase for 1-2 hours at 37 C, 5% CO2 with rotation. In some
embodiments, the tumors are
digested overnight with constant rotation. In some embodiments, the tumors are
digested overnight at
37 C, 5% CO2 with constant rotation. In some embodiments, the whole tumor is
combined with the
enzymes to form a tumor digest reaction mixture.
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[00465] In some embodiments, the tumor is reconstituted with the lyophilized
enzymes in a sterile
buffer. In some embodiments, the buffer is sterile HBSS.
[00466] In some embodiments, the enzyme mixture comprises collagenase. In some
embodiments,
the collagenase is collagenase IV. In some embodiments, the working stock for
the collagenase is a
100 mg/mL 10X working stock.
[00467] In some embodiments, the enzyme mixture comprises DNAse. In some
embodiments, the
working stock for the DNAse is a 10,000IU/mL 10X working stock.
[00468] In some embodiments, the enzyme mixture comprises hyaluronidase. In
some
embodiments, the working stock for the hyaluronidase is a 10-mg/mL 10X working
stock.
[00469] In some embodiments, the enzyme mixture comprises 10 mg/mL
collagenase, 1000 IU/mL
DNAse, and 1 mg/mL hyaluronidase.
[00470] In some embodiments, the enzyme mixture comprises 10 mg/mL
collagenase, 500 IU/mL
DNAse, and 1 mg/mL hyaluronidase.
[00471] In general, the harvested cell suspension is called a "primary cell
population" or a "freshly
harvested" cell population.
[00472] In some embodiments, fragmentation includes physical fragmentation,
including for
example, dissection as well as digestion. In some embodiments, the
fragmentation is physical
fragmentation. in some embodiments, the fragmentation is dissection. In some
embodiments, the
fragmentation is by digestion. In some embodiments, TILs can be initially
cultured from enzymatic
tumor digests and tumor fragments obtained from digesting or fragmenting a
tumor sample obtained
from a patient.
[00473] In some embodiments, where the tumor is a solid tumor, the tumor
undergoes physical
fragmentation after the tumor sample is obtained in, for example, Step A (as
provided in Figure 1). In
some embodiments, the fragmentation occurs before cryopreservation. In some
embodiments, the
fragmentation occurs after cryopreservation. In some embodiments, the
fragmentation occurs after
obtaining the tumor and in the absence of any cryopreservation. In some
embodiments, the tumor is
fragmented and 10, 20, 30, 40 or more fragments or pieces are placed in each
container for the first
expansion. in some embodiments, the tumor is fragmented and 30 or 40 fragments
or pieces are
placed in each container for the first expansion. In some embodiments, the
tumor is fragmented and
40 fragments or pieces are placed in each container for the first expansion.
In some embodiments, the
multiple fragments comprise about 4 to about 50 fragments, wherein each
fragment has a volume of
about 27 mm3. In some embodiments, the multiple fragments comprise about 30 to
about 60
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fragments with a total volume of about 1300 min3 to about 1500 min3. In some
embodiments, the
multiple fragments comprise about 50 fragments with a total volume of about
1350 min3. In some
embodiments, the multiple fragments comprise about 50 fragments with a total
mass of about 1 gram
to about 1.5 grams. In some embodiments, the multiple fragments comprise about
4 fragments.
[00474] In some embodiments, the TILs are obtained from tumor fragments. In
some embodiments,
the tumor fragment is obtained by sharp dissection. In some embodiments, the
tumor fragment is
between about 1 min3 and 10 mm3. In some embodiments, the tumor fragment is
between about 1
min3 and 8 min3. In some embodiments, the tumor fragment is about 1 mm3. In
some embodiments,
the tumor fragment is about 2 min3. In some embodiments, the tumor fragment is
about 3 min3. In
some embodiments, the tumor fragment is about 4 min3. In some embodiments, the
tumor fragment is
about 5 mini. In some embodiments, the tumor fragment is about 6 min'. In some
embodiments, the
tumor fragment is about 7 min3. In some embodiments, the tumor fragment is
about 8 min3. In some
embodiments, the tumor fragment is about 9 min3. in some embodiments, the
tumor fragment is about
min3. In some embodiments, the tumors are 1-4 mm 1-4 mm x 1-4 mm. In some
embodiments,
the tumors are 1 mm >< 1 mm x 1 mm. In some embodiments, the tumors are 2 mm x
2 mm x 2 mm. In
some embodiments, the tumors are 3 mm>< 3 mm >< 3 mm. In some embodiments, the
tumors are 4
mm x 4 mm x 4 mm.
[00475] In some embodiments, the tumors are resected in order to minimize the
amount of
hemorrhagic, necrotic, and/or fatty tissues on each piece. In some
embodiments, the tumors are
resected in order to minimize the amount of hemorrhagic tissue on each piece.
In some embodiments,
the tumors arc resected in order to minimize the amount of necrotic tissue on
each piece. In some
embodiments, the tumors are resected in order to minimize the amount of fatty
tissue on each piece.
[00476] In some embodiments, the tumor fragmentation is performed in order to
maintain the tumor
internal structure. In some embodiments, the tumor fragmentation is performed
without preforming a
sawing motion with a scalpel. In some embodiments, the TILs are obtained from
tumor digests. In
some embodiments, tumor digests were generated by incubation in enzyme media,
for example but
not limited to RPMI 1640, 2 mM GlutaMAX, 10 mg/mL gentamicin, 30 U/mL DNasc,
and 1.0
mg/mL collagenase, followed by mechanical dissociation (GentleMACS, Miltenyi
Biotec, Auburn,
CA). After placing the tumor in enzyme media, the tumor can be mechanically
dissociated for
approximately 1 minute. The solution can then be incubated for 30 minutes at
37 C in 5% CO2 and
it then mechanically disrupted again for approximately 1 minute. After being
incubated again for 30
minutes at 37 C in 5% CO2, the tumor can be mechanically disrupted a third
time for
approximately 1 minute. In some embodiments, after the third mechanical
disruption if large pieces
of tissue were present, 1 or 2 additional mechanical dissociations were
applied to the sample, with
or without 30 additional minutes of incubation at 37 C in 5% CO2. In some
embodiments, at the
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end of the final incubation if the cell suspension contained a large number of
red blood cells or dead
cells, a density gradient separation using Ficoll can be performed to remove
these cells.
[00477] In some embodiments, the harvested cell suspension prior to the first
expansion step is
called a -primary cell population" or a -freshly harvested" cell population.
[00478] In some embodiments, cells can be optionally frozen after sample
harvest and stored frozen
prior to entry into the expansion described in Step B, which is described in
further detail below, as
well as exemplified in Figure 1, as well as Figure 8.
1. Pleural effusion T-cells or TILs
[00479] In some embodiments, the sample is a pleural fluid sample. In some
embodiments, the
source of the T-cells or TILs for expansion according to the processes
described herein is a pleural
fluid sample. In some embodiments; the sample is a pleural effusion derived
sample. In some
embodiments, the source of the T-cells or TILs for expansion according to the
processes described
herein is a pleural effusion derived sample. See, for example, methods
described in U.S. Patent
Publication US 2014/0295426, incorporated herein by reference in its entirety
for all purposes.
[00480] In some embodiments, any pleural fluid or pleural effusion suspected
of and/or containing
TILs can be employed. Such a sample may be derived from a primary or
metastatic lung cancer, such
as NSCLC or SCLC. In some embodiments, the sample may be secondary metastatic
cancer cells
which originated from another organ, e.g., breast, ovary, colon or prostate.
In some embodiments, the
sample for use in the expansion methods described herein is a pleural exudate.
In some embodiments,
the sample for use in the expansion methods described herein is a pleural
transudate. Other biological
samples may include other serous fluids containing TILs, including, e.g.,
ascites fluid from the
abdomen or pancreatic cyst fluid. Ascites fluid and pleural fluids involve
very similar chemical
systems; both the abdomen and lung have mesofhelial lines and fluid forms in
the pleural space and
abdominal spaces in the same matter in malignancies and such fluids in some
embodiments contain
TILs. In some embodiments, wherein the disclosure exemplifies pleural fluid,
the same methods may
be performed vvith similar results using ascites or other cyst fluids
containing TILs.
[00481] In some embodiments, the pleural fluid is in unprocessed form,
directly as removed from
the patient. In some embodiments, the unprocessed pleural fluid is placed in a
standard blood
collection tube, such as an EDTA or Heparin tube, prior to the contacting
step. In some embodiments,
the unprocessed pleural fluid is placed in a standard CellSavek tube (Veridex)
prior to the contacting
step. In some embodiments, the sample is placed in the CellSave tube
immediately after collection
from the patient to avoid a decrease in the number of viable TILs. The number
of viable TILs can
decrease to a significant extent within 24 hours, if left in the untreated
pleural fluid, even at 4 C. In
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some embodiments, the sample is placed in the appropriate collection tube
within 1 hour, 5 hours, 10
hours, 15 hours, or up to 24 hours after removal from the patient. In some
embodiments, the sample is
placed in the appropriate collection tube within 1 hour, 5 hours, 10 hours, 15
hours, or up to 24 hours
after removal from the patient at 4 C.
[00482] In some embodiments, the pleural fluid sample from the chosen subject
may be diluted. In
one embodiment, the dilution is 1:10 pleural fluid to diluent. In some
embodiments, the dilution is 1:9
pleural fluid to diluent. In some embodiments, the dilution is 1:8 pleural
fluid to diluent. In some
embodiments, the dilution is 1.5 pleural fluid to diluent Tn some embodiments,
the dilution is 1-2
pleural fluid to diluent. In some embodiments, the dilution is 1:1 pleural
fluid to diluent. In some
embodiments, diluents include saline, phosphate buffered saline, another
buffer or a physiologically
acceptable diluent. In some embodiments, the sample is placed in the CellSave
tube immediately after
collection from the patient and dilution to avoid a decrease in the viable
TILs, which may occur to a
significant extent within 24-48 hours, if left in the untreated pleural fluid,
even at 4 C. In some
embodiments, the pleural fluid sample is placed in the appropriate collection
tube within 1 hour, 5
hours, 10 hours, 15 hours, 24 hours, 36 hours, up to 48 hours after removal
from the patient, and
dilution. In some embodiments, the pleural fluid sample is placed in the
appropriate collection tube
within 1 hour, 5 hours, 10 hours, 15 hours, 24 hours, 36 hours, up to 48 hours
after removal from the
patient, and dilution at 4 C.
[00483] In still another embodiment, pleural fluid samples are concentrated by
conventional means
prior further processing steps. In some embodiments, this pre-treatment of the
pleural fluid is
preferable in circumstances in which the pleural fluid must be cryopreservcd
for shipment to a
laboratory performing the method or for later analysis (e.g., later than 24-48
hours post-collection). In
some embodiments, the pleural fluid sample is prepared by centrifuging the
pleural fluid sample after
its withdrawal from the subject and resuspending the centrifugate or pellet in
buffer. In some
embodiments, the pleural fluid sample is subjected to multiple centrifugations
and resuspensions,
before it is cryopreserved for transport or later analysis and/or processing.
[00484] In some embodiments, pleural fluid samples are concentrated prior to
further processing
steps by using a filtration method. In some embodiments, the pleural fluid
sample used in the
contacting step is prepared by filtering the fluid through a filter containing
a known and essentially
uniform pore size that allows for passage of the pleural fluid through the
membrane but retains the
tumor cells. In some embodiments, the diameter of the pores in the membrane
may be at least 4 uM.
In some embodiments the pore diameter may be 5 M or more, and in other
embodiment, any of 6, 7,
8, 9, or 10 [t1\4. After filtration, the cells, including TILs, retained by
the membrane may be rinsed off
the membrane into a suitable physiologically acceptable buffer. Cells,
including TILs, concentrated in
this way may then be used in the contacting step of the method.
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1004851 In some embodiment, pleural fluid sample (including, for example, the
untreated pleural
fluid), diluted pleural fluid, or the resuspended cell pellet, is contacted
with a lytic reagent that
differentially lyses non-nucleated red blood cells present in the sample. In
some embodiments, this
step is performed prior to further processing steps in circumstances in which
the pleural fluid contains
substantial numbers of RBCs. Suitable lysing reagents include a single lytic
reagent or a lytic reagent
and a quench reagent, or a lytic agent, a quench reagent and a fixation
reagent. Suitable lytic systems
are marketed commercially and include the BD Pharm LyseTM system (Becton
Dickenson). Other
lytic systems include the VersalyseTM system, the FACSlyseTM system (Becton
Dickenson), the
ImmunoprepTM system or Erythrolyse II system (Beckman Coulter, Inc.), or an
ammonium chloride
system. In some embodiments, the lytic reagent can vary with the primary
requirements being
efficient lysis of the red blood cells, and the conservation of the TILs and
phenotypic properties of the
TILs in the pleural fluid. In addition to employing a single reagent for
lysis, the lytic systems useful in
methods described herein can include a second reagent, e.g., one that quenches
or retards the effect of
the lytic reagent during the remaining steps of the method, e.g., Stabilyse TM
reagent (Beckman
Coulter, Inc.). A conventional fixation reagent may also be employed depending
upon the choice of
lytic reagents or the preferred implementation of the method.
[00486] In some embodiments, the pleural fluid sample, unprocessed, diluted or
multiply
centrifuged or processed as described herein above is cryoprescrved at a
temperature of about ¨140 C
prior to being further processed and/or expanded as provided herein.
B. STEP B: First Expansion
1004871 In some embodiments, the present methods provide for obtaining young
TILs, which are
capable of increased replication cycles upon administration to a
subject/patient and as such may
provide additional therapeutic benefits over older TILs (i.e., TILs which have
further undergone more
rounds of replication prior to administration to a subject/patient). Features
of young TILs have been
described in the literature, for example Donia, et al., Seand. J.
1111171141'101. 2012, 75, 157-167, Dudley,
etal., Cl/n. Cancer Res. 2010, /6,6122-6131; Huang, et al.,' Immunother. 2005,
28, 258-267;
Besser, etal., Cl/n. Cancer Res. 2013,19, OF1-0F9; Besser, et al.,'
Immunother. 2009, 32:415-423;
Robbins, etal., I Immunol. 2004, 173, 7125-7130; Shen, etal., I Immunother,
2007, 30, 123-129;
Zhou, etal., J. Immunother. 2005, 28, 53-62; and Tran, etal., I Immunother.,
2008, 3/, 742-751,
each of which is incorporated herein by reference.
1004881 The diverse antigen receptors of T and B lymphocytes are produced by
somatic
recombination of a limited, but large number of gene segments. These gene
segments: V (variable), D
(diversity), J (joining), and C (constant), determine the binding specificity
and downstream
applications of immunoglobulins and T-cell receptors (TCRs). The present
invention provides a
CA 03195023 2023- 4- 5
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method for generating TILs which exhibit and increase the T-cell repertoire
diversity. In some
embodiments, the TILs obtained by the present method exhibit an increase in
the T-cell repertoire
diversity. In some embodiments, the TILs obtained by the present method
exhibit an increase in the T-
cell repertoire diversity as compared to freshly harvested TILs and/or TILs
prepared using other
methods than those provide herein including for example, methods other than
those embodied in
Figure 1. In some embodiments, the TILs obtained by the present method exhibit
an increase in the 1-
cell repertoire diversity as compared to freshly harvested TILs and/or TILs
prepared using methods
referred to as process 1C, as exemplified in Figure 5 and/or Figure 6. In some
embodiments, the TILs
obtained in the first expansion exhibit an increase in the T-cell repertoire
diversity. In some
embodiments, the increase in diversity is an increase in the immunoglobulin
diversity and/or the T-
cell receptor diversity. In some embodiments, the diversity is in the
immunoglobulin is in the
immunoglobulin heavy chain. In some embodiments, the diversity is in the
immunoglobulin is in the
immunoglobulin light chain. In some embodiments, the diversity is in the T-
cell receptor. In some
embodiments, the diversity is in one of the T-cell receptors selected from the
group consisting of
alpha, beta, gamma, and delta receptors. In some embodiments, there is an
increase in the expression
of T-cell receptor (TCR) alpha and/or beta. In some embodiments, there is an
increase in the
expression of T-cell receptor (TCR) alpha. In some embodiments, there is an
increase in the
expression of T-cell receptor (TCR) beta. In some embodiments, there is an
increase in the expression
of TCRab (i.e., TCRa/13).
[00489] After dissection or digestion of tumor fragments, for example such as
described in Step A
of Figure 1, the resulting cells are cultured in serum containing IL-2 under
conditions that favor the
growth of TILs over tumor and other cells. In some embodiments, the tumor
digests are incubated in 2
mL wells in media comprising inactivated human AB scrum with 6000 1U/mL of 1L-
2. This primary
cell population is cultured for a period of days, generally from 3 to 14 days,
resulting in a bulk TIL
population, generally about 1 x 108 bulk TIL cells. In some embodiments, this
primary cell population
is cultured for a period of 7 to 14 days, resulting in a bulk TIL population,
generally about 1 x 108
bulk TIL cells. In some embodiments, this primary cell population is cultured
for a period of 10 to 14
days, resulting in a bulk TIL population, generally about 1 >< 108 bulk TIL
cells. In some
embodiments, this primary cell population is cultured for a period of about 11
days, resulting in a bulk
TIL population, generally about 1 x 108 bulk TIL cells.
[00490] In some embodiments, expansion of TILs may be performed using an
initial bulk TIL
expansion step (for example such as those described in Step B of Figure 1,
which can include
processes referred to as pre-REP) as described below and herein, followed by a
second expansion
(Step D, including processes referred to as rapid expansion protocol (REP)
steps) as described below
under Step D and herein, followed by optional cryopreservation, and followed
by a second Step D
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(including processes referred to as restimulation REP steps) as described
below and herein. The TILs
obtained from this process may be optionally characterized for phenotypic
characteristics and
metabolic parameters as described herein.
[00491] In embodiments where TIL cultures are initiated in 24-well plates, for
example, using
Costar 24-well cell culture cluster, flat bottom (Coming Incorporated,
Corning, NY, each well can be
seeded with 1 >< 106 tumor digest cells or one tumor fragment in 2 mL of
complete medium (CM) with
IL-2 (6000 IU/mL; Chiron Corp., Emeryville; CA). In some embodiments, the
tumor fragment is
between about 1 mill 3 and 10 mm3.
[00492] In some embodiments, the first expansion culture medium is referred to
as "CM", an
abbreviation for culture media. In some embodiments, CM for Step B consists of
RPMI 1640 with
GlutaMAX, supplemented with 10% human AB serum, 25 mM Hepes, and 10 mg/mL
gentamicin. In
embodiments where cultures are initiated in gas-permeable flasks with a 40 mL
capacity and a 10 cm2
gas-permeable silicon bottom (for example, G-REX10; Wilson Wolf Manufacturing,
New Brighton;
MN), each flask was loaded with 10-40>< 106 viable tumor digest cells or 5-30
tumor fragments in
10-40 mL of CM with IL-2. Both the G-REX10 and 24-well plates were incubated
in a humidified
incubator at 37 C in 5% CO,, and 5 days after culture initiation, half the
media was removed and
replaced with fresh CM and IL-2 and after day 5, half the media was changed
every 2-3 days.
[00493] In some embodiments, the culture medium used in the expansion
processes disclosed herein
is a serum-free medium or a defined medium. In some embodiments, the serum-
free or defined
medium comprises a basal cell medium and a serum supplement and/or a serum
replacement. In some
embodiments, the serum-free or defined medium is used to prevent and/or
decrease experimental
variation due in part to the lot-to-lot variation of serum-containing media.
[00494] In some embodiments, the serum-free or defined medium comprises a
basal cell medium
and a serum supplement and/or serum replacement. In some embodiments, the
basal cell medium
includes, but is not limited to CTSTm OpTmizerTm T-cell Expansion Basal Medium
, CTS'
OpTmizerTm T-Cell Expansion SFM, CTSTm AIM-V Medium, CTSTm AIM-V SFM,
LymphoONETM
T-Cell Expansion )(ono-Free Medium, Dulbecco's Modified Eagle's Medium (DMEM),
Minimal
Essential Medium (MEM), Basal Medium Eagle (BME), RPM! 1640, F-10, F-12,
Minimal Essential
Medium (aMEM), Glasgow's Minimal Essential Medium (G-MEM), RPM' growth medium,
and
Iscove's Modified Dulbecco's Medium.
[00495] In some embodiments, the serum supplement or serum replacement
includes, but is not
limited to one or more of CTSTm OpTmizer T-Cell Expansion Serum Supplement,
CTSTm Immune
Cell Serum Replacement, one or more albumins or albumin substitutes, one or
more amino acids, one
or more vitamins, one or more transferrins or transferrin substitutes, one or
more antioxidants, one or
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more insulins or insulin substitutes, one or more collagen precursors, one or
more antibiotics, and one
or more trace elements. In some embodiments, the defined medium comprises
albumin and one or
more ingredients selected from the group consisting of glycine, L- hi stidine,
L-isoleucine, L-
methionine, L-phenylalanine, L-proline, L- hydroxyproline, L-serine, L-
threonine, L-tryptophan, L-
tyrosine, L-valine, thiamine, reduced glutathione, L-ascorbic acid-2-
phosphate, iron saturated
transfcrrin, insulin, and compounds containing the trace clement moieties Ag',
Al", Ba", Cd", Co",
Cr", Ge", Se", Br, T, mn2+, P. si4-% v5+, mo6-% N=2-%
I
Rh+, Sn' and Zr". In some embodiments, the
defined medium further comprises L-glutamine, sodium bicarbonate and/or 2-
mercaptoethanol.
[00496] In some embodiments, the CTST1mOpTmizerTm T-cell Immune Cell Serum
Replacement is
used with conventional growth media, including but not limited to CTSTm
OpTmizerTm T-cell
Expansion Basal Medium, CTSTm OpTmizerTm T-cell Expansion SFM, CTSTm AIM-V
Medium,
CSTTm AIM-V SFM, LymphoONETM T-Cell Expansion Xeno-Free Medium, Dulbecco's
Modified
Eagle's Medium (DMEM), Minimal Essential Medium (MEM), Basal Medium Eagle
(BME), RPMI
1640, F-10, F-12, Minimal Essential Medium (aMEM), Glasgow's Minimal Essential
Medium (G-
MEM), RPMI growth medium, and Iscove's Modified Dulbecco's Medium.
[00497] In some embodiments, the total serum replacement concentration (vol%)
in the serum-free
or defined medium is from about 1%, 2%, 3%, 4%, 5%, 6%, 7%, 8%, 9%, 10%, 11%,
12%, 13%,
14%, 15%, 16%, 17%, 18%, 19%, or 20% by volume of the total serum-free or
defined medium. In
some embodiments, the total serum replacement concentration is about 3% of the
total volume of the
serum-free or defined medium. In some embodiments, the total serum replacement
concentration is
about 5% of the total volume of the scrum-free or defined medium. In some
embodiments, the total
serum replacement concentration is about 10% of the total volume of the serum-
free or defined
medium.
[00498] In some embodiments, the serum-free or defined medium is CTSTm
OpTmizerTm T-cell
Expansion SFM (ThermoFisher Scientific). Any formulation of CTSTm OpTmizerTm
is useful in the
present invention. CTSTm OpTmizerTm T-cell Expansion SFM is a combination of
1L CTSTm
OpTmizerTm T-cell Expansion Basal Medium and 26 mL CTSTm OpTmizerTm T-Cell
Expansion
Supplement, which are mixed together prior to use. In some embodiments, the
CTSTm OpTrnizerTm T-
cell Expansion SFM is supplemented with about 3% of the CTSTm Immune Cell
Serum Replacement
(SR) (ThermoFisher Scientific). In some embodiments, the CTSTm OpTmizerTm T-
cell Expansion
SFM is supplemented with about 3% of the CTSTm Immune Cell Serum Replacement
(SR)
(ThermoFisher Scientific), along with 2-mercaptoethanol at 55mM. In some
embodiments, the CTSTm
OpTmizerTm T-cell Expansion SFM is supplemented with about 3% of the CTSTm
Immune Cell
Serum Replacement (SR) (ThermoFisher Scientific) and the final concentration
of 2-mercaptoethanol
in the media is 551iM.
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[00499] In some embodiments, the defined medium is CTSTm OpTmizerTm T-cell
Expansion SFM
(ThermoFisher Scientific). Any formulation of CTSTm OpTmizerTm is useful in
the present invention.
CTSTm OpTmizerTm T-cell Expansion SFM is a combination of 1L CTSTm OpTmizerTm
T-cell
Expansion Basal Medium and 26 mL CTSTm OpTmizerTm T-Cell Expansion Supplement,
which are
mixed together prior to use. In some embodiments, the CTSTm OpTmizerTm T-cell
Expansion SFM is
supplemented with about 3% of the CTSTm Immune Cell Serum Replacement (SR)
(ThennoFisher
Scientific), along with 2-mercaptoethanol at 55mM. In some embodiments, the
CTSTmOpTmizerTm T-
cell Expansion SFM is supplemented with about 3% of the CTSTm Immune Cell
Serum Replacement
(SR) (ThermoFisher Scientific), 55mM of 2-mercaptoethanol, and 2mM of L-
glutamine. In some
embodiments, the CTSTmOpTmizerTm T-cell Expansion SFM is supplemented with
about 3% of the
CTSTm Immune Cell Serum Replacement (SR) (ThermoFisher Scientific), 55mM of 2-
mercaptoethanol, and 2mM of L-glutamine, and further comprises about 1000
IU/mL to about 8000
IU/mL of 1L-2. In some embodiments, the CTSTmOpTinizerTm T-cell Expansion SFM
is
supplemented with about 3% of the CTSTm Immune Cell Serum Replacement (SR)
(ThermoFisher
Scientific), 55mM of 2-mercaptoethanol, and 2mM of L-glutamine, and further
comprises about 3000
IU/mL of 1L-2. In some embodiments, the CTSTmOpTmizerTm T-cell Expansion SFM
is
supplemented with about 3% of the CTSTm Immune Cell Serum Replacement (SR)
(ThermoFisher
Scientific), 55mM of 2-mcrcaptocthanol, and 2mM of L-glutaminc, and further
comprises about 6000
IU/mL of IL-2. In some embodiments, the CTSTmOpTmizerTm T-cell Expansion SFM
is
supplemented with about 3% of the CTSTm Immune Cell Serum Replacement (SR)
(ThermoFisher
Scientific) and 55mM of 2-mercaptoethanol, and further comprises about 1000
IU/mL to about 8000
IU/mL of IL-2. In some embodiments, the CTSTmOpTmizerTm T-cell Expansion SFM
is
supplemented with about 3% of the CTSTm Immune Cell Serum Replacement (SR)
(ThermoFisher
Scientific) and 55mM of 2-mercaptoethanol, and further comprises about 3000
IU/mL of IL-2. In
some embodiments, the CTSTmOpTinizerTm T-cell Expansion SFM is supplemented
with about 3% of
the CTSTm Immune Cell Serum Replacement (SR) (ThermoFisher Scientific) and
55mM of 2-
mercaptoethanol, and further comprises about 1000 IU/mL to about 6000 IU/mL of
IL-2. In some
embodiments, the CTS"OpTmizerTm T-cell Expansion SFM is supplemented with
about 3% of the
CTSTm Immune Cell Serum Replacement (SR) (ThermoFisher Scientific) and about
2mM glutamine,
and further comprises about 1000 IU/mL to about 8000 IU/mL of IL-2. In some
embodiments, the
CTSTmOpTmizerTm T-cell Expansion SFM is supplemented with about 3% of the
CTSTm Immune
Cell Serum Replacement (SR) (ThermoFisher Scientific) and about 2mM glutamine,
and further
comprises about 3000 IU/mL of IL-2. In some embodiments, the CTSTmOpTmizerTm T-
cell
Expansion SFM is supplemented with about 3% of the CTSTm Immune Cell Serum
Replacement (SR)
(ThermoFisher Scientific) and about 2mM glutamine, and further comprises about
6000 IU/mL of IL-
2. In some embodiments, the CTSTm OpTmizerTm T-cell Expansion SFM is
supplemented with about
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3% of the CTSTm Immune Cell Serum Replacement (SR) (ThermoFisher Scientific)
and the final
concentration of 2-mercaptoethanol in the media is 55 M.
[00500] In some embodiments, the serum-free medium or defined medium is
supplemented with
glutamine (i.e., GlutaMAX0) at a concentration of from about 0.1mM to about
10mM, 0.5mM to
about 9mM, 1mM to about 8mM, 2mM to about 7mM, 3mM to about 6mM, or 4mM to
about 5 mM.
In some embodiments, the serum-free medium or defined medium is supplemented
with glutamine
(i.e.. GlutaMAX0) at a concentration of about 2mM.
[00501] In some embodiments, the serum-free medium or defined medium is
supplemented with 2-
mercaptoethanol at a concentration of from about 5mM to about 150mM, 10mM to
about 140mM,
15mM to about 130mM, 20mM to about 120mM, 25mM to about 110mM, 30mM to about
100mM,
35mM to about 95mM, 40mM to about 90mM, 45mM to about 85mM, 50mM to about
80mM, 55mM
to about 75mM, 60mM to about 70mM, or about 65mM. In some embodiments, the
serum-free
medium or defined medium is supplemented with 2-mercaptoethanol at a
concentration of about
55mM. In some embodiments, the final concentration of 2-mercaptoethanol in the
media is 55 M.
[00502] In some embodiments, the defined media described in International PCT
Publication No.
WO/1998/030679, which is herein incorporated by reference, are useful in the
present invention. In
that publication, serum-free eukarvotic cell culture media are described. The
scrum-free, cukarvotic
cell culture medium includes a basal cell culture medium supplemented with a
serum-free supplement
capable of supporting the growth of cells in serum- free culture. The scrum-
free cukaryotic cell
culture medium supplement comprises or is obtained by combining, one or more
ingredients selected.
from the group consisting of one or more altiumins or albumin substitutes, one
or more amino a.eids,
one or more vitamins, one or more transferrins or transferrin substitutes, one
or more antioxidants,
one or more insulins or insulin substitutes, one or more collagen precursors,
one or more trace
elements, and one or more antibiotics. In some embodiments, the defined medium
further comprises
L-gintamine, sochnin bicarbonate and/or beta-mereaptoethanol, in some
embodiments, the defined
medium comprises an albumin or an albumin substitute and one or more
ingredients selected from
group consisting of one or more amino acids, one or morc vitamins, one or more
transfenins or
transferrin substitutes, one or more antioxidants, one or more ii-isulus or
insulin substitutes, one or
more collagen precursors, and one or more trace elements. In soiric
embodiments, the defined medium
comprises albumin and one or more ingredients selected from the group
consisting of glycine, L-
histidine, L-isoleucine, L-methionine, L-phertylalanine, L-proline, L-
hydroxyproline, L-serine, L-
threonine, L-tryptophan, L-tyrosine, L--valine, thiamine, reduced glutathione,
L-ascorbic acid-2-
phosphate, iron saturated transferrin., insulin, and compounds containing the
trace element moieties
.Ag ' , Ba2', cd2,, ce2 1, 0.31, Ge4 se4 ,, Br,
st) se 1, v51 , rvic,6!, .Nri2:, Rh Sn.2 and 2374 I .
in sonic embodiments, the basal cell media is selected from the group
consisting of Di ilbecco's
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Modified Eagle's Medium (DMEM), Minimal Essential Medium (MEM), Basal Medium
Eagle
(RAE), RPM! 1640, F-10, F-12, Minimal Essential Medium (aMEM), Glasgow's
Minimal Essential
Medium (G-MIEM), RPMT growth medium, and iseove's Modified Du 4-Kx:co's Medium
[00503] In some embodiments, the concentration of glycine in the defined
medium is in the range of
from about 5-200 mg/L, the concentration of L- histidine is about 5-250 mg/L,
the concentration of L-
isoleucine is about 5-300 mg/L, the concentration of L-methionine is about 5-
200 mg/L, the
concentration of L-phenylalanine is about 5-400 mg/L, the concentration of L-
proline is about 1-1000
mg/Tõ the concentration of I.- hydroxyproline is about 1-45 mg/L, the
concentration of L-serine is
about 1-250 mg/L, the concentration of L-threonine is about 10-500 mg/L, the
concentration of L-
tryptophan is about 2-110 mg/L, the concentration of L-tyrosine is about 3-175
mg/L, the
concentration of L-valine is about 5-500 mg/L, the concentration of thiamine
is about 1-20 mg/L, the
concentration of reduced glutathione is about 1-20 mg/L, the concentration of
L-ascorbic acid-2-
phosphate is about 1-200 mg/L, the concentration of iron saturated transfen-in
is about 1-50 mg/L, the
concentration of insulin is about 1-100 mg/L, the concentration of sodium
selenite is about 0.000001-
0.0001 mg/L, and the concentration of albumin (e.g., AlbuMAX I) is about 5000-
50,000 mg/L.
[00504] In some embodiments, the non-trace element moiety ingredients in the
defined medium are
present in the concentration ranges listed in the column under the heading
"Concentration Range in
1X Medium" in Table 4. In other embodiments, the non-trace element moiety
ingredients in the
defined medium are present in the final concentrations listed in the column
under the heading "A
Preferred Embodiment of the 1X Medium" in Table 4. In other embodiments, the
defined medium is a
basal cell medium comprising a scrum free supplement. In some of these
embodiments, the scrum free
supplement comprises non-trace moiety ingredients of the type and in the
concentrations listed in the
column under the heading "A Preferred Embodiment in Supplement" in Table 4
below.
Table 4: Concentrations of Non-Trace Element Moiety Ingredients
Ingredient A preferred. Concentration range in A
preferred
embodiment in. IX medium (mg/L)
embodiment in I X
supplement (ing/L) medium
(mg/L)
(About)
(About)
(About)
Glyetne 150 5-200 53
040 5-250
183
L-IsoicucinQ 3400 5-300
615
L-Methioninc,' 90 44
L-Phenyialanine 1800 5-400
336
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L-Proline 4000 14000
600
L-Hydroxyproline 100 145
15
L-Seritie 800 1-250
162
L-Threorii Be 2200 10-500
425
I...-Tiyptophan 440 2410
82
1,-Tyrosine 77 3-175
L-Valine 2400 5-500
454
Thiamine 33 1-20 9
Reduced Gil:Etat-hi one 10 1-20
LS
Ascorbic Acid-2-1304 330 1-200
50
(Mg Salt)
1'mi-113R:inn (iron 55 1-50 8
saturated)
Insulin 100 1400
10
Sodium Selc,mitt-; 0.07 0.00000141.0001
0.00001
AibuMAXi83,000 5000-50,000
1.2,500
[00505] In some embodiments, the osmolarity of the defined medium is between
about 260 and 350
mOsmol. In some embodiments, the osmolarity is between about 280 and 310
mOsmol. In some
embodiments, the defined medium is supplemented with up to about 3.7 g/L, or
about 2.2 g/L sodium
bicarbonate. The defined medium can be further supplemented with L-glutamine
(final concentration
of about 2 mM), one or more antibiotics, non-essential amino acids (NEAA;
final concentration of
about 100 uM), 2-mercaptoethanol (final concentration of about 100 M).
[00506] In some embodiments, the defined media described in Smith, et al.,
Clin Trans?
Immunology, 4(1) 2015 (doi: 10.1038/cti.2014.31) are useful in the present
invention. Briefly, RPMI
or CTSTm OpTmizerTm was used as the basal cell medium, and supplemented with
either 0, 2%, 5%,
or 10% CTSTm Immune Cell Serum Replacement.
[00507] In some embodiments, the cell medium in the first and/or second gas
permeable container is
unfiltered. The use of unfiltered cell medium may simplify the procedures
necessary to expand the
number of cells. In some embodiments, the cell medium in the first and/or
second gas permeable
container lacks beta-mercaptoethanol (BME or PME; also known as 2-
mercaptoethanol, CAS 60-24-
2).
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[00508] After preparation of the tumor fragments, the resulting cells (i.e.,
fragments) are cultured in
serum containing IL-2 under conditions that favor the growth of TILs over
tumor and other cells. In
some embodiments, the tumor digests are incubated in 2 mL wells in media
comprising inactivated
human AB serum (or, in some cases, as outlined herein, in the presence of an
APC cell population)
with 6000 IU/mL of IL-2. This primary cell population is cultured for a period
of days, generally from
to 14 days, resulting in a bulk TIL population, generally about lx 10 bulk TIL
cells. In some
embodiments, the growth media during the first expansion comprises IL-2 or a
variant thereof. In
some embodiments, the IL is recombinant human IL-2 (rhIL-2). In some
embodiments the IL-2 stock
solution has a specific activity of 20-30x 1061U/mg for a 1 mg vial. In some
embodiments the 1L-2
stock solution has a specific activity of 20x106IU/mg for a 1 mg vial. In some
embodiments the IL-2
stock solution has a specific activity of 25 x106 IU/mg for a 1 mg vial. In
some embodiments the IL-2
stock solution has a specific activity of 30x106IU/mg for a 1 mg vial. In some
embodiments, the IL- 2
stock solution has a final concentration of 4-8x106 IU/mg of IL-2. In some
embodiments, the IL- 2
stock solution has a final concentration of 5-7x106 IU/mg of IL-2. In some
embodiments, the IL- 2
stock solution has a final concentration of 6x106 TU/mg of IL-2. In some
embodiments, the 1L-2 stock
solution is prepare as described in Example 5. In some embodiments, the first
expansion culture
media comprises about 10,000 IU/mL of IL-2, about 9,000 IU/mL of IL-2, about
8,000 IU/mL of IL-
2, about 7,000 IU/mL of IL-2, about 6000 IU/mL of IL-2 or about 5,000 IU/mL of
IL-2. In some
embodiments, the first expansion culture media comprises about 9,000 IU/mL of
IL-2 to about 5,000
IU/mL of IL-2. In some embodiments, the first expansion culture media
comprises about 8,000 IU/mL
of IL-2 to about 6,000 IU/mL of IL-2. In some embodiments, the first expansion
culture media
comprises about 7,000 IU/mL of IL-2 to about 6,000 IU/mL of IL-2. In some
embodiments, the first
expansion culture media comprises about 6,000 IU/mL of IL-2. In some
embodiments, the cell culture
medium further comprises IL-2. In some embodiments, the cell culture medium
comprises about 3000
IU/mL of IL-2. In some embodiments, the cell culture medium further comprises
1L-2. In some
embodiments, the cell culture medium comprises about 3000 IU/mL of IL-2. In
some embodiments,
the cell culture medium comprises about 1000 IU/mL, about 1500 IU/mL, about
2000 IU/mL, about
2500 IU/mL, about 3000 IU/mL, about 3500 IU/mL, about 4000 IU/mL, about 4500
IU/mL, about
5000 IU/mL, about 5500 IU/mL, about 6000 IU/mL, about 6500 IU/mL, about 7000
IU/mL, about
7500 IU/mL, or about 8000 IU/mL of IL-2. In some embodiments, the cell culture
medium comprises
between 1000 and 2000 IU/mL, between 2000 and 3000 IU/mL, between 3000 and
4000 IU/mL,
between 4000 and 5000 IU/mL, between 5000 and 6000 IU/mL, between 6000 and
7000 IU/mL,
between 7000 and 8000 IU/mL, or about 8000 IU/mL of IL-2.
[00509] In some embodiments, first expansion culture media comprises about 500
IU/mL of IL-15,
about 400 IU/mL of TL-15, about 300 IU/mL of IL-15, about 200 TU/mL of IL-15,
about 180 IU/mL
of IL-15, about 160 IU/mL of IL-15, about 140 IU/mL of IL-15, about 120 IU/mL
of IL-15, or about
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100 IU/mL of IL-15. In some embodiments, the first expansion culture media
comprises about 500
IU/mL of IL-15 to about 100 IU/mL of IL-15. In some embodiments, the first
expansion culture
media comprises about 400 IU/mL of IL-15 to about 100 IU/mL of TL-15. In some
embodiments, the
first expansion culture media comprises about 300 IU/mL of IL-15 to about 100
IU/mL of IL-15. In
some embodiments, the first expansion culture media comprises about 200 IU/mL
of IL-15. In some
embodiments, the cell culture medium comprises about 180 IU/mL of IL-15. In
some embodiments,
the cell culture medium further comprises IL-15. In some embodiments, the cell
culture medium
comprises about 180 IU/mL of IL-15.
[00510] In some embodiments, first expansion culture media comprises about 20
IU/mL of IL-2I,
about 15 IU/mL of IL-21, about 12 IU/mL of IL-21, about 10 IU/mL of IL-21,
about 5 IU/mL of IL-
21, about 4 IU/mL of IL-21, about 3 IU/mL of IL-21, about 2 IU/mL of IL-21,
about 1 IU/mL of IL-
21, or about 0.5 IU/mL of IL-21. In some embodiments, the first expansion
culture media comprises
about 20 IU/mL of IL-21 to about 0.5 IU/mL of 1L-21. In some embodiments, the
first expansion
culture media comprises about 15 IU/mL of IL-21 to about 0.5 IU/mL of IL-21.
In some
embodiments, the first expansion culture media comprises about 12 IU/mL of IL-
21 to about 0.5
IU/mL of IL-21. In some embodiments, the first expansion culture media
comprises about 10 IU/mL
of IL-21 to about 0.5 IU/mL of IL-21. In some embodiments, the first expansion
culture media
comprises about 5 IU/mL of IL-21 to about 1 IU/mL of IL-21. In some
embodiments, the first
expansion culture media comprises about 2 IU/mL of IL-21. In some embodiments,
the cell culture
medium comprises about 1 IU/mL of TL-21. In some embodiments, the cell culture
medium comprises
about 0.5 IU/mL of IL-21. In some embodiments, the cell culture medium further
comprises IL-21. In
some embodiments, the cell culture medium comprises about 1 IU/mL of IL-21.
[00511] In some embodiments, the cell culture medium comprises an anti-CD3
agonist antibody,
e.g., OKT-3 antibody. In some embodiments, the cell culture medium comprises
about 30 ng/mL of
OKT-3 antibody. In some embodiments, the cell culture medium comprises about
0.1 ng/mL, about
0.5 ng/mL, about 1 ng/mL, about 2.5 ng/mL, about 5 ng/mL, about 7.5 ng/mL,
about 10 ng/mL, about
15 ng/mL, about 20 ng/mL, about 25 ng/mL, about 30 ng/mL, about 35 ng/mL,
about 40 ng/mL,
about 50 ng/mL, about 60 ng/mL, about 70 ng/mL, about 80 ng/mL, about 90
ng/mL, about 100
ng/mL, about 200 ng/mL, about 500 ng/mL, and about liAg/mL of OKT-3 antibody.
In some
embodiments, the cell culture medium comprises between 0.1 ng/mL and 1 ng/mL,
between 1 ng/mL
and 5 ng/mL, between 5 ng/mL and 10 ng/mL, between 10 ng/mL and 20 ng/mL,
between 20 ng/mL
and 30 ng/mL, between 30 ng/mL and 40 ng/mL, between 40 ng/mL and 50 ng/mL,
and between 50
ng/mL and 100 ng/mL of OKT-3 antibody. In some embodiments, the cell culture
medium does not
comprise OKT-3 antibody. In some embodiments, the OKT-3 antibody is muromonab.
See, for
example, Table 1.
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[00512] In some embodiments, the cell culture medium comprises one or more
TNFRSF agonists in
a cell culture medium. In some embodiments, the TNFRSF agonist comprises a 4-
1BB agonist. In
some embodiments, the TNFRSF agonist is a 4-1BB agonist, and the 4-1BB agonist
is selected from
the group consisting of urelumab, utomilumab, EU-101, a fusion protein, and
fragments, derivatives,
variants, biosimilars, and combinations thereof. In some embodiments, the
TNFRSF agonist is added
at a concentration sufficient to achieve a concentration in the cell culture
medium of between 0.1
ng/mL and 100 pg/mL. In some embodiments, the TNFRSF agonist is added at a
concentration
sufficient to achieve a concentration in the cell culture medium of between 20
us/mL and 40 us/mL.
[00513] In some embodiments, in addition to one or more TNFRSF agonists, the
cell culture
medium further comprises IL-2 at an initial concentration of about 3000 IU/mL
and OKT-3 antibody
at an initial concentration of about 30 ng/mL, and wherein the one or more
'TNFRSF agonists
comprises a 4-1BB agonist.
[00514] In some embodiments, the first expansion culture medium is referred to
as -CM-, an
abbreviation for culture media. In some embodiments, it is referred to as CM1
(culture medium 1). In
some embodiments, CM consists of RPMI 1640 with GlutaIVIAX, supplemented with
10% human AB
serum, 25 mM Hepes, and 10 mg/mL gentamicin. In embodiments where cultures are
initiated in gas-
permeable flasks with a 40 mL capacity and a 10m' gas-permeable silicon bottom
(for example, G-
REX10; Wilson Wolf Manufacturing, New Brighton, MN), each flask was loaded
with 10-40x106
viable tumor digest cells or 5-30 tumor fragments in 10-40mL of CM with IL-2.
Both the G-REX10
and 24-well plates were incubated in a humidified incubator at 37 C in 5% CO2
and 5 days after
culture initiation, half the media was removed and replaced with fresh CM and
1L-2 and after day 5,
half the media was changed every 2-3 days. In some embodiments, the CM is the
CM1 described in
the Examples, see, Example 1. In some embodiments, the first expansion occurs
in an initial cell
culture medium or a first cell culture medium. In some embodiments, the
initial cell culture medium
or the first cell culture medium comprises IL-2.
[00515] In some embodiments, the first expansion (including processes such as
for example those
described in Step B of Figure 1, which can include those sometimes referred to
as the pre-REP)
process is shortened to 3-14 days, as discussed in the examples and figures.
In some embodiments, the
first expansion (including processes such as for example those described in
Step B of Figure 1, which
can include those sometimes referred to as the pre-REP) is shortened to 7 to
14 days, as discussed in
the Examples and shown in Figures 4 and 5, as well as including for example,
an expansion as
described in Step B of Figure 1. In some embodiments, the first expansion of
Step B is shortened to
10-14 days. In some embodiments, the first expansion is shortened to 11 days,
as discussed in, for
example, an expansion as described in Step B of Figure 1.
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[00516] In some embodiments, the first TIL expansion can proceed for 1 day, 2
days, 3 days, 4 days,
days, 6 days, 7 days, 8 days, 9 days, 10 days, 11 days, 12 days, 13 days, or
14 days. In some
embodiments, the first TIL expansion can proceed for 1 day to 14 days. In some
embodiments, the
first TIL expansion can proceed for 2 days to 14 days. In some embodiments,
the first TIL expansion
can proceed for 3 days to 14 days. In some embodiments, the first TIL
expansion can proceed for 4
days to 14 days. In some embodiments, the first TIL expansion can proceed for
5 days to 14 days. In
some embodiments, the first TIL expansion can proceed for 6 days to 14 days.
In some embodiments,
the first TIL expansion can proceed for 7 days to 14 days. In some
embodiments, the first TIL
expansion can proceed for 8 days to 14 days. In some embodiments, the first
TIL expansion can
proceed for 9 days to 14 days. In some embodiments, the first TIL expansion
can proceed for 10 days
to 14 days. In some embodiments, the first TIL expansion can proceed for 11
days to 14 days. In some
embodiments, the first TIL expansion can proceed for 12 days to 14 days. In
some embodiments, the
first TIL expansion can proceed for 13 days to 14 days. In some embodiments,
the first TIL expansion
can proceed for 14 days. In some embodiments, the first TIL expansion can
proceed for 1 day to 11
days. In some embodiments, the first TIL expansion can proceed for 2 days to
11 days. In some
embodiments, the first TIL expansion can proceed for 3 days to 11 days. In
some embodiments, the
first TIL expansion can proceed for 4 days to 11 days. In some embodiments,
the first TIL expansion
can proceed for 5 days to 11 days. In some embodiments, the first TIL
expansion can proceed for 6
days to 11 days. In some embodiments, the first TIL expansion can proceed for
7 days to 11 days. In
some embodiments, the first TIL expansion can proceed for 8 days to 11 days.
In some embodiments,
the first TIL expansion can proceed for 9 days to 11 days. In some
embodiments, the first TIL
expansion can proceed for 10 days to 11 days. In some embodiments, the first
TIL expansion can
proceed for 11 days.
[00517] In some embodiments, a combination of IL-2, IL-7, IL-15, and/or IL-21
are employed as a
combination during the first expansion. In some embodiments, IL-2, IL-7, IL-
15, and/or IL-21 as well
as any combinations thereof can be included during the first expansion,
including for example during
a Step B processes according to Figure 1, as well as described herein. In some
embodiments, a
combination of IL-2, IL-15, and IL-21 are employed as a combination during the
first expansion. In
some embodiments, IL-2, IL-15, and IL-21 as well as any combinations thereof
can be included
during Step B processes according to Figure 1 and as described herein.
[00518] In some embodiments, the first expansion (including processes referred
to as the pre-REP;
for example, Step B according to Figure 1) process is shortened to 3 to 14
days, as discussed in the
examples and figures. In some embodiments, the first expansion of Step B is
shortened to 7 to 14
days. In some embodiments, the first expansion of Step B is shortened to 10 to
14 days. In some
embodiments, the first expansion is shortened to 11 days.
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[00519] In some embodiments, the first expansion, for example, Step B
according to Figure 1, is
performed in a closed system bioreactor. In some embodiments, a closed system
is employed for the
TIL expansion, as described herein. In some embodiments, a single bioreactor
is employed. In some
embodiments, the single bioreactor employed is for example a G-REX-10 or a G-
REX-100. In some
embodiments, the closed system bioreactor is a single bioreactor.
1. Cytokincs and Other Additives
[00520] The expansion methods described herein generally use culture media
with high doses of a
cytokine, in particular IL-2, as is known in the art.
Alternatively, using combinations of cytokines for the rapid expansion and or
second expansion of
TILs is additionally possible, with combinations of two or more of IL-2, IL-15
and IL-21 as is
described in U.S. Patent Application Publication No. US 2017/0107490 Al, the
disclosure of which is
incorporated by reference herein. Thus, possible combinations include 1L-2 and
1L-15, 1L-2 and IL-
21, IL-15 and IL-21 and IL-2, or IL-15 and IL-21, with the latter finding
particular use in many
embodiments. The use of combinations of cytokines specifically favors the
generation of
lymphocytes, and in particular T-cells as described therein.
[0001] In some embodiments, Step B may also include the addition of OKT-3
antibody or
muromonab to the culture media, as described elsewhere herein. In some
embodiments, Step B may
also include the addition of a 4-1BB agonist to the culture media, as
described elsewhere herein. In
some embodiments, Step B may also include the addition of an OX-40 agonist to
the culture media, as
described elsewhere herein. In other embodiments, additives such as peroxisome
proliferator-activated
receptor gamma coactivator 1-alpha agonists, including proliferator-activated
receptor (PPAR)-gamma
agonists such as a thiazolidinedione compound, may be used in the culture
media during Step B, as
described in U.S. Patent Application Publication No. US 2019/0307796 Al, the
disclosure of which is
incorporated by reference herein.
C. STEP C: First Expansion to Second Expansion Transition
[00521] In some cases, the bulk TIL population obtained from the first
expansion, including for
example the TIL population obtained from for example, Step B as indicated in
Figure 1, can be
cryopreserved immediately, using the protocols discussed herein below.
Alternatively, the TIL
population obtained from the first expansion, referred to as the second TIL
population, can be
subjected to a second expansion (which can include expansions sometimes
referred to as REP) and
then cryopreserved as discussed below. Similarly, in the case where
genetically modified TILs will be
used in therapy, the first TIL population (sometimes referred to as the bulk
TIL population) or the
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second TIL population (which can in some embodiments include populations
referred to as the REP
TIL populations) can be subjected to genetic modifications for suitable
treatments prior to expansion
or after the first expansion and prior to the second expansion.
[00522] In some embodiments, the TILs obtained from the first expansion (for
example, from Step
B as indicated in Figure 1) are stored until phenotyped for selection. In some
embodiments, the TILs
obtained from the first expansion (for example, from Step B as indicated in
Figure 1) are not stored
and proceed directly to the second expansion. In some embodiments, the TILs
obtained from the first
expansion are not cryopreserved after the first expansion and prior to the
second expansion. In some
embodiments, the transition from the first expansion to the second expansion
occurs at about 3 days,
4, days, 5 days, 6 days, 7 days, 8 days, 9 days, 10 days, 11 days, 12 days, 13
days, or 14 days from
when fragmentation occurs. In some embodiments, the transition from the first
expansion to the
second expansion occurs at about 3 days to 14 days from when fragmentation
occurs. In some
embodiments, the transition from the first expansion to the second expansion
occurs at about 4 days to
14 days from when fragmentation occurs. In some embodiments, the transition
from the first
expansion to the second expansion occurs at about 4 days to 10 days from when
fragmentation occurs.
In some embodiments, the transition from the first expansion to the second
expansion occurs at about
7 days to 14 days from when fragmentation occurs. In some embodiments, the
transition from the first
expansion to the second expansion occurs at about 14 days from when
fragmentation occurs.
[00523] In some embodiments, the transition from the first expansion to the
second expansion
occurs at 1 day, 2 days, 3 days, 4 days, 5 days, 6 days, 7 days, 8 days, 9
days, 10 days, 11 days, 12
days, 13 days, or 14 days from when fragmentation occurs. In some embodiments,
the transition from
the first expansion to the second expansion occurs 1 day to 14 days from when
fragmentation occurs.
In some embodiments, the first TIL expansion can proceed for 2 days to 14
days. In some
embodiments, the transition from the first expansion to the second expansion
occurs 3 days to 14 days
from when fragmentation occurs. In some embodiments, the transition from the
first expansion to the
second expansion occurs 4 days to 14 days from when fragmentation occurs. In
some embodiments,
the transition from the first expansion to the second expansion occurs 5 days
to 14 days from when
fragmentation occurs. In some embodiments, the transition from the first
expansion to the second
expansion occurs 6 days to 14 days from when fragmentation occurs. In some
embodiments, the
transition from the first expansion to the second expansion occurs 7 days to
14 days from when
fragmentation occurs. In some embodiments, the transition from the first
expansion to the second
expansion occurs 8 days to 14 days from when fragmentation occurs. In some
embodiments, the
transition from the first expansion to the second expansion occurs 9 days to
14 days from when
fragmentation occurs. In some embodiments, the transition from the first
expansion to the second
expansion occurs 10 days to 14 days from when fragmentation occurs. In some
embodiments, the
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transition from the first expansion to the second expansion occurs 11 days to
14 days from when
fragmentation occurs. In some embodiments, the transition from the first
expansion to the second
expansion occurs 12 days to 14 days from when fragmentation occurs. In some
embodiments, the
transition from the first expansion to the second expansion occurs 13 days to
14 days from when
fragmentation occurs. In some embodiments, the transition from the first
expansion to the second
expansion occurs 14 days from when fragmentation occurs. In some embodiments,
the transition from
the first expansion to the second expansion occurs 1 day to 11 days from when
fragmentation occurs.
In some embodiments, the transition from the first expansion to the second
expansion occurs 2 days to
11 days from when fragmentation occurs. In some embodiments, the transition
from the first
expansion to the second expansion occurs 3 days to 11 days from when
fragmentation occurs. In some
embodiments, the transition from the first expansion to the second expansion
occurs 4 days to 11 days
from when fragmentation occurs. In some embodiments, the transition from the
first expansion to the
second expansion occurs 5 days to 11 days from when fragmentation occurs. In
some embodiments,
the transition from the first expansion to the second expansion occurs 6 days
to 11 days from when
fragmentation occurs. In some embodiments, the transition from the first
expansion to the second
expansion occurs 7 days to 11 days from when fragmentation occurs. In some
embodiments, the
transition from the first expansion to the second expansion occurs 8 days to
11 days from when
fragmentation occurs. In some embodiments, the transition from the first
expansion to the second
expansion occurs 9 days to 11 days from when fragmentation occurs. In some
embodiments, the
transition from the first expansion to the second expansion occurs 10 days to
11 days from when
fragmentation occurs. In some embodiments, the transition from the first
expansion to the second
expansion occurs 11 days from when fragmentation occurs.
1005241 In some embodiments, the TILs are not stored after the first expansion
and prior to the
second expansion, and the TILs proceed directly to the second expansion (for
example, in some
embodiments, there is no storage during the transition from Step B to Step D
as shown in Figure 1). In
some embodiments, the transition occurs in closed system, as described herein.
In some embodiments,
the TILs from the first expansion, the second population of TILs, proceeds
directly into the second
expansion with no transition period.
1005251 In some embodiments, the transition from the first expansion to the
second expansion, for
example, Step C according to Figure 1, is performed in a closed system
bioreactor. In some
embodiments, a closed system is employed for the TIL expansion, as described
herein. In some
embodiments, a single bioreactor is employed. In some embodiments, the single
bioreactor employed
is for example a G-REX -10 or a G-REX -100. In some embodiments, the closed
system bioreactor is
a single bioreactor.
D. STEP D: Second Expansion
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1005261 In some embodiments, the TIL cell population is expanded in number
after harvest and
initial bulk processing for example, after Step A and Step B, and the
transition referred to as Step C,
as indicated in Figure 1). This further expansion is referred to herein as the
second expansion, which
can include expansion processes generally referred to in the art as a rapid
expansion process (REP; as
well as processes as indicated in Step D of Figure 1). The second expansion is
generally accomplished
using a culture media comprising a number of components, including feeder
cells, a cytokine source,
and an anti-CD3 antibody, in a gas-permeable container.
1005271 In some embodiments, the second expansion or second TIL expansion
(which can include
expansions sometimes referred to as REP; as well as processes as indicated in
Step D of Figure 1) of
TIL can be performed using any TIL flasks or containers known by those of
skill in the art. In some
embodiments, the second TIL expansion can proceed for 7 days, 8 days, 9 days,
10 days, 11 days, 12
days, 13 days, or 14 days. In some embodiments, the second TIL expansion can
proceed for about 7
days to about 14 days. In some embodiments, the second TIL expansion can
proceed for about 8 days
to about 14 days. In some embodiments, the second TIL expansion can proceed
for about 9 days to
about 14 days. in some embodiments, the second TIL expansion can proceed for
about 10 days to
about 14 days. In some embodiments, the second TIL expansion can proceed for
about 11 days to
about 14 days. In some embodiments, the second TIL expansion can proceed for
about 12 days to
about 14 days. In some embodiments, the second TIL expansion can proceed for
about 13 days to
about 14 days. In some embodiments, the second TIL expansion can proceed for
about 14 days.
1005281 In some embodiments, the second expansion can be performed in a gas
permeable container
using the methods of the present disclosure (including for example, expansions
referred to as REP; as
well as processes as indicated in Step D of Figure 1). For example, TILs can
be rapidly expanded
using non-specific T-cell receptor stimulation in the presence of interleukin-
2 (IL-2) or interleukin-15
(IL-15). The non-specific T-cell receptor stimulus can include, for example,
an anti-CD3 antibody,
such as about 30 ng/mL of OKT3, a mouse monoclonal anti-CD3 antibody
(commercially available
from Ortho-McNeil, Raritan, NJ or Miltenyi Biotech, Auburn, CA) or UHCT-1
(commercially
available from BioLegend, San Diego, CA, USA). TILs can be expanded to induce
further stimulation
of the TILs in vitro by including one or more antigens during the second
expansion, including
antigenic portions thereof, such as epitope(s), of the cancer, which can be
optionally expressed from a
vector, such as a human leukocyte antigen A2 (HLA-A2) binding peptide, e.g.,
0.3 gM MART-1 :26-
35(27 L) or gpl 00:209-217 (210M), optionally in the presence of a T-cell
growth factor, such as 300
IU/mL IL-2 or IL-15. Other suitable antigens may include, e.g., NY-ES0-1, TRP-
1, TRP-2,
tyrosinase cancer antigen, MAGE-A3, SSX-2, and VEGFR2, or antigenic portions
thereof. TIL may
also be rapidly expanded by re-stimulation with the same antigen(s) of the
cancer pulsed onto HLA-
A2-expressing antigen-presenting cells. Alternatively, the TILs can be further
re-stimulated with, e.g.,
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example, irradiated, autologous lymphocytes or with irradiated HLA-A2+
allogeneic lymphocytes and
IL-2. In some embodiments, the re-stimulation occurs as part of the second
expansion. In some
embodiments, the second expansion occurs in the presence of irradiated,
autologous lymphocytes or
with irradiated HLA-A2+ allogeneic lymphocytes and IL-2.
1005291 In some embodiments, the cell culture medium further comprises IL-2.
In some
embodiments, the cell culture medium comprises about 3000 IU/mL of IL-2. In
some embodiments,
the cell culture medium comprises about 1000 IU/mL, about 1500 IU/mL, about
2000 IU/mL, about
2500 IU/mLõ about 3000 IllimIõ about 3500 TU/mIõ about 4000 ILJ/rnTõ about
4500 IU/mL, about
5000 IU/mL, about 5500 IU/mL, about 6000 IU/mL, about 6500 IU/mL, about 7000
IU/mL, about
7500 IU/mL, or about 8000 IU/mL of IL-2. In some embodiments, the cell culture
medium comprises
between 1000 and 2000 IU/mL, between 2000 and 3000 IU/mL, between 3000 and
4000 IU/mL,
between 4000 and 5000 IU/mL, between 5000 and 6000 IU/mL, between 6000 and
7000 IU/mL,
between 7000 and 8000 IU/mL, or between 8000 IU/mL of' IL-2.
[00530] In some embodiments, the cell culture medium comprises OKT-3 antibody.
In some
embodiments, the cell culture medium comprises about 30 ng/mL of OKT-3
antibody. In some
embodiments, the cell culture medium comprises about 0.1 ng/mL, about 0.5
ng/mL, about 1 ng/mL,
about 2.5 ng/mL, about 5 ng/mL, about 7.5 ng/mL, about 10 ng/mL, about 15
ng/mL, about 20
ng/mL, about 25 ng/mL, about 30 ng/mL, about 35 ng/mL, about 40 ng/mL, about
50 ng/mL, about
60 ng/mL, about 70 ng/mL, about 80 ng/mL, about 90 ng/mL, about 100 ng/mL,
about 200 ng/mL,
about 500 ng/mL, and about 1 iag/mL of OKT-3 antibody. In some embodiments,
the cell culture
medium comprises between 0.1 ng/mL and 1 ng/mL, between 1 ng/mL and 5 ng/mL,
between 5
ng/mL and 10 ng/mL, between 10 ng/mL and 20 ng/mL, between 20 ng/mL and 30
ng/mL, between
30 ng/mL and 40 ng/mL, between 40 ng/mL and 50 ng/mL, and between 50 ng/mL and
100 ng/mL of
OKT-3 antibody. In some embodiments, the cell culture medium does not comprise
OKT-3
antibody. In some embodiments, the OKT-3 antibody is muromonab.
[00531] In some embodiments, the cell culture medium comprises one or more
TNFRSF agonists in
a cell culture medium. In some embodiments, the TNFRSF agonist comprises a 4-
1BB agonist. In
sonic embodiments, the 'TNFRSF agonist is a 4-1BB agonist, and the 4-1BB
agonist is selected from
the group consisting of urelumab, utomilumab, EU-101, a fusion protein, and
fragments, derivatives,
variants, biosimilars, and combinations thereof. In some embodiments, the
TNFRSF agonist is added
at a concentration sufficient to achieve a concentration in the cell culture
medium of between 0.1
1.ig/mL and 100 iag/mL. In some embodiments, the TNFRSF agonist is added at a
concentration
sufficient to achieve a concentration in the cell culture medium of between 20
i_ig/mL and 40 i_ig/mL.
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[00532] In some embodiments, in addition to one or more TNFRSF agonists, the
cell culture
medium further comprises IL-2 at an initial concentration of about 3000 IU/mL
and OKT-3 antibody
at an initial concentration of about 30 ng/mL, and wherein the one or more
TNFRSF agonists
comprises a 4-1BB agonist.
[00533] In some embodiments, a combination of IL-2, IL-7, IL-15, and/or IL-21
are employed as a
combination during the second expansion. In some embodiments, IL-2, IL-7, IL-
15, and/or IL-21 as
well as any combinations thereof can be included during the second expansion,
including for example
during a Step D processes according to Figure 1, as well as described herein.
In some embodiments, a
combination of IL-2, IL-15, and IL-21 are employed as a combination during the
second expansion.
In some embodiments, IL-2, IL-15, and IL-21 as well as any combinations
thereof can be included
during Step D processes according to Figure 1 and as described herein.
[00534] In some embodiments, the second expansion can be conducted in a
supplemented cell
culture medium comprising IL-2, OKT-3, antigen-presenting feeder cells, and
optionally a TNFRSF
agonist. In some embodiments, the second expansion occurs in a supplemented
cell culture medium.
In some embodiments, the supplemented cell culture medium comprises 1L-2, OKT-
3, and antigen-
presenting feeder cells. In some embodiments, the second cell culture medium
comprises IL-2, OKT-
3, and antigen-presenting cells (APCs; also referred to as antigen-presenting
feeder cells). In some
embodiments, the second expansion occurs in a cell culture medium comprising
IL-2, OKT-3, and
antigen-presenting feeder cells (i.e., antigen presenting cells).
[00535] In some embodiments, the second expansion culture media comprises
about 500 IU/mL of
IL-15, about 400 IU/mL of IL-15, about 300 IU/mL of IL-15, about 200 IU/mL of
IL-15, about 180
IU/mL of IL-15, about 160 IU/mL of IL-15, about 140 IU/mL of IL-15, about 120
IU/mL of IL-15, or
about 100 IU/mL of IL-15. In some embodiments, the second expansion culture
media comprises
about 500 IU/mL of IL-15 to about 100 IU/mL of IL-15. In some embodiments, the
second expansion
culture media comprises about 400 IU/mL of IL-15 to about 100 IU/mL of IL-15.
In some
embodiments, the second expansion culture media comprises about 300 IU/mL of
IL-15 to about 100
IU/mL of IL-15. In some embodiments, the second expansion culture media
comprises about 200
IU/mL of IL-15. In some embodiments, the cell culture medium comprises about
180 IU/mL of IL-15.
In some embodiments, the cell culture medium further comprises IL-15. In some
embodiments, the
cell culture medium comprises about 180 IU/mL of 1L-15.
[00536] In some embodiments, the second expansion culture media comprises
about 20 IU/mL of
IL-21, about 15 IU/mL of IL-21, about 12 IU/mL of IL-21, about 10 IU/mL of IL-
21, about 5 IU/mL
of IL-21, about 4 IU/mL of IL-21, about 3 IU/mL of IL-21, about 2 IU/mL of IL-
21, about 1 IU/mL
of IL-21, or about 0.5 IU/mL of IL-21. In some embodiments, the second
expansion culture media
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comprises about 20 IU/mL of IL-21 to about 0.5 IU/mL of IL-21. In some
embodiments, the second
expansion culture media comprises about 15 IU/mL of IL-21 to about 0.5 IU/mL
of IL-21. In some
embodiments, the second expansion culture media comprises about 12 IU/mL of IL-
21 to about 0.5
IU/mL of IL-21. In some embodiments, the second expansion culture media
comprises about 10
IU/mL of IL-21 to about 0.5 IU/mL of IL-21. In some embodiments, the second
expansion culture
media comprises about 5 IU/mL of 1L-21 to about 1 1U/mL of IL-21. In some
embodiments, the
second expansion culture media comprises about 2 IU/mL of IL-21. In some
embodiments, the cell
culture medium comprises about 1 IU/mL of IL-21. In some embodiments, the cell
culture medium
comprises about 0.5 IU/mL of IL-21. In some embodiments, the cell culture
medium further
comprises IL-21. In some embodiments, the cell culture medium comprises about
1 IU/mL of IL-21.
[00537] In some embodiments the antigen-presenting feeder cells (APCs) are
PBMCs. In some
embodiments, the ratio of TILs to PBMCs and/or antigen-presenting cells in the
rapid expansion
and/or the second expansion is about 1 to 25, about 1 to 50, about 1 to 100,
about 1 to 125, about 1 to
150, about 1 to 175, about 1 to 200, about 1 to 225, about 1 to 250, about 1
to 275, about 1 to 300,
about 1 to 325, about 1 to 350, about 1 to 375, about 1 to 400, or about 1 to
500. In some
embodiments, the ratio of TILs to PBMCs in the rapid expansion and/or the
second expansion is
between 1 to 50 and 1 to 300. In some embodiments, the ratio of TILs to PBMCs
in the rapid
expansion and/or the second expansion is between 1 to 100 and 1 to 200.
[00538] In some embodiments, REP and/or the second expansion is performed in
flasks with the
bulk TILs being mixed with a 100- or 200-fold excess of inactivated feeder
cells, 30 mg/mL OKT3
anti-CD3 antibody and 3000 IU/mL 1L-2 in 150 mL media. Media replacement is
done (generally 2/3
media replacement via respiration with fresh media) until the cells are
transferred to an alternative
growth chamber. Alternative growth chambers include G-REX flasks and gas
permeable containers as
more fully discussed below.
[00539] In some embodiments, the second expansion (which can include processes
referred to as the
REP process) is shortened to 7-14 days, as discussed in the examples and
figures. In some
embodiments, the second expansion is shortened to 11 days.
[00540] In some embodiments, REP and/or the second expansion may be performed
using T-175
flasks and gas permeable bags as previously described (Tran, et at., J
lmmunother. 2008, 31, 742-51;
Dudley, et at., J. Immunother. 2003, 26, 332-42) or gas permeable cultureware
(G-REX flasks). In
some embodiments, the second expansion (including expansions referred to as
rapid expansions) is
performed in T-175 flasks, and about 1 x 106 TILs suspended in 150 mL of media
may be added to
each T-175 flask. The TILs may be cultured in a 1 to 1 mixture of CM and AIM-V
medium,
supplemented with 3000 IU per mL of IL-2 and 30 ng per mL of anti-CD3. The T-
175 flasks may be
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incubated at 37 C in 5% CO2. Half the media may be exchanged on day 5 using
50/50 medium with
3000 IU per mL of IL-2. In some embodiments, on day 7 cells from two T-175
flasks may be
combined in a 3 L bag and 300 mL of AIM V with 5% human AB serum and 3000 IU
per mL of 1L-2
was added to the 300 mL of TIL suspension. The number of cells in each bag was
counted every day
or two and fresh media was added to keep the cell count between 0.5 and 2.0 x
106 cells/mL.
[00541] In some embodiments, the second expansion (which can include
expansions referred to as
REP, as well as those referred to in Step D of Figure 1) may be performed in
500 mL capacity gas
permeable flasks with 100 cm gas-permeable silicon bottoms (G-REX 100,
commercially available
from Wilson Wolf Manufacturing Corporation, New Brighton, MN, USA), 5>< 106 or
10>< 106 TIL
may be cultured with PBMCs in 400 mL of 50/50 medium, supplemented with 5%
human AB serum,
3000 IU per mL of IL-2 and 30 ng per mL of anti-CD3 (OKT3). The G-REX 100
flasks may be
incubated at 37 C in 5% CO2. On day 5, 250 mL of supernatant may be removed
and placed into
centrifuge bottles and centrifuged at 1500 rpm (491 x g) for 10 minutes. The
TIL pellets may be re-
suspended with 150 mL of fresh medium with 5% human AB serum, 3000 IU per mL
of IL-2, and
added back to the original G-REX 100 flasks. When TIL are expanded serially in
G-REX 100 flasks,
on day 7 the TIL in each G-REX 100 may be suspended in the 300 mL of media
present in each flask
and the cell suspension may be divided into 3 100 mL aliquots that may be used
to seed 3 G-REX 100
flasks. Then 150 mL of AIM-V with 5% human AB serum and 3000 IU per mL of IL-2
may be added
to each flask. The G-REX 100 flasks may be incubated at 37 C in 5% CO2 and
after 4 days 150 mL
of AIM-V with 3000 IU per mL of-IL-2 may be added to each G-REX 100 flask. The
cells may be
harvested on day 14 of culture.
[00542] In some embodiments, the second expansion (including expansions
referred to as REP) is
performed in flasks with the bulk TILs being mixed with a 100- or 200-fold
excess of inactivated
feeder cells, 30 mg/mL OKT3 anti-CD3 antibody and 3000 IU/mL IL-2 in 150 mL
media. In some
embodiments, media replacement is done until the cells are transferred to an
alternative growth
chamber. In some embodiments, 2/3 of the media is replaced by respiration with
fresh media. In some
embodiments, alternative growth chambers include G-REX flasks and gas
permeable containers as
more fully discussed below.
[00543] In some embodiments, the second expansion (including expansions
referred to as REP) is
performed and further comprises a step wherein TILs are selected for superior
tumor reactivity. Any
selection method known in the art may be used. For example, the methods
described in U.S. Patent
Application Publication No. 2016/0010058 Al, the disclosures of which are
incorporated herein by
reference, may be used for selection of TILs for superior tumor reactivity.
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[00544] Optionally, a cell viability assay can be performed after the second
expansion (including
expansions referred to as the REP expansion), using standard assays known in
the art. For example, a
trypan blue exclusion assay can be done on a sample of the bulk TILs, which
selectively labels dead
cells and allows a viability assessment. In some embodiments, TIL samples can
be counted and
viability determined using a Cellometer K2 automated cell counter (Nexcelom
Bioscience, Lawrence,
MA). In some embodiments, viability is deterrnined according to the standard
Cellometer K2 Image
Cytometer Automatic Cell Counter protocol.
[00545] In some embodiments, the second expansion (including expansions
referred to as REP) of
TIL can be performed using T-175 flasks and gas-permeable bags as previously
described (Tran, et
al., 2008, J Immunother.,31:7 42-7 51, and Dudley, et al., 2003, J
Immunother., 26:332-342) or gas-
permeable G-REX flasks. In some embodiments, the second expansion is performed
using flasks. In
some embodiments, the second expansion is performed using gas-permeable G-REX
flasks. In some
embodiments, the second expansion is performed in T-175 flasks, and about 1 x
106 TIL arc
suspended in about 150 mL of media and this is added to each T-175 flask. The
TIL are cultured with
irradiated (50 Gy) allogeneic PBMC as "feeder" cells at a ratio of Ito 100 and
the cells were
cultured in a 1 to 1 mixture of CM and AIM-V medium (50/50 medium),
supplemented with 3000
III/mL of 1L-2 and 30 ng/mL of anti-CD3. The T-175 flasks are incubated at 37
C in 5% CO, In
some embodiments, half the media is changed on day 5 using 50/50 medium with
3000 IU/mL of
IL-2. In some embodiments, on day 7, cells from 2 T-175 flasks are combined in
a 3 L bag and
300 mL of AIM-V with 5% human AB serum and 3000 IU/mL of IL-2 is added to the
300 mL of
TIL suspension. The number of cells in each bag can be counted every day or
two and fresh media
can be added to keep the cell count between about 0.5 and about 2.0 x 10'
cells/mL.
[00546] In some embodiments, the second expansion (including expansions
referred to as REP) are
performed in 500 mL capacity flasks with 100 cm2 gas-permeable silicon bottoms
(G-REX 100,
Wilson Wolf), about 5 x 10' or 10 x 10' TIL are cultured with irradiated
allogeneic PBMC at a ratio
of 1 to 100 in 400 mL of 50/50 medium, supplemented with 3000 IU/mL of IL-2
and 30 ng/ mL of
anti-CD3. The G-REX 100 flasks are incubated at 37 C in 5% CO2. In some
embodiments, on day 5,
250mL of supernatant is removed and placed into centrifuge bottles and
centrifuged at 1500 rpm
(491g) for 10 minutes. The TIL pellets can then be resuspended with 150 mL of
fresh 50/50 medium
with 3000 1U/ mL of IL-2 and added back to the original G-REX 100 flasks. In
embodiments where
TILs are expanded serially in G-REX 100 flasks, on day 7 the TIL in each G-REX
100 are suspended
in the 300 mL of media present in each flask and the cell suspension was
divided into three 100 mL
aliquots that are used to seed 3 G-REX 100 flasks. Then 150 mL of AIM-V with
5% human AB
serum and 3000 IU/mL of IL-2 is added to each flask. The G-REX 100 flasks are
incubated at 37 C in
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5% CO2 and after 4 days 150 mL of AIM-V with 3000 IU/mL of IL-2 is added to
each G-REX 100
flask. The cells are harvested on day 14 of culture.
[00547] The diverse antigen receptors of T and B lymphocytes are produced by
somatic
recombination of a limited, but large number of gene segments. These gene
segments: V (variable), D
(diversity), J (joining), and C (constant), determine the binding specificity
and downstream
applications of immunoglobulins and T-cell receptors (TCRs). The present
invention provides a
method for generating TILs which exhibit and increase the T-cell repertoire
diversity. In some
embodiments, the TILs obtained by the present method exhibit an increase in
the T-cell repertoire
diversity. In some embodiments, the TILs obtained in the second expansion
exhibit an increase in the
T-cell repertoire diversity. In some embodiments, the increase in diversity is
an increase in the
immunoglobulin diversity and/or the T-cell receptor diversity. In some
embodiments, the diversity is
in the immunoglobulin is in the immunoglobulin heavy chain. In some
embodiments, the diversity is
in the immunoglobulin is in the immunoglobulin light chain. In some
embodiments, the diversity is in
the T-cell receptor. In some embodiments, the diversity is in one of the T-
cell receptors selected from
the group consisting of alpha, beta, gamma, and delta receptors. In some
embodiments, there is an
increase in the expression of T-cell receptor (TCR) alpha and/or beta. In some
embodiments, there is
an increase in the expression of T-cell receptor (TCR) alpha. In some
embodiments, there is an
increase in the expression of T-cell receptor (TCR) beta. In some embodiments,
there is an increase in
the expression of TCRab (i.e., TCRa/13).
[00548] In SOITIC embodiments, the second expansion culture medium (e.g.,
sometimes referred to
as CM2 or the second cell culture medium), comprises 1L-2, OKT-3, as well as
the antigen-
presenting feeder cells (APCs), as discussed in more detail below.
[00549] In some embodiments, the culture medium used in the expansion
processes disclosed herein
is a serum-free medium or a defined medium. In some embodiments, the serum-
free or defined
medium comprises a basal cell medium and a serum supplement and/or a serum
replacement. In some
embodiments, the serum-free or defined medium is used to prevent and/or
decrease experimental
variation due in part to the lot-to-lot variation of scrum-containing media.
[00550] In some embodiments, the serum-free or defined medium comprises a
basal cell medium
and a serum supplement and/or serum replacement. In some embodiments, the
basal cell medium
includes, but is not limited to CTSTm OpTmizerTm T-cell Expansion Basal Medium
, CTSTm
OpTmizerTm T-Cell Expansion SFM, CTS'im AIM-V Medium, CTS" AIM-V SFM,
LymphoONE"
T-Cell Expansion Xeno-Free Medium, Dulbecco's Modified Eagle's Medium (DMEM),
Minimal
Essential Medium (MEM), Basal Medium Eagle (BME), RPM! 1640, F-10, F-12,
Minimal Essential
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Medium (aMEM), Glasgow's Minimal Essential Medium (G-MEM), RPMI growth medium,
and
Iscove's Modified Dulbecco's Medium.
[00551] In some embodiments, the serum supplement or serum replacement
includes, but is not
limited to one or more of CTSTm OpTmizer T-Cell Expansion Serum Supplement,
CTSTm Immune
Cell Serum Replacement, one or more albumins or albumin substitutes, one or
more amino acids, one
or more vitamins, one or more transferrins or transferrin substitutes, one or
more antioxidants, one or
more insulins or insulin substitutes, one or more collagen precursors, one or
more antibiotics, and one
or more trace elements. In some embodiments, the defined medium comprises
albumin and one or
more ingredients selected from the group consisting of glycine, L- histidine,
L-isoleucine, L-
methionine, L-phenylalanine, L-proline, L- hydroxyproline, L-serine, L-
threonine, L-tryptophan, L-
tyrosine, L-valine, thiamine, reduced glutathione, L-ascorbic acid-2-
phosphate, iron saturated
transferrin, insulin, and compounds containing the trace element moieties Ag',
Al", Ba", Cd', Co',
Cr", Ge", Se", Br, T, Mn", P. Si", V', Mo", Ni", Rb', Sn" and Zr". in some
embodiments, the
defined medium further comprises L-glutamine, sodium bicarbonate and/or 2-
mercaptoethanol.
[00552] In some embodiments, the CTSTmOpTmizerTm T-cell Immune Cell Serum
Replacement is
used with conventional growth media, including but not limited to CTSTm
OpTmizerTm T-cell
Expansion Basal Medium, CTSTm OpTmizerTm T-cell Expansion SFM, CTSTm AIM-V
Medium,
CSTTm ATM-V SFM, LymphoONETM T-Cell Expansion Xeno-Free Medium, Dulbecco's
Modified
Eagle's Medium (DMEM), Minimal Essential Medium (MEM), Basal Medium Eagle
(BME), RPMI
1640, F-10, F-12, Minimal Essential Medium (aMEM), Glasgow's Minimal Essential
Medium (G-
MEM), RPM' growth medium, and Iscove's Modified Dulbccco's Medium.
[00553] In some embodiments, the total serum replacement concentration (vol%)
in the serum-free
or defined medium is from about 1%, 2%, 3%, 4%, 5%, 6%, 7%, 8%, 9%, 10%, 11%,
12%, 13%,
14%, 15%, 16%, 17%, 18%, 19%, or 20% by volume of the total serum-free or
defined medium. In
some embodiments, the total serum replacement concentration is about 3% of the
total volume of the
serum-free or defined medium. In some embodiments, the total serum replacement
concentration is
about 5% of the total volume of the serum-free or defined medium. In some
embodiments, the total
serum replacement concentration is about 10% of the total volume of the serum-
free or defined
medium.
[00554] In some embodiments, the serum-free or defined medium is CTSTm
OpTmizerTm T-cell
Expansion SFM (ThermoFisher Scientific). Any formulation of CTSTm OpTmizerTm
is useful in the
present invention. CTSTm OpTmizerTm T-cell Expansion SFM is a combination of
1L CTSTm
OpTmizerTm T-cell Expansion Basal Medium and 26 mL CTSTm OpTmizerTm T-Cell
Expansion
Supplement, which are mixed together prior to use. In some embodiments, the
CTSTm OpTmizerTm T-
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cell Expansion SFM is supplemented with about 3% of the CTSTm Immune Cell
Serum Replacement
(SR) (ThermoFisher Scientific). In some embodiments, the CTSTm OpTmizerTm T-
cell Expansion
SFM is supplemented with about 3% of the CTSTm Immune Cell Serum Replacement
(SR)
(ThermoFisher Scientific), along with 2-mercaptoethanol at 55mM. In some
embodiments. the CTSTm
OpTmizerTm T-cell Expansion SFM is supplemented with about 3% of the CTSTm
Immune Cell
Serum Replacement (SR) (ThermoFisher Scientific) and the final concentration
of 2-mercaptoethanol
in the media is 55 ,M.
[00555] In some embodiments, the defined medium is CTSTm OpTmizerTm T-cell
Expansion SFM
(ThermoFisher Scientific). Any formulation of CTSTm OpTmizerTm is useful in
the present invention.
CTSTm OpTmizerTm T-cell Expansion SFM is a combination of 1L CTSTm OpTmizerTm
T-cell
Expansion Basal Medium and 26 mL CTSTm OpTmizerTm T-Cell Expansion Supplement,
which are
mixed together prior to use. In some embodiments, the CTSTm OpTmizerTm T-cell
Expansion SFM is
supplemented with about 3% of the CTSTm Immune Cell Serum Replacement (SR)
(ThermoFisher
Scientific), along with 2-mercaptoethanol at 55mM. In some embodiments, the
CTSTmOpTmizerTm T-
cell Expansion SFM is supplemented with about 3% of the CTSTm Immune Cell
Serum Replacement
(SR) (ThermoFisher Scientific), 55mM of 2-mercaptoethanol, and 2mM of L-
glutamine. In some
embodiments, the CTSTmOpTmizerTm T-cell Expansion SFM is supplemented with
about 3% of the
CTSTm Immune Cell Serum Replacement (SR) (ThermoFisher Scientific), 55mM of 2-
mercaptoethanol, and 2mM of L-glutamine, and further comprises about 1000
IU/mL to about 8000
of TL-2. hi some embodiments, the CTSTmOpTtnizerTM T-cell Expansion SFM is
supplemented with about 3% of the CTSTm Immune Cell Serum Replacement (SR)
(ThermoFisher
Scientific), 55mM of 2-mercaptoethanol, and 2mM of L-glutamine, and further
comprises about 3000
IU/mL of IL-2. In some embodiments, the CTSTmOpTmizerfm T-cell Expansion SFM
is
supplemented with about 3% of the CTSTm Immune Cell Serum Replacement (SR)
(ThermoFisher
Scientific), 55mM of 2-mercaptoethanol, and 2mM of L-glutamine, and further
comprises about 6000
IU/mL of IL-2. In some embodiments, the CTSTmOpTmizerTm T-cell Expansion SFM
is
supplemented with about 3% of the CTSTm Immune Cell Serum Replacement (SR)
(ThermoFisher
Scientific) and 55mM of 2-mercaptoethanol, and further comprises about 1000
IU/mL to about 8000
IU/mL of IL-2. In some embodiments, the CTSTmOpTmizerTm T-cell Expansion SFM
is
supplemented with about 3% of the CTSTm Immune Cell Serum Replacement (SR)
(ThermoFisher
Scientific) and 55mM of 2-mercaptoethanol, and further comprises about 3000
IU/mL of IL-2. In
some embodiments, the CTSTmOpTinizerTm T-cell Expansion SFM is supplemented
with about 3% of
the CTSTm Immune Cell Serum Replacement (SR) (ThermoFisher Scientific) and
55mM of 2-
mercaptoethanol, and further comprises about 1000 IU/mL to about 6000 IU/mL of
IL-2. In some
embodiments, the CTSTmOpTmizerfm T-ccll Expansion SFM is supplemented with
about 3% of the
CTSTm Immune Cell Serum Replacement (SR) (ThermoFisher Scientific) and about
2mM glutamine,
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and further comprises about 1000 IU/mL to about 8000 IU/mL of IL-2. In some
embodiments, the
CTSTmOpTmizerTm T-cell Expansion SFM is supplemented with about 3% of the
CTSTm Immune
Cell Serum Replacement (SR) (ThennoFisher Scientific) and about 2mM glutamine,
and further
comprises about 3000 IU/mL of IL-2. In some embodiments, the CTSTmOpTmizerTm T-
cell
Expansion SFM is supplemented with about 3% of the CTSTm Immune Cell Serum
Replacement (SR)
(Thermaisher Scientific) and about 2mM glutamine, and further comprises about
6000 IU/mL of IL-
2. In some embodiments, the CTSTm OpTmizerTm T-cell Expansion SFM is
supplemented with about
3% of the CTSTm Immune Cell Serum Replacement (SR) (ThermoFisher Scientific)
and the final
concentration of 2-mercaptoethanol in the media is 55 M.
[00556] In some embodiments, the serum-free medium or defined medium is
supplemented with
glutamine (i.e., GlutaMAXE10 at a concentration of from about 0.1mM to about
10mM, 0.5mM to
about 9mM, 1mM to about 8mM, 2mM to about 7mM, 3mM to about 6mM, or 4mM to
about 5 mM.
In some embodiments, the serum-free medium or defined medium is supplemented
with glutamine
(i.e., GlutaMAXV) at a concentration of about 2mM.
[00557] In some embodiments, the serum-free medium or defined medium is
supplemented with 2-
mercaptoethanol at a concentration of from about 5mM to about 150mM, 10mM to
about 140mM,
15mM to about 130mM, 20mM to about 120mM, 25mM to about 110mM, 30mM to about
100mM,
35mM to about 95mM, 40mM to about 90mM, 45mM to about 85mM, 50mM to about
80mM, 55mM
to about 75mM, 60mM to about 70mM, or about 65mM. In some embodiments, the
serum-free
medium or defined medium is supplemented with 2-mercaptoethanol at a
concentration of about
55mM. In some embodiments, the final concentration of 2-mercaptoethanol in the
media is 55 M.
[00558] In some embodiments, the defined media described in International PCT
Publication No.
WO/1998/030679, which is herein incorporated by reference, are useful in the
present invention. In
that publication, serum-free eukaryotic cell culture media are described. The
serum-free, eukaryotic
cell culture medium includes a basal cell culture medium supplemented with a
serum-free supplement
capable of supporting the growth of cells in serum- free culture, The serum-
free eukaryotic cell
culture medium supplement comprises or is obtained by combining one or more
ingredients selected
fi-OE31 the group consisting of one Of more albumins or albumin substitutes,
one Of More amino a.cids,
one or more vitamins, one or more transferrins or transferrin substitutes, one
or more antioxidants,
one or More insulins Or insulin substitutes, one or more collagen precursors,
one or more trace
elements, and one or more antibiotics. In some embodiments, the defined medium
further comprises
L-alutamine, sodium bicarbonate and/or beta-mercaptoetbanol in some
embodiments, the defined
medium comprises an albumin or an albumin substitute and one or more
ingredients selected from
group consisting of one or more amino acids, one or more vitamins, one or more
transferrins or
transferrin substitutes, one or more antioxidants, one or more insulins or
insulin substitutes, one or
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more collagen precursors, and One or more trace elements. In sonic
embodiments, the defined medium
comprises albumin and one or more ingredients selected from the group
consisting of glycine, L-
histidine, L-isolencine, L-inethioninc, Iphenylalanine, L-proline, hydi-
oxyproline, L-serine, L.--
-threoninQ, L-tryptophart, le-tyrosine, L-valine, thiamine, reduced
glutathione,L-ascothie acid-2-
phosphate, iron saturated transferrin, insulin, and compounds containing the
trace element moieties
Ag õAI', Cd", Co', Cr', Gel', Se', Br, T, Mn, P,
Sn and Zr'.
In some embodiments, the basal cell media is selected from the group
consisting of Dulbeeco's
Modified Eagle's Medium (DMEM), Minimal Essential Medium (MEM), Basal Medium
Eagle
(BME), RPM! 1640, F-I0, F-12, Minimal Essential Medium (aMEM), Glasgow's
Minimal Essential
Medium (G-MEM), RPMI growth medium, and Iscove's Modified Dulhecco's Medium.
[00559] In sonic embodiments, the concentration of glycine in the defined
medium is in the range of
from about 5-200 mg/L, the concentration of L- histidine is about 5-250 mg/L,
the concentration of L-
isoleucine is about 5-300 mg/L, the concentration of L-methionine is about 5-
200 mg/L, the
concentration of L-phenylalanine is about 5-400 mg/L, the concentration of L-
proline is about 1-1000
mg/L, the concentration of L- hydroxyproline is about 1-45 mg/L, the
concentration of L-serine is
about 1-250 mg/L, the concentration of L-threonine is about 10-500 mg/L, the
concentration of L-
tryptophan is about 2-110 mg/L, the concentration of L-tyrosine is about 3-175
mg/L, the
concentration of L-valine is about 5-500 mg/L, the concentration of thiamine
is about 1-20 mg/L, the
concentration of reduced glutathione is about 1-20 mg/L, the concentration of
L-ascorbic acid-2-
phosphate is about 1-200 mg/L, the concentration of iron saturated transferrin
is about 1-50 mg/L, the
concentration of insulin is about 1-100 mg/L, the concentration of sodium
selenite is about 0.000001-
0.0001 mg/L, and the concentration of albumin (e.g., AlbuMAX I) is about 5000-
50,000 mg/L.
[00560] In some embodiments, the non-trace element moiety ingredients in the
defined medium are
present in the concentration ranges listed in the column under the heading
"Concentration Range in
1X Medium" in Table 4. In other embodiments, the non-trace element moiety
ingredients in the
defined medium are present in the final concentrations listed in the column
under the heading "A
Preferred Embodiment of the 1X Medium" in Table 4. In other embodiments, the
defined medium is a
basal cell medium comprising a serum free supplement. In some of these
embodiments, the serum free
supplement comprises non-trace moiety ingredients of the type and in the
concentrations listed in the
column under the heading "A Preferred Embodiment in Supplement" in Table 4.
[00561] In some embodiments, the osmolarity of the defined medium is between
about 260 and 350
mOsmol. In some embodiments, the osmolarity is between about 280 and 310
mOsmol. In some
embodiments, the defined medium is supplemented with up to about 3.7 g/L, or
about 2.2 g/L sodium
bicarbonate. The defined medium can be further supplemented with L-glutamine
(final concentration
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of about 2 mM), one or more antibiotics, non-essential amino acids (NEAA;
final concentration of
about 1001AM), 2-mercaptoethanol (final concentration of about 100 IIM).
[00562] In some embodiments, the defined media described in Smith, et al.,
Clin Trans?
Immunology, 4(1) 2015 (doi: 10.1038/cti.2014.31) are useful in the present
invention. Briefly, RPMI
or CTSTm OpTmizerTm was used as the basal cell medium, and supplemented with
either 0, 2%, 5%,
or 10% CTSTm Immune Cell Serum Replacement.
[00563] In some embodiments, the cell medium in the first and/or second gas
permeable container is
unfiltered. The use of unfiltered cell medium may simplify the procedures
necessary to expand the
number of cells. In some embodiments, the cell medium in the first and/or
second gas permeable
container lacks beta-mercaptoethanol (BME or PME; also known as 2-
mercaptoethanol, CAS 60-24-
2).
[00564] In some embodiments, the second expansion, for example, Step D
according to Figure 1, is
performed in a closed system bioreactor. In some embodiments, a closed system
is employed for the
TIL expansion, as described herein. In some embodiments, a single bioreactor
is employed. In some
embodiments, the single bioreactor employed is for example a G-REX -10 or a G-
REX -100. In some
embodiments, the closed system bioreactor is a single bioreactor.
[00565] In some embodiments, the step of rapid or second
expansion is split into a plurality of
steps to achieve a scaling up of the culture by: (a) performing the rapid or
second expansion by
culturing TILs in a small scale culture in a first container, e.g., a G-REX-
100 MCS container, for a
period of about 3 to 7 days, and then (b) effecting the transfer of the TILs
in the small scale culture to
a second container larger than the first container, e.g., a G-REX-500-MCS
container, and culturing the
TILs from the small scale culture in a larger scale culture in the second
container for a period of about
4 to 7 days.
[00566] In some embodiments, the step of rapid or second
expansion is split into a plurality of
steps to achieve a scaling out of the culture by: (a) performing the rapid or
second expansion by
culturing TILs in a first small scale culture in a first container, e.g., a G-
REX-100 MCS container, for
a period of about 3 to 7 days, and then (b) effecting the transfer and
apportioning of the TILs from the
first small scale culture into and amongst at least 2, 3, 4, 5, 6, 7, 8, 9,
10, 11, 12, 13, 14, 15, 16, 17, 18,
19, or 20 second containers that are equal in size to the first container,
wherein in each second
container the portion of the TILs from first small scale culture transferred
to such second container is
cultured in a second small scale culture for a period of about 4 to 7 days.
[00567] In some embodiments, the first small scale TIL culture is
apportioned into a plurality
of about 2 to 5 subpopulations of TILs.
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[00568] In some embodiments, the step of rapid or second
expansion is split into a plurality of
steps to achieve a scaling out and scaling up of the culture by: (a)
performing the rapid or second
expansion by culturing TILs in a small scale culture in a first container,
e.g., a G-REX-100 MCS
container, for a period of about 3 to 7 days, and then (b) effecting the
transfer and apportioning of the
TILs from the small scale culture into and amongst at least 2, 3, 4, 5, 6, 7,
8, 9, 10, 11, 12, 13, 14, 15,
16, 17, 18, 19, or 20 second containers that are larger in size than the first
container, e.g., G-REX-
500MCS containers, wherein in each second container the portion of the TILs
from the small scale
culture transferred to such second container is cultured in a larger scale
culture for a period of about 4
to 7 days.
[00569] In some embodiments, the step of rapid or second
expansion is split into a plurality of
steps to achieve a scaling out and scaling up of the culture by: (a)
performing the rapid or second
expansion by culturing TILs in a small scale culture in a first container,
e.g., a G-REX-100 MCS
container, for a period of about 5 days, and then (b) effecting the transfer
and apportioning of the TILs
from the small scale culture into and amongst 2, 3 or 4 second containers that
are larger in size than
the first container, e.g., G-REX-500 MCS containers, wherein in each second
container the portion of
the TILs from the small scale culture transferred to such second container is
cultured in a larger scale
culture for a period of about 6 days.
[00570] In some embodiments, upon the splitting of the rapid or
second expansion, each
second container comprises at least 108 TILs. In some embodiments, upon the
splitting of the rapid or
second expansion, each second container comprises at least 108 TILs, at least
109 TILs, or at least 101
TILs. In one exemplary embodiment, each second container comprises at least
101" TILs.
[00571] In some embodiments, the first small scale TIL culture is
apportioned into a plurality
of subpopulations. In some embodiments, the first small scale TIL culture is
apportioned into a
plurality of about 2 to 5 subpopulations. In some embodiments, the first small
scale TIL culture is
apportioned into a plurality of about 2, 3, 4, or 5 subpopulations.
[00572] In some embodiments, after the completion of the rapid or
second expansion, the
plurality of subpopulations comprises a therapeutically effective amount of
TILs. In some
embodiments, after the completion of the rapid or second expansion, one or
more subpopulations of
TILs are pooled together to produce a therapeutically effective amount of
TILs. In some
embodiments, after the completion of the rapid expansion, each subpopulation
of TILs comprises a
therapeutically effective amount of TILs.
[00573] In some embodiments, the rapid or second expansion is
performed for a period of
about 3 to 7 days before being split into a plurality of steps. In some
embodiments, the splitting of the
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rapid or second expansion occurs at about day 3, day 4, day 5, day 6, or day 7
after the initiation of
the rapid or second expansion.
[00574] In some embodiments, the splitting of the rapid or second
expansion occurs at about
day 7, day 8, day 9, day 10, day 11, day 12, day 13, day 14, day 15, or day 16
day 17, or day 18 after
the initiation of the first expansion (i.e., pre-REP expansion). In one
exemplary embodiment, the
splitting of the rapid or second expansion occurs at about day 16 after the
initiation of the first
expansion.
[00575] In some embodiments, the rapid or second expansion is
further performed for a period
of about 7 to 11 days after the splitting. In some embodiments, the rapid or
second expansion is
further performed for a period of about 5 days, 6 days, 7 days, 8 days, 9
days, 10 days, or 11 days
after the splitting.
[00576] In some embodiments, the cell culture medium used for the
rapid or second expansion
before the splitting comprises the same components as the cell culture medium
used for the rapid or
second expansion after the splitting. In some embodiments, the cell culture
medium used for the rapid
or second expansion before the splitting comprises different components from
the cell culture medium
used for the rapid or second expansicm after the splitting.
[00577] In some embodiments, the cell culture medium used for the
rapid or second expansion
before the splitting comprises IL-2, optionally OKT-3 and further optionally
APCs. In some
embodiments, the cell culture medium used for the rapid or second expansion
before the splitting
comprises IL-2, OKT-3, and further optionally APCs. In some embodiments, the
cell culture medium
used for the rapid or second expansion before the splitting comprises IL-2,
OKT-3 and APCs.
[00578] In some embodiments, the cell culture medium used for the
rapid or second expansion
before the splitting is generated by supplementing the cell culture medium in
the first expansion with
fresh culture medium comprising IL-2, optionally OKT-3 and further optionally
APCs. In some
embodiments, the cell culture medium used for the rapid or second expansion
before the splitting is
generated by supplementing the cell culture medium in the first expansion with
fresh culture medium
comprising IL-2, OKT-3 and APCs. In some embodiments, the cell culture medium
used for the rapid
or second expansion before the splitting is generated by replacing the cell
culture medium in the first
expansion with fresh cell culture medium comprising IL-2, optionally OKT-3 and
further optionally
APCs. In some embodiments, the cell culture medium used for the rapid or
second expansion before
the splitting is generated by replacing the cell culture medium in the first
expansion with fresh cell
culture medium comprising IL-2, OKT-3 and APCs.
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[00579] In some embodiments, the cell culture medium used for the
rapid or second expansion
after the splitting comprises IL-2, and optionally OKT-3. In some embodiments,
the cell culture
medium used for the rapid or second expansion after the splitting comprises IL-
2, and OKT-3. In
some embodiments, the cell culture medium used for the rapid or second
expansion after the splitting
is generated by replacing the cell culture medium used for the rapid or second
expansion before the
splitting with fresh culture medium comprising IL-2 and optionally OKT-3. In
some embodiments,
the cell culture medium used for the rapid or second expansion after the
splitting is generated by
replacing the cell culture medium used for the rapid or second expansion
before the splitting with
fresh culture medium comprising IL-2 and OKT-3.
[00580] In some embodiments, the splitting of the rapid expansion
occurs in a closed system.
[00581] In some embodiments, the scaling up of the TIL culture
during the rapid or second
expansion comprises adding fresh cell culture medium to the TIL culture (also
referred to as feeding
the TILs). In some embodiments, the feeding comprises adding fresh cell
culture medium to the TIL
culture frequently. In some embodiments, the feeding comprises adding fresh
cell culture medium to
the TIL culture at a regular interval. In some embodiments, the fresh cell
culture medium is supplied
to the TILs via a constant flow. In some embodiments, an automated cell
expansion system such as
Xuri W25 is used for the rapid expansion and feeding.
1. Feeder Cells and Antigen Presenting Cells
[00582] In some embodiments, the second expansion procedures described herein
(for example
including expansion such as those described in Step D from Figure 1, as well
as those referred to as
REP) require an excess of feeder cells during REP TIL expansion and/or during
the second expansion_
In many embodiments, the feeder cells are peripheral blood mononuclear cells
(PBMCs) obtained
from standard whole blood units from healthy blood donors. The PBMCs are
obtained using standard
methods such as Ficoll-Paque gradient separation.
[00583] In general, the allogenic PBMCs are inactivated, either via
irradiation or heat treatment, and
used in the REP procedures, as described in the examples, which provides an
exemplary protocol for
evaluating the replication incompetence of irradiate allogeneic PBMCs.
[00584] In some embodiments, PBMCs are considered replication incompetent and
accepted for use
in the TIL expansion procedures described herein if the total number of viable
cells on day 14 is less
than the initial viable cell number put into culture on day 0 of the REP
and/or day 0 of the second
expansion (i.e. , the start day of the second expansion).
[00585] In some embodiments, PBMCs are considered replication incompetent and
accepted for use
in the TIL expansion procedures described herein if the total number of viable
cells, cultured in the
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presence of OKT3 and IL-2, on day 7 and day 14 has not increased from the
initial viable cell number
put into culture on day 0 of the REP and/or day 0 of the second expansion
(i.e., the start day of the
second expansion). In some embodiments, the PBMCs are cultured in the presence
of 30 ng/mL
OKT3 antibody and 3000 IU/mL IL-2.
[00586] In some embodiments, PBMCs are considered replication incompetent and
accepted for use
in the TIL expansion procedures described herein if the total number of viable
cells, cultured in the
presence of OKT3 and IL-2, on day 7 and day 14 has not increased from the
initial viable cell number
put into culture on day 0 of the REP and/or day 0 of the second expansion
(i.e., the start day of the
second expansion). In some embodiments, the PBMCs are cultured in the presence
of 5-60 ng/mL
OKT3 antibody and 1000-6000 IU/mL IL-2. In some embodiments, the PBMCs are
cultured in the
presence of 10-50 ng/mL OKT3 antibody and 2000-5000 IU/mL IL-2. In some
embodiments, the
PBMCs are cultured in the presence of 20-40 ng/mL OKT3 antibody and 2000-4000
IU/mL IL-2. In
some embodiments, the PBMCs are cultured in the presence of 25-35 ng/mL OKT3
antibody and
2500-3500 IU/mL IL-2.
[00587] In some embodiments, the antigen-presenting feeder cells are PBMCs. In
some
embodiments, the antigen-presenting feeder cells are artificial antigen-
presenting feeder cells. In some
embodiments, the ratio of TILs to antigen-presenting feeder cells in the
second expansion is about 1 to
25, about 1 to 50, about 1 to 100, about 1 to 125, about 1 to 150, about 1 to
175, about 1 to 200, about
1 to 225, about 1 to 250, about 1 to 275, about 1 to 300, about I to 325,
about 1 to 350, about 1 to 375,
about 1 to 400, or about 1 to 500. In some embodiments, the ratio of TILs to
antigen-presenting feeder
cells in the second expansion is between 1 to 50 and 1 to 300. In some
embodiments, the ratio of T1L
to antigen-presenting feeder cells in the second expansion is between 1 to 100
and 1 to 200.
[00588] In some embodiments, the second expansion procedures described herein
require a ratio of
about 2.5x109 feeder cells to about 100x106 TIL. In some embodiments, the
second expansion
procedures described herein require a ratio of about 2.5x109 feeder cells to
about 50x106 TIL. In yet
another embodiment, the second expansion procedures described herein require
about 2.5x109 feeder
cells to about 25x106 TIL.
[00589] In some embodiments, the second expansion procedures described herein
require an excess
of feeder cells during the second expansion. In many embodiments, the feeder
cells are peripheral
blood mononuclear cells (PBMCs) obtained from standard whole blood units from
healthy blood
donors. The PBMCs are obtained using standard methods such as Ficoll-Paque
gradient separation. In
some embodiments, artificial antigen-presenting (aAPC) cells are used in place
of PBMCs.
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[00590] In general, the allogenic PBMCs are inactivated, either via
irradiation or heat treatment, and
used in the TIL expansion procedures described herein, including the exemplary
procedures described
in the figures and examples.
[00591] In some embodiments, artificial antigen presenting cells are used in
the second expansion as
a replacement for, or in combination with, PBMCs.
2. Cytokines
[00592] The expansion methods described herein generally use culture media
with high doses of a
cytokine, in particular IL-2, as is known in the art.
[00593] Alternatively, using combinations of cytokines for the rapid expansion
and or second
expansion of TILs is additionally possible, with combinations of two or more
of IL-2, IL-15 and IL-
21 as is described in U.S. Patent Application US 2017/0107490 Al, the
disclosure of which is
incorporated by reference herein. Thus, possible combinations include IL-2 and
IL-15, IL-2 and IL-
21, IL-15 and IL-21 and IL-2, IL-15 and IL-21, with the latter finding
particular use in many
embodiments. The use of combinations of cytokines specifically favors the
generation of
lymphocytes, and in particular T-cells as described therein.
[00594] In some embodiments, Step D may also include the addition of OKT-3
antibody or
muromonab to the culture media, as described elsewhere herein. In some
embodiments, Step D may
also include the addition of a 4-1BB agonist to the culture media, as
described elsewhere herein. In
some embodiments, Step D may also include the addition of an OX-40 agonist to
the culture media, as
described elsewhere herein. In addition, additives such as peroxisome
proliferator-activated receptor
gamma coactivator I-alpha agonists, including proliferator-activated receptor
(PPAR)-gamma agonists
such as a thiazolidinedione compound, may be used in the culture media during
Step D, as described
in U.S. Patcnt Application Publication No. US 2019/0307796 Al, the disclosure
of which is
incorporated by reference herein.
E. STEP E: Harvest TILs
[00595] After the second expansion step, cells can be harvested. In some
embodiments the TILs are
harvested after one, two, three, four or more expansion steps, for example as
provided in Figure 1. In
some embodiments the TILs are harvested after two expansion steps, for example
as provided in
Figure 1.
[00596] TILs can be harvested in any appropriate and sterile manner, including
for example by
centrifugation. Methods for TIL harvesting are well known in the art and any
such know methods can
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be employed with the present process. In some embodiments, TILs are harvested
using an automated
system.
[00597] Cell harvesters and/or cell processing systems are commercially
available from a variety of
sources, including, for example, Fresenius Kabi, Tomtec Life Science, Perkin
Elmer, and Inotech
Biosystems International, Inc. Any cell based harvester can be employed with
the present methods. In
some embodiments, the cell harvester and/or cell processing systems is a
membrane-based cell
harvester. In some embodiments, cell harvesting is via a cell processing
system, such as the LOVO
system (manufactured by Fresenius Kabi). The term "LOVO cell processing
system" also refers to
any instalment or device manufactured by any vendor that can pump a solution
comprising cells
through a membrane or filter such as a spinning membrane or spinning filter in
a sterile and/or closed
system environment, allowing for continuous flow and cell processing to remove
supernatant or cell
culture media without pelletization. In some embodiments, the cell harvester
and/or cell processing
system can perform cell separation, washing, fluid-exchange, concentration,
and/or other cell
processing steps in a closed, sterile system.
[00598] In some embodiments, the harvest, for example, Step E according to
Figure 1, is performed
from a closed system bioreactor. In some embodiments, a closed system is
employed for the T1L
expansion, as described herein. In some embodiments, a single bioreactor is
employed. In some
embodiments, the single bioreactor employed is for example a G-REX -10 or a G-
REX -100. In some
embodiments, the closed system bioreactor is a single bioreactor.
[00599] In some embodiments, Step E according to Figure 1, is performed
according to the
processes described herein. In some embodiments, the closed system is accessed
via syringes under
sterile conditions in order to maintain the sterility and closed nature of the
system. In some
embodiments, a closed system as described in the Examples is employed.
[00600] In some embodiments, TILs are harvested according to the methods
described in the
Examples. In some embodiments, TILs between days 1 and 11 are harvested using
the methods as
described in the steps referred herein, such as in the day 11 TIL harvest in
the Examples. In some
embodiments, TILs between days 12 and 24 are harvested using the methods as
described in the steps
referred herein, such as in the Day 22 TIL harvest in the Examples. In some
embodiments, TILs
between days 12 and 22 are harvested using the methods as described in the
steps referred herein,
such as in the Day 22 TIL harvest in the Examples.
F. STEP F: Final Formulation and Transfer to Infusion
Container
[00601] After Steps A through E as provided in an exemplary order in Figure 1
and as outlined in
detailed above and herein are complete, cells are transferred to a container
for use in administration to
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a patient, such as an infusion bag or sterile vial. In some embodiments, once
a therapeutically
sufficient number of TILs are obtained using the expansion methods described
above, they are
transferred to a container for use in administration to a patient.
[00602] In some embodiments, TILs expanded using APCs of the present
disclosure are
administered to a patient as a pharmaceutical composition. In some
embodiments, the pharmaceutical
composition is a suspension of TILs in a sterile buffer. TILs expanded using
PBMCs of the present
disclosure may be administered by any suitable route as known in the art. In
some embodiments, the
T-cells are administered as a single intra-arterial or intravenous infusion,
which preferably lasts
approximately 30 to 60 minutes. Other suitable routes of administration
include intraperitoneal,
intrathecal, and intralymphatic administration.
IV. Gen 3 TIL Manufacturing Processes
1006031 Without being limited to any particular theory, it is believed that
the priming first expansion
that primes an activation of T cells followed by the rapid second expansion
that boosts the activation
of T cells as described in the methods of the invention allows the preparation
of expanded T cells that
retain a "younger" phenotype, and as such the expanded T cells of the
invention are expected to
exhibit greater cytotoxicity against cancer cells than T cells expanded by
other methods. In particular,
it is believed that an activation of T cells that is primed by exposure to an
anti-CD3 antibody (e.g.
OKT-3), IL-2 and optionally antigen-presenting cells (APCs) and then boosted
by subsequent
exposure to additional anti-CD-3 antibody (e.g. OKT-3), IL-2 and APCs as
taught by the methods of
the invention limits or avoids the maturation of T cells in culture, yielding
a population of T cells with
a less mature phenotype, which T cells are less exhausted by expansion in
culture and exhibit greater
cytotoxicity against cancer cells. In some embodiments, the step of rapid
second expansion is split
into a plurality of steps to achieve a scaling up of the culture by: (a)
performing the rapid second
expansion by culturing T cells in a small scale culture in a first container,
e.g., a G-REX 100MCS
container, for a period of about 3 to 4 days, and then (b) effecting the
transfer of the T cells in the
small scale culture to a second container larger than the first container,
e.g., a G-REX 500MCS
container, and culturing the T cells from the small scale culture in a larger
scale culture in the second
container for a period of about 4 to 7 days. In some embodiments, the step of
rapid expansion is split
into a plurality of steps to achieve a scaling out of the culture by: (a)
performing the rapid second
expansion by culturing T cells in a first small scale culture in a first
container, e.g., a G-REX 100MCS
container, for a period of about 3 to 4 days, and then (b) effecting the
transfer and apportioning of the
T cells from the first small scale culture into and amongst at least 2, 3, 4,
5,6, 7, 8, 9, 10, 11, 12, 13,
14, 15, 16, 17, 18, 19, or 20 second containers that are equal in size to the
first container, wherein in
each second container the portion of the T cells from first small scale
culture transferred to such
second container is cultured in a second small scale culture for a period of
about 4 to 7 days. In some
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embodiments, the step of rapid expansion is split into a plurality of steps to
achieve a scaling out and
scaling up of the culture by: (a) performing the rapid second expansion by
culturing T cells in a small
scale culture in a first container, e.g., a G-REX 100MCS container, for a
period of about 3 to 4 days,
and then (b) effecting the transfer and apportioning of the T cells from the
small scale culture into and
amongst at least 2, 3,4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18,
19, or 20 second containers that
arc larger in size than the first container, e.g., G-REX 500MCS containers,
wherein in each second
container the portion of the T cells from the small scale culture transferred
to such second container is
cultured in a larger scale culture for a period of about 4 to 7 days. In some
embodiments, the step of
rapid expansion is split into a plurality of steps to achieve a scaling out
and scaling up of the culture
by: (a) performing the rapid second expansion by culturing T cells in a small
scale culture in a first
container, e.g., a G-REX 100MCS container, for a period of about 4 days, and
then (b) effecting the
transfer and apportioning of the T cells from the small scale culture into and
amongst 2, 3 or 4 second
containers that are larger in size than the first container, e.g., G-REX
500MCS containers, wherein in
each second container the portion of the T cells from the small scale culture
transferred to such second
container is cultured in a larger scale culture for a period of about 5 days.
[00604] In some embodiments, upon the splitting of the rapid
expansion, each second
container comprises at least 108 TILs. In some embodiments, upon the splitting
of the rapid
expansion, each second container comprises at least 108 TILs, at least 109
TILs, or at least 101 TILs.
In one exemplary embodiment, each second container comprises at least 1010
TILs.
[00605] In some embodiments, the first small scale TIL culture is
apportioned into a plurality
of subpopulations. In some embodiments, the first small scale TIL culture is
apportioned into a
plurality of about 2 to 5 subpopulations. In some embodiments, the first small
scale TIL culture is
apportioned into a plurality of about 2, 3, 4, or 5 subpopulations.
[00606] In some embodiments, after the completion of the rapid
expansion, the plurality of
subpopulations comprises a therapeutically effective amount of TILs. In some
embodiments, after the
completion of the rapid expansion, one or more subpopulations of TILs are
pooled together to
produce a therapeutically effective amount of TILs. In some embodiments, after
the completion of the
rapid expansion, each subpopulation of TiLs comprises a therapeutically
effective amount of TILs.
[00607] In some embodiments, the rapid expansion is performed for
a period of about 1 to 5
days before being split into a plurality of steps. In some embodiments, the
splitting of the rapid
expansion occurs at about day 1, day 2, day 3, day 4, or day 5 after the
initiation of the rapid
expansion.
[00608] In some embodiments, the splitting of the rapid expansion
occurs at about day 8, day
9, day 10, day 11, day 12, or day 13 after the initiation of the first
expansion (i.e., pre-REP
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expansion). In one exemplary embodiment, the splitting of the rapid expansion
occurs at about day 10
after the initiation of the priming first expansion. In another exemplary
embodiment, the splitting of
the rapid expansion occurs at about day 11 after the initiation of the priming
first expansion.
[00609] In some embodiments, the rapid expansion is further
performed for a period of about
4 to 11 days after the splitting. In some embodiments, the rapid expansion is
further performed for a
period of about 3 days, 4 days, 5 days, 6 days, 7 days, 8 days, 9 days, 10
days, or ii days after the
splitting.
[00610] In some embodiments, the cell culture medium used for the
rapid expansion before
the splitting comprises the same components as the cell culture medium used
for the rapid expansion
after the splitting. In some embodiments, the cell culture medium used for the
rapid expansion before
the splitting comprises different components from the cell culture medium used
for the rapid
expansion after the splitting.
[00611] In some embodiments, the cell culture medium used for the
rapid expansion before
the splitting comprises IL-2, optionally OKT-3 and further optionally APCs. In
some embodiments,
the cell culture medium used for the rapid expansion before the splitting
comprises IL-2, OKT-3, and
further optionally APCs hi some embodiments, the cell culture medium used for
the rapid expansion
before the splitting comprises IL-2, OKT-3 and APCs.
[00612] In some embodiments, the cell culture medium used for the
rapid expansion before
the splitting is generated by supplementing the cell culture medium in the
first expansion with fresh
culture medium comprising IL-2, optionally OKT-3 and further optionally APCs.
In some
embodiments, the cell culture medium used for the rapid expansion before the
splitting is generated
by supplementing the cell culture medium in the first expansion with fresh
culture medium
comprising IL-2, OKT-3 and APCs. In some embodiments, the cell culture medium
used for the rapid
expansion before the splitting is generated by replacing the cell culture
medium in the first expansion
with fresh cell culture medium comprising IL-2, optionally OKT-3 and further
optionally APCs. In
some embodiments, the cell culture medium used for the rapid expansion before
the splitting is
generated by replacing the cell culture medium in the first expansion with
fresh cell culture medium
comprising IL-2, OKT-3 and APCs.
[00613] In some embodiments, the cell culture medium used for the
rapid expansion after the
splitting comprises IL-2, and optionally OKT-3. In some embodiments, the cell
culture medium used
for the rapid expansion after the splitting comprises 1L-2, and OKT-3. In some
embodiments, the cell
culture medium used for the rapid expansion after the splitting is generated
by replacing the cell
culture medium used for the rapid expansion before the splitting with fresh
culture medium
comprising IL-2 and optionally OKT-3. In some embodiments, the cell culture
medium used for the
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rapid expansion after the splitting is generated by replacing the cell culture
medium used for the rapid
expansion before the splitting with fresh culture medium comprising IL-2 and
OKT-3.
[00614] In some embodiments, the splitting of the rapid expansion
occurs in a closed system.
1006151 In some embodiments, the scaling up of the TIL culture
during the rapid expansion
comprises adding fresh cell culture medium to the TIL culture (also referred
to as feeding the TILs).
In some embodiments, the feeding comprises adding fresh cell culture medium to
the TIL culture
frequently. In some embodiments, the feeding comprises adding fresh cell
culture medium to the TIL
culture at a regular interval. In some embodiments, the fresh cell culture
medium is supplied to the
TILs via a constant flow. In some embodiments, an automated cell expansion
system such as Xuri
W25 is used for the rapid expansion and feeding.
[00616] In some embodiments, the rapid second expansion is performed after the
activation of T
cells effected by the priming first expansion begins to decrease, abate, decay
or subside.
1006171 In some embodiments, the rapid second expansion is performed after the
activation of T
cells effected by the priming first expansion has decreased by at or about 1,
2, 3, 4, 5, 6, 7, 8, 9, 10,
11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29,
30, 31, 32, 33, 34, 35, 36, 37,
38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50, 51, 52, 53, 54, 55, 56,
57, 58, 59, 60, 61, 62, 63, 64,
65, 66, 67, 68, 69, 70, 71, 72, 73, 74, 75, 76, 77, 78, 79, 80, 81, 82, 83,
84, 85, 86, 87, 88, 89, 90, 91,
92, 93, 94, 95, 96, 97, 98, 99, or 100%.
[00618] In some embodiments, the rapid second expansion is performed after the
activation of T
cells effected by the priming first expansion has decreased by a percentage in
the range of at or about
1% to 100%.
[00619] In some embodiments, the rapid second expansion is performed after the
activation of T
cells effected by the priming first expansion has decreased by a percentage in
the range of at or about
1% to 10%, 10% to 20%, 20% to 30%, 30% to 40%, 40% to 50%, 50% to 60%, 60% to
70%, 70% to
80%, 80% to 90%, or 90% to 100%.
[00620] In some embodiments, the rapid second expansion is performed after the
activation of T
cells effected by the priming first expansion has decreased by at least at or
about 1, 2, 3, 4, 5, 6, 7, 8,
9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28,
29, 30, 31, 32, 33, 34, 35,
36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50, 51, 52, 53, 54,
55, 56, 57, 58, 59, 60, 61, 62,
63, 64, 65, 66, 67, 68, 69, 70, 71, 72, 73, 74, 75, 76, 77, 78, 79, 80, 81,
82, 83, 84, 85, 86, 87, 88, 89,
90, 91, 92, 93, 94, 95, 96, 97, 98, or 99%.
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[00621] In some embodiments, the rapid second expansion is performed after the
activation of T
cells effected by the priming first expansion has decreased by up to at or
about 1, 2, 3, 4, 5, 6, 7, 8, 9,
10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28,
29, 30, 31, 32, 33, 34, 35, 36,
37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50, 51, 52, 53, 54, 55,
56, 57, 58, 59, 60, 61, 62, 63,
64, 65, 66, 67, 68, 69, 70, 71, 72, 73, 74, 75, 76, 77, 78, 79, 80, 81, 82,
83, 84, 85, 86, 87, 88, 89, 90,
91, 92, 93, 94, 95, 96, 97, 98, 99 or 100%.
[00622] In some embodiments, the decrease in the activation of T cells
effected by the priming first
expansion is determined by a reduction in the amount of interferon gamma
released by the T cells in
response to stimulation with antigen.
[00623] In some embodiments, the priming first expansion of T cells is
performed during a period of
up to at or about 7 days or about 8 days.
[00624] In some embodiments, the priming first expansion of T cells is
performed during a period of
up to at or about 1 day, 2 days, 3 days, 4 days, 5 days, 6 days, 7 days, or 8
days.
[00625] In some embodiments, the priming first expansion of T cells is
performed during a period of
1 day, 2 days, 3 days, 4 days, 5 days, 6 days, 7 days, or 8 days.
[00626] In some embodiments, the rapid second expansion of T cells is
performed during a period
of up to at or about 11 days.
[00627] In some embodiments, the rapid second expansion of T cells is
performed during a period
of up to at or about 1 day, 2 days, 3 days, 4 days, 5 days, 6 days, 7 days, 8
days, 9 days, 10 days or 11
days.
[00628] In some embodiments, the rapid second expansion of T cells is
performed during a period
of 1 day, 2 days, 3 days, 4 days, 5 days, 6 days, 7 days, 8 days, 9 days, 10
days or 11 days.
[00629] In some embodiments, the priming first expansion of T cells is
performed during a period of
from at or about 1 day to at or about 7 days and the rapid second expansion of
T cells is performed
during a period of from at or about 1 day to at or about 11 days.
[00630] In some embodiments, the priming first expansion of T cells is
performed during a period of
up to at or about 1 day, 2 days, 3 days, 4 days, 5 days, 6 days, 7 days, or 8
days and the rapid second
expansion of T cells is performed during a period of up to at or about 1 day,
2 days, 3 days, 4 days, 5
days, 6 days, 7 days, 8 days, 9 days, 10 days or 11 days.
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[00631] In some embodiments, the priming first expansion of T cells is
performed during a period of
from at or about 1 day to at or about 8 days and the rapid second expansion of
T cells is performed
during a period of from at or about 1 day to at or about 9 days.
[00632] In some embodiments, the priming first expansion of T cells is
performed during a period of
8 days and the rapid second expansion of T cells is performed during a period
of 9 days.
[00633] In some embodiments, the priming first expansion of T cells is
performed during a period of
from at or about 1 day to at or about 7 days and the rapid second expansion of
T cells is performed
during a period of from at or about 1 day to at or about 9 days.
[00634] In some embodiments, the priming first expansion of T cells is
performed during a period of
7 days and the rapid second expansion of T cells is performed during a period
of 9 days.
[00635] In some embodiments, the T cells are tumor infiltrating lymphocytes
(TILs).
[00636] In some embodiments, the T cells are marrow infiltrating lymphocytes
(MILs).
[00637] In some embodiments, the T cells are peripheral blood lymphocytes
(PBLs).
[00638] In some embodiments, the T cells are obtained from a donor suffering
from a cancer.
[00639] In some embodiments, the T cells are TILs obtained from a tumor
excised from a patient
suffering from a cancer.
[00640] In some embodiments, the T cells are MILs obtained from bone marrow of
a patient
suffering from a hematologic malignancy.
[00641] In some embodiments, the T cells are PBLs obtained from peripheral
blood mononuclear
cells (PBMCs) from a donor. In some embodiments, the donor is suffering from a
cancer. In some
embodiments, the cancer is the cancer is selected from the group consisting of
melanoma, ovarian
cancer, endometrial cancer, thyroid cancer, cervical cancer, non-small-cell
lung cancer (NSCLC),
lung cancer, bladder cancer, breast cancer, cancer caused by human papilloma
virus, head and neck
cancer (including head and neck squamous cell carcinoma (1-INSCC)),
glioblastoma (including GBM),
gastrointestinal cancer, renal cancer, and renal cell carcinoma. In some
embodiments, the cancer is
selected from the group consisting of melanoma, ovarian cancer, cervical
cancer, non-small-cell lung
cancer (NSCLC), lung cancer, bladder cancer, breast cancer, cancer caused by
human papilloma virus,
head and neck cancer (including head and neck squamous cell carcinoma
(HNSCC)), glioblastoma
(including GBM), gastrointestinal cancer, renal cancer, and renal cell
carcinoma. In some
embodments, the donor is suffering from a tumor. In some embodiments, the
tumor is a liquid tumor.
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In some embodiments, the tumor is a solid tumor. In some embodiments, the
donor is suffering from a
hematologic malignancy.
[00642] In certain aspects of the present disclosure, immune effector cells,
e.g., T cells, can be
obtained from a unit of blood collected from a subject using any number of
techniques known to the
skilled artisan, such as FICOLL separation. In one preferred aspect, cells
from the circulating blood of
an individual are obtained by apheresis. The apheresis product typically
contains lymphocytes,
including T cells, monocytes, granulocytes, B cells, other nucleated white
blood cells, red blood cells,
and platelets. In one aspect, the cells collected by apheresis may be washed
to remove the plasma
fraction and, optionally, to place the cells in an appropriate buffer or media
for subsequent processing
steps. In one embodiment, the cells are washed with phosphate buffered saline
(PBS). In an
alternative embodiment, the wash solution lacks calcium and may lack magnesium
or may lack many
if not all divalent cations. In one aspect, T cells are isolated from
peripheral blood lymphocytes by
lysing the red blood cells and depleting the monocytes, for example, by
centrifugation through a
PERCOLL gradient or by counterflovv centrifugal elutriation.
[00643] In some embodiments, the T cells are PBLs separated from whole blood
or apheresis
product enriched for lymphocytes from a donor. In some embodiments, the donor
is suffering from a
cancer. In some embodiments, the cancer is the cancer is selected from the
group consisting of
melanoma, ovarian cancer, endometrial cancer, thyroid cancer, cervical cancer,
non-small-cell lung
cancer (NSCLC), lung cancer, bladder cancer, breast cancer, cancer caused by
human papilloma virus,
head and neck cancer (including head and neck squamous cell carcinoma
(HNSCC)), glioblastoma
(including GBM), gastrointestinal cancer, renal cancer, and renal cell
carcinoma. In some
embodiments, the cancer is selected from the group consisting of melanoma,
ovarian cancer, cervical
cancer, non-small-cell lung cancer (NSCLC), lung cancer, bladder cancer,
breast cancer, cancer
caused by human papilloma virus, head and neck cancer (including head and neck
squamous cell
carcinoma (HNSCC)), glioblastoma (including GBM), gastrointestinal cancer,
renal cancer, and renal
cell carcinoma. In some embodments, the donor is suffering from a tumor. In
some embodiments, the
tumor is a liquid tumor. In some embodiments, the tumor is a solid tumor. In
some embodiments, the
donor is suffering from a hematologic malignancy. In some embodiments, the
PBLs are isolated from
whole blood or apheresis product enriched for lymphocytes by using positive or
negative selection
methods, i.e., removing the PBLs using a marker(s), e.g., CD3+ CD45 , for T
cell phenotype, or
removing non-T cell phenotype cells, leaving PBLs. In other embodiments, the
PBLs are isolated by
gradient centrifugation. Upon isolation of PBLs from donor tissue, the priming
first expansion of
PBLs can be initiated by seeding a suitable number of isolated PBLs (in some
embodiments,
approximately 1 x 107 PBLs) in the priming first expansion culture according
to the priming first
expansion step of any of the methods described herein.
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[00644] An exemplary TIL process known as process 3 (also referred to herein
as Gen 3) containing
some of these features is depicted in Figure 8 (in particular, e.g., Figure 8A
and/or Figure 8B and/or
Figure 8C and/or Figure 8D), and some of the advantages of this embodiment of
the present invention
over Gen 2 are described in Figures 1, 2, 8, 30, and 31 (in particular, e.g.,
Figure 8A and/or Figure 8B
and/or Figure 8C and/or Figure 8D). Embodiments of Gen 3 are shown in Figures
1, 8, and 30 (in
particular, e.g., Figure 8A and/or Figure 8B and/or Figure 8C and/or Figure
8D). Process 2A or Gen 2
or Gen 2A is also described in U.S. Patent Publication No. 2018/0280436,
incorporated by reference
herein in its entirety. The Gen 3 process is also described in International
Patent Publication WO
2020/096988.
[00645] As discussed and generally outlined herein, TILs are taken from a
patient sample and
manipulated to expand their number prior to transplant into a patient using
the TIL expansion process
described herein and referred to as Gen 3. In some embodiments, the TILs may
be optionally
genetically manipulated as discussed below. In some embodiments, the TILs may
be cryopreserved
prior to or after expansion. Once thawed, they may also be restimulated to
increase their metabolism
prior to infusion into a patient.
[00646] In some embodiments, the priming first expansion (including processes
referred herein as
the pre-Rapid Expansion (Pre-REP), as well as processes shown in Figure 8 (in
particular, e.g., Figure
8A and/or Figure 8B and/or Figure 8C and/or Figure 8D) as Step B) is shortened
to 1 to 8 days and
the rapid second expansion (including processes referred to herein as Rapid
Expansion Protocol
(REP) as well as processes shown in Figure 8 (in particular, e.g., Figure 8A
and/or Figure 8B and/or
Figure 8C and/or Figure 8D) as Step D) is shortened to 1 to 9 days, as
discussed in detail below as
well as in the examples and figures. In some embodiments, the priming first
expansion (including
processes referred herein as the pre-Rapid Expansion (Pre-REP), as well as
processes shown in Figure
8 (in particular, e.g., Figure 8A and/or Figure 8B and/or Figure 8C and/or
Figure 8D) as Step B) is
shortened to 1 to 8 days and the rapid second expansion (including processes
referred to herein as
Rapid Expansion Protocol (REP) as well as processes shown in Figure 8 (in
particular, e.g., Figure 8A
and/or Figure 8B and/or Figure 8C and/or Figure 8D) as Step D) is shortened to
1 to 8 days, as
discussed in detail below as well as in the examples and figures. In some
embodiments, the priming
first expansion (including processes referred herein as the pre-Rapid
Expansion (Pre-REP), as well as
processes shown in Figure 8 (in particular, e.g., Figure 8A and/or Figure 8B
and/or Figure 8C and/or
Figure 8D) as Step B) is shortened to 1 to 7 days and the rapid second
expansion (including processes
referred to herein as Rapid Expansion Protocol (REP) as well as processes
shown in Figure 8 (in
particular, e.g., Figure 8A and/or Figure 8B and/or Figure 8C and/or Figure
8D) as Step D) is
shortened to 1 to 9 days, as discussed in detail below as well as in the
examples and figures. In some
embodiments, the priming first expansion (including processes referred herein
as the pre-Rapid
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Expansion (Pre-REP), as well as processes shown in Figure 8 (in particular,
e.g., Figure 1B and/or
Figure 8C) as Step B) is 1 to 7 days and the rapid second expansion (including
processes referred to
herein as Rapid Expansion Protocol (REP) as well as processes shown in Figure
8 (in particular, e.g.,
Figure 8A and/or Figure 8B and/or Figure 8C and/or Figure 8D) as Step D) is 1
to 10 days, as
discussed in detail below as well as in the examples and figures. In some
embodiments, the priming
first expansion (for example, an expansion described as Step 13 in Figure 8
(in particular, e.g., Figure
8A and/or Figure 8B and/or Figure 8C and/or Figure 8D)) is shortened to 8 days
and the rapid second
expansion (for example, an expansion as described in Step D in Figure 8 (in
particular, e.g., Figure 8A
and/or Figure 8B and/or Figure 8C and/or Figure 8D)) is 7 to 9 days. In some
embodiments, the
priming first expansion (for example, an expansion described as Step B in
Figure 8 (in particular, e.g.,
Figure 8A and/or Figure 8B and/or Figure 8C and/or Figure 8D)) is 8 days and
the rapid second
expansion (for example, an expansion as described in Step D in Figure 8 (in
particular, e.g., Figure 8A
and/or Figure 8B and/or Figure 8C and/or Figure 8D)) is 8 to 9 days. In some
embodiments, the
priming first expansion (for example, an expansion described as Step B in
Figure 8 (in particular, e.g.,
Figure 8A and/or Figure 8B and/or Figure 8C and/or Figure 8D)) is shortened to
7 days and the rapid
second expansion (for example, an expansion as described in Step D in Figure 8
(in particular, e.g.,
Figure 8A and/or Figure 8B and/or Figure 8C and/or Figure 8D)) is 7 to 8 days.
In some
embodiments, the priming first expansion (for example, an expansion described
as Step B in Figure 8
(in particular, e.g., Figure 8A and/or Figure 8B and/or Figure 8C and/or
Figure 8D)) is shortened to 8
days and the rapid second expansion (for example, an expansion as described in
Step D in Figure 8 (in
particular, e.g., Figure 8A and/or Figure 8B and/or Figure 8C and/or Figure
8D)) is 8 days. In some
embodiments, the priming first expansion (for example, an expansion described
as Step B in Figure 8
(in particular, e.g., Figure 8A and/or Figure 8B and/or Figure 8C and/or
Figure 8D)) is 8 days and the
rapid second expansion (for example, an expansion as described in Step D in
Figure 8 (in particular,
e.g.. Figure 8A and/or Figure 8B and/or Figure 8C and/or Figure 8D)) is 9
days. In some
embodiments, the priming first expansion (for example, an expansion described
as Step B in Figure 8
(in particular, e.g., Figure 8A and/or Figure 8B and/or Figure 8C and/or
Figure 8D)) is 8 days and the
rapid second expansion (for example, an expansion as described in Step D in
Figure 8 (in particular,
e.g., Figure 8A and/or Figure 8B and/or Figure 8C and/or Figure 8D)) is 10
days. In some
embodiments, the priming first expansion (for example, an expansion described
as Step B in Figure 8
(in particular, e.g., Figure 8A and/or Figure 8B and/or Figure 8C and/or
Figure 8D)) is 7 days and the
rapid second expansion (for example, an expansion as described in Step D in
Figure 8 (in particular,
e.g., Figure 8A and/or Figure 8B and/or Figure 8C and/or Figure 8D)) is 7 to
10 days. In some
embodiments, the priming first expansion (for example, an expansion described
as Step B in Figure 8
(in particular, e.g., Figure 8A and/or Figure 8B and/or Figure 8C and/or
Figure 8D)) is 7 days and the
rapid second expansion (for example, an expansion as described in Step D in
Figure 8 (in particular,
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e.g., Figure 8A and/or Figure 8B and/or Figure 8C and/or Figure 8D)) is 8 to
10 days. In some
embodiments, the priming first expansion (for example, an expansion described
as Step B in Figure 8
(in particular, e.g., Figure 8A and/or Figure 8B and/or Figure 8C and/or
Figure 8D)) is 7 days and the
rapid second expansion (for example, an expansion as described in Step D in
Figure 8 (in particular,
e.g., Figure 8A and/or Figure 8B and/or Figure 8C and/or Figure 8D)) is 9 to
10 days. In some
embodiments, the priming first expansion (for example, an expansion described
as Step B in Figure 8
(in particular, e.g., Figure 8A and/or Figure 8B and/or Figure 8C and/or
Figure 8D)) is shortened to 7
days and the rapid second expansion (for example, an expansion as described in
Step D in Figure 8 (in
particular, e.g., Figure 8A and/or Figure 8B and/or Figure 8C and/or Figure
8D)) is 7 to 9 days. In
some embodiments, the combination of the priming first expansion and rapid
second expansion (for
example, expansions described as Step B and Step D in Figure 8 (in particular,
e.g., Figure 1B and/or
Figure 8C)) is 14-16 days, as discussed in detail below and in the examples
and figures. Particularly,
it is considered that certain embodiments of the present invention comprise a
priming first expansion
step in which TILs are activated by exposure to an anti-CD3 antibody, e.g.,
OKT-3 in the presence of
1L-2 or exposure to an antigen in the presence of at least 1L-2 and an anti-
CD3 antibody e.g. OKT-3.
In certain embodiments, the TILs which are activated in the priming first
expansion step as described
above are a first population of TILs i.e., which are a primary cell
population.
[00647] The "Step" Designations A, B, C, etc., below are in reference to the
non-limiting example
in Figure 8 (in particular, e.g., Figure 8A and/or Figure 8B and/or Figure 8C
and/or Figure 8D) and in
terefenCe to certain non-limiting embodiments described herein. The ordering
or the Steps below and
in Figure 8 (in particular, e.g., Figure 8A and/or Figure 8B and/or Figure 8C
and/or Figure 8D) is
exemplary and any combination or order of steps, as well as additional steps,
repetition of steps,
and/or omission of steps is contemplated by the present application and the
methods disclosed herein.
A. STEP A: Obtain Patient Tumor Sample
[00648] In general, TILs are initially obtained from a patient tumor sample
("primary TILs") or
from circulating lymphocytes, such as peripheral blood lymphocytes, including
peripheral blood
lymphocytes having TIL-like characteristics, and are then expanded into a
larger population for
further manipulation as described herein, optionally cryopreserved, and
optionally evaluated for
phenotype and metabolic parameters as an indication of TIL health.
[00649] A patient tumor sample may be obtained using methods known in the art,
generally via
surgical resection, needle biopsy or other means for obtaining a sample that
contains a mixture of
tumor and TIL cells. In general, the tumor sample may be from any solid tumor,
including primary
tumors, invasive tumors or metastatic tumors. The tumor sample may also be a
liquid tumor, such as a
tumor obtained from a hematological malignancy. The solid tumor may be of any
cancer type,
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including, but not limited to, breast, pancreatic, prostate, colorectal, lung,
brain, renal, stomach, and
skin (including but not limited to squamous cell carcinoma, basal cell
carcinoma, and melanoma). In
sonic embodiments, the cancer is selected from cervical cancer, head and neck
cancer (including, for
example, head and neck squamous cell carcinoma (HNSCC)), glioblastoma (GBM),
gastrointestinal
cancer, ovarian cancer, sarcoma, pancreatic cancer, bladder cancer, breast
cancer, triple negative
breast cancer, and non-small cell lung carcinoma. In some embodiments, the
cancer is melanoma. In
some embodiments, useful TILs are obtained from malignant melanoma tumors, as
these have been
reported to have particularly high levels of TILs.
[00650] Once obtained, the tumor sample is generally fragmented using sharp
dissection into small
pieces of between 1 to about 8 mm3, with from about 2-3 mm3 being particularly
useful. The TILs are
cultured from these fragments using enzymatic tumor digests. Such tumor
digests may be produced by
incubation in enzymatic media (e.g., Roswell Park Memorial Institute (RPMI)
1640 buffer, 2 mM
glutamate, 10 mcg/mL gentamicine, 30 units/mL of DNase and 1.0 nig/mL of
collagenase) followed
by mechanical dissociation (e.g., using a tissue dissociator). Tumor digests
may be produced by
placing the tumor in enzymatic media and mechanically dissociating the tumor
for approximately 1
minute, followed by incubation for 30 minutes at 37 C in 5% CO,, followed by
repeated cycles of
mechanical dissociation and incubation under the foregoing conditions until
only small tissue pieces
are present. At the end of this process, if the cell suspension contains a
large number of red blood cells
or dead cells, a density gradient separation using FICOLL branched hydrophilic
polysaccharide may
be performed to remove these cells. Alternative methods known in the art may
be used, such as those
described in U.S. Patent Application Publication No. 2012/0244133 Al, the
disclosure of which is
incorporated by reference herein. Any of the foregoing methods may be used in
any of the
embodiments described herein for methods of expanding TILs or methods treating
a cancer.
[00651] As indicated above, in some embodiments, the TILs are derived from
solid tumors. In some
embodiments, the solid tumors are not fragmented. In some embodiments, the
solid tumors are not
fragmented and are subjected to enzymatic digestion as whole tumors. In some
embodiments, the
tumors arc digested in in an enzyme mixture comprising collagcnase, DNase, and
hyaluronidase. In
some embodiments, the tumors are digested in in an enzyme mixture comprising
collagenase, DNase,
and hyaluronidase for 1-2 hours. In some embodiments, the tumors are digested
in in an enzyme
mixture comprising collagenase, DNase, and hyaluronidase for 1-2 'hours at 37
C, 5% CO?. In some
embodiments, the tumors are digested in in an enzyme mixture comprising
collagenase, DNase, and
hyaluronidase for 1-2 hours at 37 C, 5% CO2 with rotation. In some
embodiments, the tumors are
digested overnight with constant rotation. In some embodiments, the tumors arc
digested overnight at
37 C, 5% CO2 with constant rotation. In some embodiments, the whole tumor is
combined with the
enzymes to form a tumor digest reaction mixture.
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[00652] In some embodiments, the tumor is reconstituted with the lyophilized
enzymes in a sterile
buffer. In some embodiments, the buffer is sterile HBSS.
[00653] In some embodiments, the enzyme mixture comprises collagenase. In some
embodiments,
the collagenase is collagenase IV. In some embodiments, the working stock for
the collagenase is a
100 mg/mL 10X working stock.
[00654] In some embodiments, the enzyme mixture comprises DNAse. In some
embodiments, the
working stock for the DNAse is a 10,000IU/mL 10X working stock.
[00655] In some embodiments, the enzyme mixture comprises hyaluronidase. In
some
embodiments, the working stock for the hyaluronidase is a 10-mg/mL 10X working
stock.
[00656] In some embodiments, the enzyme mixture comprises 10 mg/mL
collagenase, 1000 IU/mL
DNAse, and 1 mg/mL hyaluronidase.
[00657] In some embodiments, the enzyme mixture comprises 10 mg/mL
collagenase, 500 IU/mL
DNAse, and 1 mg/mL hyaluronidase.
[00658] In general, the cell suspension obtained from the tumor is called a
"primary cell population"
or a "freshly obtained" or a "freshly isolated" cell population_ In certain
embodiments, the freshly
obtained cell population of TILs is exposed to a cell culture medium
comprising antigen presenting
cells, IL-12 and OKT-3.
[00659] In some embodiments, fragmentation includes physical fragmentation,
including, for
example, dissection as well as digestion. In some embodiments, the
fragmentation is physical
fragmentation. In some embodiments, the fragmentation is dissection. In some
embodiments, the
fragmentation is by digestion. In some embodiments, TILs can be initially
cultured from enzymatic
tumor digests and tumor fragments obtained from patients. In some embodiments,
TILs can be
initially cultured from enzymatic tumor digests and tumor fragments obtained
from patients.
[00660] In some embodiments, where the tumor is a solid tumor, the tumor
undergoes physical
fragmentation after the tumor sample is obtained in, for example, Step A (as
provided in Figure 8 (in
particular, e.g., Figure 8A and/or Figure 8B and/or Figure 8C and/or Figure
8D)). In some
embodiments, the fragmentation occurs before cryopreservation. In some
embodiments, the
fragmentation occurs after cryopreservation. In some embodiments, the
fragmentation occurs after
obtaining the tumor and in the absence of any cryopreservation. In some
embodiments, the step of
fragmentation is an in vitro or ex-vivo process. In some embodiments, the
tumor is fragmented and 10,
20, 30, 40 or more fragments or pieces arc placed in each container for the
priming first expansion. In
some embodiments, the tumor is fragmented and 30 or 40 fragments or pieces are
placed in each
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container for the priming first expansion. In some embodiments, the tumor is
fragmented and 40
fragments or pieces are placed in each container for the priming first
expansion. In some
embodiments, the multiple fragments comprise about 4 to about 50 fragments,
wherein each fragment
has a volume of about 27 mm3. In some embodiments, the multiple fragments
comprise about 30 to
about 60 fragments with a total volume of about 1300 mm3 to about 1500 mm3. In
some
embodiments, the multiple fragments comprise about 50 fragments with a total
volume of about 1350
mm3. In some embodiments, the multiple fragments comprise about 50 fragments
with a total mass of
about 1 gram to about 1.5 grams. In some embodiments, the multiple fragments
comprise about 4
fragments.
[00661] In some embodiments, the TILs are obtained from tumor fragments. In
some embodiments,
the tumor fragment is obtained by sharp dissection. In some embodiments, the
tumor fragment is
between about 1 mm3 and 10 mm3. In some embodiments, the tumor fragment is
between about 1
mms and 8 min3. In some embodiments, the tumor fragment is about 1 mm3. In
some embodiments,
the tumor fragment is about 2 mm3. In some embodiments, the tumor fragment is
about 3 mm3. In
some embodiments, the tumor fragment is about 4 mm3. In some embodiments, the
tumor fragment is
about 5 mm3. In some embodiments, the tumor fragment is about 6 mm3. In some
embodiments, the
tumor fragment is about 7 mm3. In some embodiments, the tumor fragment is
about 8 mm3. In some
embodiments, the tumor fragment is about 9 mm3. In some embodiments, the tumor
fragment is about
mm3. In some embodiments, the tumor fragments are 1-4 mm " 1-4 mm x 1-4 mm. In
some
embodiments, the tumor fragments are 1 mm " 1 mm A 1 mm. In some embodiments,
the tumor
fragments are 2 mm 2 mm x 2 mm. In some embodiments, the tumor fragments are 3
mm '3 mm x 3
mm. In some embodiments, the tumor fragments are 4 mm 4 mm x 4 mm.
[00662] In some embodiments, the tumors are fragmented in order to minimize
the amount of
hemorrhagic, necrotic, and/or fatty tissues on each piece. In some
embodiments, the tumors are
fragmented in order to minimize the amount of hemorrhagic tissue on each
piece. In some
embodiments, the tumors are fragmented in order to minimize the amount of
necrotic tissue on each
piece. In some embodiments, the tumors are fragmented in order to minimize the
amount of fatty
tissue on each piece. In certain embodiments, the step of fragmentation of the
tumor is an in vitro or
ex-vivo method.
[00663] In some embodiments, the tumor fragmentation is performed in order to
maintain the tumor
internal structure. In some embodiments, the tumor fragmentation is performed
without performing a
sawing motion with a scalpel. In some embodiments, the TILs are obtained from
tumor digests. In
some embodiments, tumor digests were generated by incubation in enzyme media,
for example but
not limited to RPMI 1640, 2 mM GlutaMAX, 10 mg/mL gentamicin, 30 U/mL DNase,
and 1.0
mg/mL collagenase, followed by mechanical dissociation (GentleMACS, Miltenyi
Biotec, Auburn,
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CA). After placing the tumor in enzyme media, the tumor can be mechanically
dissociated for
approximately 1 minute. The solution can then be incubated for 30 minutes at
37 C in 5% CO2 and
it then mechanically disrupted again for approximately 1 minute. After being
incubated again for 30
minutes at 37 'V in 5% CO2, the tumor can be mechanically disrupted a third
time for
approximately 1 minute. In some embodiments, after the third mechanical
disruption if large pieces
of tissue were present, 1 or 2 additional mechanical dissociations were
applied to the sample, with
or without 30 additional minutes of incubation at 37 C in 5% CO2. In some
embodiments, at the
end of the final incubation if the cell suspension contains a large number of
red blood cells or dead
cells, a density gradient separation using Ficoll can be performed to remove
these cells.
[00664] In some embodiments, the cell suspension prior to the priming first
expansion step is called
a "primary cell population" or a "freshly obtained" or "freshly isolated" cell
population.
[00665] In some embodiments, cells can be optionally frozen after sample
isolation (e.g., after
obtaining the tumor sample and/or after obtaining the cell suspension from the
tumor sample) and
stored frozen prior to entry into the expansion described in Step B, which is
described in further detail
below, as well as exemplified in Figure 8 (in particular, e.g., Figure 8B).
1. Core/Small Biopsy Derived TILs
1006661 In some embodiments, TILs are initially obtained from a patient tumor
sample ("primary
TILs") obtained by a core biopsy or similar procedure and then expanded into a
larger population for
further manipulation as described herein, optionally cryopreserved, and
optionally evaluated for
phenotype and metabolic parameters.
[00667] In some embodiments, a patient tumor sample may be obtained using
methods known in the
art, generally via small biopsy, core biopsy, needle biopsy or other means for
obtaining a sample that
contains a mixture of tumor and TIL cells. In general, the tumor sample may be
from any solid tumor,
including primary tumors, invasive tumors or metastatic tumors. The tumor
sample may also be a
liquid tumor, such as a tumor obtained from a hematological malignancy. In
some embodiments, the
sample can be from multiple small tumor samples or biopsies. In some
embodiments, the sample can
comprise multiple tumor samples from a single tumor from the same patient. In
some embodiments,
the sample can comprise multiple tumor samples from one, two, three, or four
tumors from the same
patient. In some embodiments, the sample can comprise multiple tumor samples
from multiple tumors
from the same patient. The solid tumor may of lung and/or non-small cell lung
carcinoma (NSCLC).
[00668] In general, the cell suspension obtained from the tumor core or
fragment is called a
"primary cell population" or a "freshly obtained" or a "freshly isolated" cell
population. In certain
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embodiments, the freshly obtained cell population of TILs is exposed to a cell
culture medium
comprising antigen presenting cells, IL-2 and OKT-3.
[00669] In some embodiments, if the tumor is metastatic and the primary lesion
has been efficiently
treated/removed in the past, removal of one of the metastatic lesions may be
needed. In some
embodiments, the least invasive approach is to remove a skin lesion, or a
lymph node on the neck or
axillary area when available. In some embodiments, a skin lesion is removed or
small biopsy thereof
is removed. In some embodiments, a lymph node or small biopsy thereof is
removed. In some
embodiments, a lung or liver metastatic lesion, or an intra-abdom inal or
thoracic lymph node or small
biopsy is removed. In some embodiments, the tumor is a melanoma. In some
embodiments, the small
biopsy for a melanoma comprises a mole or portion thereof.
[00670] In some embodiments, the small biopsy is a punch biopsy. In some
embodiments, the punch
biopsy is obtained with a circular blade pressed into the skin. In some
embodiments, the punch biopsy
is obtained with a circular blade pressed into the skin, around a suspicious
mole. In some
embodiments, the punch biopsy is obtained with a circular blade pressed into
the skin, and a round
piece of skin is removed. In some embodiments, the small biopsy is a punch
biopsy and round portion
of the tumor is removed.
[00671] In some embodiments, the small biopsy is an excisional biopsy. In some
embodiments, the
small biopsy is an excisional biopsy and the entire mole or growth is removed.
In some embodiments,
the small biopsy is an excisional biopsy and the entire mole or growth is
removed along with a small
border of normal-appearing skin.
[00672] In some embodiments, the small biopsy is an incisional biopsy. In some
embodiments, the
small biopsy is an incisional biopsy and only the most irregular part of a
mole or growth is taken. In
some embodiments, the small biopsy is an incisional biopsy and the incisional
biopsy is used when
other techniques can't be completed, such as if a suspicious mole is very
large.
[00673] In some embodiments, the small biopsy is a lung biopsy. In some
embodiments, the small
biopsy is obtained by bronchoscopy. Generally, bronchoscopy, the patient is
put under anesthesia, and
a small tool goes through the nose or mouth, down the throat, and into the
bronchial passages, where
small tools are used to remove some tissue. In some embodiments, where the
tumor or growth cannot
be reached via bronchoscopy, a transthoracic needle biopsy can be employed.
Generally, for a
transthoracic needle biopsy, the patient is also under anesthesia and a needle
is inserted through the
skin directly into the suspicious spot to remove a small sample of tissue. In
some embodiments, a
transthoracic needle biopsy may require interventional radiology (for example,
the use of x-rays or CT
scan to guide the needle). In some embodiments, the small biopsy is obtained
by needle biopsy. In
some embodiments, the small biopsy is obtained endoscopic ultrasound (for
example, an endoscope
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with a light and is placed through the mouth into the esophagus). In some
embodiments, the small
biopsy is obtained surgically.
[00674] In some embodiments, the small biopsy is a head and neck biopsy. In
some embodiments,
the small biopsy is an incisional biopsy. In some embodiments, the small
biopsy is an incisional
biopsy, wherein a small piece of tissue is cut from an abnormal-looking area.
In some embodiments, if
the abnormal region is easily accessed, the sample may be taken without
hospitalization. In some
embodiments, if the tumor is deeper inside the mouth or throat, the biopsy may
need to be done in an
operating room, with general anesthesia. In some embodiments, the small biopsy
is an excisional
biopsy. In some embodiments, the small biopsy is an excisional biopsy, wherein
the whole area is
removed. In some embodiments, the small biopsy is a fine needle aspiration
(FNA). In some
embodiments, the small biopsy is a fine needle aspiration (FNA), wherein a
very thin needle attached
to a syringe is used to extract (aspirate) cells from a tumor or lump. In some
embodiments, the small
biopsy is a punch biopsy. In some embodiments, the small biopsy is a punch
biopsy, wherein punch
forceps are used to remove a piece of the suspicious area.
[00675] In some embodiments, the small biopsy is a cervical biopsy. In some
embodiments, the
small biopsy is obtained via colposcopy. Generally, colposcopy methods employ
the use of a lighted
magnifying instrument attached to magnifying binoculars (a colposcope) which
is then used to biopsy
a small section of the surface of the cervix. In some embodiments, the small
biopsy is a
conization/cone biopsy. In some embodiments, the small biopsy is a
conization/cone biopsy, wherein
an outpatient surgery may be needed to remove a larger piece of tissue from
the cervix. In some
embodiments, the cone biopsy, in addition to helping to confirm a diagnosis, a
cone biopsy can serve
as an initial treatment.
[00676] The term "solid tumor" refers to an abnormal mass of tissue that
usually does not contain
cysts or liquid areas. Solid tumors may be benign or malignant. The term -
solid tumor cancer refers to
malignant, neoplastic, or cancerous solid tumors. Solid tumor cancers include
cancers of the lung. In
some embodiments, the cancer is melanoma. In some embodiments,the cancer is
non-small cell lung
carcinoma (NSCLC). The tissue structure of solid tumors includes
interdependent tissue
compartments including the parenchyma (cancer cells) and the supporting
stromal cells in which the
cancer cells are dispersed and which may provide a supporting
microenvironment.
[00677] In some embodiments, the sample from the tumor is obtained as a fine
needle aspirate
(FNA), a core biopsy, a small biopsy (including, for example, a punch biopsy).
In some embodiments,
sample is placed first into a G-REX 10. In some embodiments, sample is placed
first into a G-REX 10
when there are 1 or 2 core biopsy and/or small biopsy samples. In some
embodiments, sample is
placed first into a G-REX 100 when there are 3, 4, 5, 6, 8, 9, or 10 or more
core biopsy and/or small
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biopsy samples. In some embodiments, sample is placed first into a G-REX 500
when there are 3, 4,
5, 6, 8, 9, or 10 or more core biopsy and/or small biopsy samples.
[00678] The FNA can be obtained from a skin tumor, including, for example, a
melanoma. In some
embodiments, the FNA is obtained from a skin tumor, such as a skin tumor from
a patient with
metastatic melanoma. In some cases, the patient with melanoma has previously
undergone a surgical
treatment.
[00679] The FNA can be obtained from a lung tumor, including, for example, an
NSCLC. In some
embodiments, the FNA is obtained from a lung tumor, such as a lung tumor from
a patient with non-
small cell lung cancer (NSCLC). In some cases, the patient with NSCLC has
previously undergone a
surgical treatment.
[00680] TILs described herein can be obtained from an FNA sample. In some
cases, the FNA
sample is obtained or isolated from the patient using a fine gauge needle
ranging from an 18 gauge
needle to a 25 gauge needle. The fine gauge needle can be 18 gauge, 19 gauge,
20 gauge, 21 gauge,
22 gauge, 23 gauge, 24 gauge, or 25 gauge. In some embodiments, the FNA sample
from the patient
can contain at least 400,000 TILs, e.g., 400,000 TILs, 450,000 TILs, 500,000
TILs, 550,000 TILs,
600,000 TILs, 650,000 TILs, 700,000 TILs, 750,000 'TILs, 800,000 Tits, 850,000
TILs, 900,000
TILs, 950,000 TILs, or more.
[00681] In some cases, the TILs described herein are obtained from a core
biopsy sample. In some
cases, the core biopsy sample is obtained or isolated from the patient using a
surgical or medical
needle ranging from an 11 gauge needle to a 16 gauge needle. The needle can be
11 gauge, 12 gauge,
13 gauge, 14 gauge, 15 gauge, or 16 gauge. In some embodiments, the core
biopsy sample from the
patient can contain at least 400,000 TILs, e.g., 400,000 TILs, 450,000 IILs,
500,000 TILs, 550,000
TILs, 600,000 TILs, 650,000 TILs, 700,000 TILs, 750,000 TILs, 800,000 TILs,
850,000 TILs.
900,000 TILs, 950,000 TILs, or more.
[00682] In general, the harvested cell suspension is called a "primary cell
population" or a "freshly
harvested" cell population.
[00683] In some embodiments, the TILs are not obtained from tumor digests. In
some embodiments,
the solid tumor cores are not fragmented.
[00684] In some embodiments, the TILs are obtained from tumor digests. In some
embodiments,
tumor digests were generated by incubation in enzyme media, for example but
not limited to RPMI
1640, 2mM GlutaMAX, 10 mg/mL gentamicin, 30 U/mL DNase, and 1.0 mg/mL
collagenase, fol-
lowed by mechanical dissociation (GentleMACS, Miltenyi Biotec, Auburn, CA).
After placing the
tumor in enzyme media, the tumor can be mechanically dissociated for
approximately 1 minute. The
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solution can then be incubated for 30 minutes at 37 C in 5% CO2 and it then
mechanically disrupted
again for approximately 1 minute. After being incubated again for 30 minutes
at 37 C in 5% CO2, the
tumor can be mechanically disrupted a third time for approximately 1 minute.
In some embodiments,
after the third mechanical disruption if large pieces of tissue were present,
1 or 2 additional
mechanical dissociations were applied to the sample, with or without 30
additional minutes of
incubation at 37 C in 5% CO2. In some embodiments, at the end of the final
incubation if the cell
suspension contained a large number of red blood cells or dead cells, a
density gradient separation
using Ficoll can be performed to remove these cells.
[00685] In some embodiments, obtaining the first population of TILs comprises
a multilesional
sampling method.
[00686] Tumor dissociating enzyme mixtures can include one or more
dissociating (digesting)
enzymes such as, but not limited to, collagenase (including any blend or type
of collagenase),
AccutaseTM, AccumaxTm, hyaktronidase, neutral protease (dispase),
chymotrypsin, chymopapain,
trypsin, caseinase, elastase, papain, protease type XIV (pronase),
deoxyribonuclease I (DNase),
trypsin inhibitor, any other dissociating or proteolytic enzyme, and any
combination thereof
[00687] hi some embodiments, the dissociating enzymes are reconstituted from
lyophilized
enzymes. In some embodiments, lyophilized enzymes are reconstituted in an
amount of sterile buffer
such as Hank's balance salt solution (HBSS).
[00688] In some instances, collagenasc (such as animal free- type 1
collagenasc) is reconstituted in
mL of sterile HBSS or another buffer. The lyophilized stock enzyme may be at a
concentration of
2892 PZ U/vial. In some embodiments, collagenase is reconstituted in 5 mL to
15 mL buffer. In some
embodiment, after reconstitution the collagenase stock ranges from about 100
PZ U/mL-about 400 PZ
U/mL, e.g., about 100 PZ U/mL-about 400 PZ U/mL, about 100 PZ U/mL-about 350
PZ U/mL, about
100 PZ U/mL-about 300 PZ U/mL, about 150 PZ U/mL-about 400 PZ U/mL, about 100
PZ U/mL,
about 150 PZ U/mL, about 200 PZ U/mL, about 210 PZ U/mL, about 220 PZ U/mL,
about 230 PZ
U/mL, about 240 PZ U/mL, about 250 PZ U/mL, about 260 PZ U/mL, about 270 PZ
U/mL, about 280
PZ U/mL, about 289.2 PZ U/mL, about 300 PZ U/mL, about 350 PZ U/mL, or about
400 PZ U/mL.
[00689] In some embodiments neutral protease is reconstituted in 1-ml of
sterile HBSS or another
buffer. The lyophilized stock enzyme may be at a concentration of 175 DMC
U/vial. In some
embodiments, after reconstitution the neutral protease stock ranges from about
100 DMC/mL-about
400 DMC/mL, e.g., about 100 DMC/mL-about 400 DMC/mL, about 100 DMC/mL-about
350
DMC/mL, about 100 DMC/mL-about 300 DMC/mL, about 150 DMC/mL-about 400 DMC/mL,
about
100 DMC/mL, about 110 DMC/mL, about 120 DMC/mL, about 130 DMC/mL, about 140
DMC/mL,
about 150 DMC/mL, about 160 DMC/mL, about 170 DMC/mL, about 175 DMC/mL, about
180
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DMC/mL, about 190 DMC/mL, about 200 DMC/mL, about 250 DMC/mL, about 300
DMC/mL,
about 350 DMC/mL, or about 400 DMC/mL.
[00690] In some embodiments, DNAse I is reconstituted in 1-ml of sterile HBSS
or another buffer.
The lyophilized stock enzyme was at a concentration of 4 KU/vial. In some
embodiments, after
reconstitution the DNase I stock ranges from about 1 KU/mL-10 KU/mL, e.g.,
about 1 KU/mL, about
2 KU/mL, about 3 KU/mL, about 4 KU/mL, about 5 KU/mL, about 6 KU/mL, about 7
KU/mL, about
8 KU/mL, about 9 KU/mL, or about 10 KU/mL.
[00691] In some embodiments, the stock of enzymes could change so verify the
concentration of the
lyophilized stock and amend the final amount of enzyme added to the digest
cocktail accordingly.
[00692] In some embodiments, the enzyme mixture includes about 10.2-ul of
neutral protease (0.36
DMC U/mL), 21.3-ul of collagenase (1.2 PZ/mL) and 250-ul of DNAse 1(200 U/mL)
in about 4.7-ml
of sterile HBSS.
2. Pleural effusion T-cells and TILs
[00693] In some embodiments, the sample is a pleural fluid sample. In some
embodiments, the
source of the T-cells or TILs for expansion according to the processes
described herein is a pleural
fluid sample. In some embodiments, the sample is a pleural effusion derived
sample. In some
embodiments, the source of the T-cells or TILs for expansion according to the
processes described
herein is a pleural effusion derived sample. See, for example, methods
described in U.S. Patent
Publication US 2014/0295426, incorporated herein by reference in its entirety
for all purposes.
[00694] In some embodiments, any pleural fluid or pleural effusion suspected
of and/or containing
TILs can be employed. Such a sample may be derived from a primary or
metastatic lung cancer, such
as NSCLC or SCLC. In some embodiments, the sample may be derived from
secondary metastatic
cancer cells which originated from another organ, e.g., breast, ovary, colon
or prostate. In some
embodiments, the sample for use in the expansion methods described herein is a
pleural exudate. In
some embodiments, the sample for use in the expansion methods described herein
is a pleural
transudate. Other biological samples may include other serous fluids
containing TILs, including, e.g.,
ascites fluid from the abdomen or pancreatic cyst fluid. Ascites fluid and
pleural fluids involve very
similar chemical systems; both the abdomen and lung have mesothelial lines and
fluid forms in the
pleural space and abdominal spaces in the same matter in malignancies and such
fluids in some
embodiments contain TILs. In some embodiments, wherein the disclosed methods
utilize pleural
fluid, the same methods may be performed with similar results using ascites or
other cyst fluids
containing TILs.
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[00695] In some embodiments, the pleural fluid is in unprocessed form,
directly as removed from
the patient. In some embodiments, the unprocessed pleural fluid is placed in a
standard blood
collection tube, such as an EDTA or Heparin tube, prior to further processing
steps. In some
embodiments, the unprocessed pleural fluid is placed in a standard CellSave
tube (Veridex) prior to
the further processing steps. In some embodiments, the sample is placed in the
CellSave tube
immediately after collection from the patient to avoid a decrease in the
number of viable TILs. The
number of viable TILs can decrease to a significant extent within 24 hours, if
left in the untreated
pleural fluid, even at 4 C. In some embodiments, the sample is placed in the
appropriate collection
tube within 1 hour, 5 hours, 10 hours, 15 hours, or up to 24 hours after
removal from the patient. In
some embodiments, the sample is placed in the appropriate collection tube
within 1 hour, 5 hours, 10
hours, 15 hours, or up to 24 hours after removal from the patient at 4 C.
[00696] In some embodiments, the pleural fluid sample from the chosen subject
may be diluted. In
one embodiment, the dilution is 1:10 pleural fluid to diluent. In some
embodiments, the dilution is 1:9
pleural fluid to diluent. In some embodiments, the dilution is 1:8 pleural
fluid to diluent. In some
embodiments, the dilution is 1:5 pleural fluid to diluent. in some
embodiments, the dilution is 1:2
pleural fluid to diluent. In some embodiments, the dilution is 1:1 pleural
fluid to diluent. In some
embodiments, diluents include saline, phosphate buffered saline, another
buffer or a physiologically
acceptable diluent. In some embodiments, the sample is placed in the CellSave
tube immediately after
collection from the patient and dilution to avoid a decrease in the viable
TILs, which may occur to a
significant extent within 24-48 hours, if left in the untreated pleural fluid,
even at 4 C. In some
embodiments, the pleural fluid sample is placed in the appropriate collection
tube within 1 hour, 5
hours, 10 hours, 15 hours, 24 hours, 36 hours, up to 48 hours after removal
from the patient, and
dilution. In some embodiments, the pleural fluid sample is placed in the
appropriate collection tube
within 1 hour, 5 hours, 10 hours, 15 hours, 24 hours, 36 hours, up to 48 hours
after removal from the
patient, and dilution at 4 C.
[00697] In still another embodiment, pleural fluid samples are concentrated by
conventional means
prior to further processing steps. In some embodiments, this pre-treatment of
the pleural fluid is
preferable in circumstances in which the pleural fluid must be cryopreserved
for shipment to a
laboratory performing the method or for later analysis (e.g., later than 24-48
hours post-collection). In
some embodiments, the pleural fluid sample is prepared by centrifuging the
pleural fluid sample after
its withdrawal from the subject and resuspending the centrifugate or pellet in
buffer. In some
embodiments, the pleural fluid sample is subjected to multiple centrifugations
and resuspensions,
before it is cryopreserved for transport or later analysis and/or processing.
[00698] In some embodiments, pleural fluid samples are concentrated prior to
further processing
steps by using a filtration method. In some embodiments, the pleural fluid
sample used in the further
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processing, is prepared by filtering the fluid through a filter containing a
known and essentially
uniform pore size that allows for passage of the pleural fluid through the
membrane but retains the
tumor cells, hi some embodiments, the diameter of the pores in the membrane
may be at least 4 M.
In some embodiments, the pore diameter may be 5 RM or more, and in other
embodiment, any of 6, 7,
8, 9, or 10 viM. After filtration, the cells, including TILs, retained by the
membrane may be rinsed off
the membrane into a suitable physiologically acceptable buffer. Cells,
including Ls, concentrated in
this way may then be used in the further processing steps of the method.
[00699] In some embodiment, pleural fluid sample (including, for example, the
untreated pleural
fluid), diluted pleural fluid, or the resuspended cell pellet, is contacted
with a lytic reagent that
differentially lyses non-nucleated red blood cells present in the sample. In
some embodiments, this
step is performed prior to further processing steps in circumstances in which
the pleural fluid contains
substantial numbers of RBCs. Suitable lysing reagents include a single lytic
reagent or a lytic reagent
and a quench reagent, or a lytic agent, a quench reagent and a fixation
reagent. Suitable lytic systems
are marketed commercially and include the BD Pharm LyseTM system (Becton
Dickenson). Other
lytic systems include the VersalyseTM system, the FACSlyseTM system (Becton
Dickenson), the
ImmunoprepTm system or Erythrolyse 11 system (Beckman Coulter, Inc.), or an
ammonium chloride
system. In some embodiments, the lytic reagent can vary with the primary
requirements being
efficient lysis of the red blood cells, and the conservation of the TILs and
phenotypic properties of the
TILs in the pleural fluid. In addition to employing a single reagent for
lysis, the lytic systems useful in
methods described herein can include a second reagent, e.g., one that quenches
or retards the effect of
the lytic reagent during the remaining steps of the method, e.g., Stabilyse TM
reagent (Beckman
Coulter, Inc.). A conventional fixation reagent may also be employed depending
upon the choice of
lytic reagents or the preferred implementation of the method.
[00700] In some embodiments, the pleural fluid sample, unprocessed, diluted or
multiply
centrifuged or processed as described herein above is cryopreserved at a
temperature of about ¨140 C
prior to being further processed and/or expanded as provided herein.
Methods of Expanding Peripheral Blood Lymphocytes (PBLs) from Peripheral Blood
[00701] PBL Method 1. In some embodiments of the invention, PBLs arc expanded
using the
processes described herein. In some embodiments of the invention, the method
comprises obtaining a
PBMC sample from whole blood_ In some embodiments, the method comprises
enriching T-cells by
isolating pure T-cells from PBMCs using negative selection of a non-CD19+
fraction. In some
embodiments, the method comprises enriching T-cells by isolating pure T-cells
from PBMCs using
magnetic bead-based negative selection of a non-CD19+ fraction.
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[00702] In some embodiments of the invention, PBL Method 1 is performed as
follows: On Day 0, a
cryopreserved PBMC sample is thawed and PBMCs are counted. T-cells are
isolated using a Human
Pan T-Cell Isolation Kit and LS columns (Miltenyi Biotec).
[00703] PBL Method 2. In some embodiments of the invention, PBLs are expanded
using PBL
Method 2, which comprises obtaining a PBMC sample from whole blood. The T-
cells from the
PBMCs are enriched by incubating the PBMCs for at least three hours at 37 C
and then isolating the
non-adherent cells.
[00704] In some embodiments of the invention, PBL Method 2 is performed as
follows: On Day 0,
the cryopreserved PMBC sample is thawed and the PBMC cells are seeded at 6
million cells per well
in a 6 well plate in CM-2 media and incubated for 3 hours at 37 degrees
Celsius. After 3 hours, the
non-adherent cells, which are the PBLs, are removed and counted.
[00705] PBL Method 3. In some embodiments of the invention, PBLs are expanded
using PBL
Method 3, which comprises obtaining a PBMC sample from peripheral blood. B-
cells are isolated
using a CD19+ selection and T-cells are selected using negative selection of
the non-CD19+ fraction
of the PBMC sample.
[00706] In some embodiments of the invention, PBL Method 3 is performed as
follows: On Day 0,
cryopreserved PBMCs derived from peripheral blood are thawed and counted.
CD19+ B-cells are
sorted using a CD19 Multisort Kit, Human (Miltenyi Biotcc). Of the non-CD19+
cell fraction, T-cells
are purified using the Human Pan T-cell Isolation Kit and LS Columns (Miltenyi
Biotec).
[00707] In some embodiments, PBMCs are isolated from a whole blood sample. In
some
embodiments, the PBMC sample is used as the starting material to expand the
PBLs. In some
embodiments, the sample is cryopreserved prior to the expansion process. In
other embodiments, a
fresh sample is used as the starting material to expand the PBLs. In some
embodiments of the
invention, T-cells are isolated from PBMCs using methods known in the art. In
some embodiments,
the T-cells are isolated using a Human Pan T-cell isolation kit and LS
columns. In some embodiments
of the invention, T-cells are isolated from PBMCs using antibody selection
methods known in the art,
for example, CD19 negative selection.
[00708] In some embodiments of the invention, the PBMC sample is incubated for
a period of time
at a desired temperature effective to identify the non-adherent cells. In some
embodiments of the
invention, the incubation time is about 3 hours. In some embodiments of the
invention, the
temperature is about 37 Celsius. The non-adherent cells are then expanded
using the process
described above.
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[00709] In some embodiments, the PBMC sample is from a subject or patient who
has been
optionally pre-treated with a regimen comprising a kinase inhibitor or an ITK
inhibitor. In some
embodiments, the tumor sample is from a subject or patient who has been pre-
treated with a regimen
comprising a kinase inhibitor or an ITK inhibitor. In some embodiments, the
PBMC sample is from a
subject or patient who has been pre-treated with a regimen comprising a kinase
inhibitor or an ITK
inhibitor, has undergone treatment for at least 1 month, at least 2 months, at
least 3 months, at least 4
months, at least 5 months, at least 6 months, or 1 year or more. In other
embodiments, the PBMCs are
derived from a patient who is currently on an ITK inhibitor regimen, such as
ibrutinib.
[00710] In some embodiments, the PBMC sample is from a subject or patient who
has been pre-
treated with a regimen comprising a kinase inhibitor or an ITK inhibitor and
is refractory to treatment
with a kinase inhibitor or an ITK inhibitor, such as ibrutinib.
[00711] In some embodiments, the PBMC sample is from a subject or patient who
has been pre-
treated with a regimen comprising a kinase inhibitor or an ITK inhibitor but
is no longer undergoing
treatment with a kinase inhibitor or an ITK inhibitor. In some embodiments,
the PBMC sample is
from a subject or patient who has been pre-treated with a regimen comprising a
kinase inhibitor or an
ITK inhibitor but is no longer undergoing treatment with a kinase inhibitor or
an ITK inhibitor and
has not undergone treatment for at least 1 month, at least 2 months, at least
3 months, at least 4
months, at least 5 months, at least 6 months, or at least 1 year or more. In
other embodiments, the
PBMCs are derived from a patient who has prior exposure to an ITK inhibitor,
but has not been
treated in at least 3 months, at least 6 months, at least 9 months, or at
least 1 year.
[00712] In some embodiments of the invention, at Day 0, cells are selected for
CD19+ and sorted
accordingly. In some embodiments of the invention, the selection is made using
antibody binding
beads. In some embodiments of the invention, pure T-cells are isolated on Day
0 from the PBMCs.
[00713] In some embodiments of the invention, for patients that are not pre-
treated with ibrutinib or
other ITK inhibitor, 10-15 mL of Buffy Coat will yield about 5 x109 PBMC,
which, in turn, will yield
about 5.5 x107 PBLs.
[00714] In some embodiments of the invention, for patients that are pre-
treated with ibrutinib or
other ITK inhibitor, the expansion process will yield about 20x 109 PBLs. In
some embodiments of the
invention, 40.3x 106 PBMCs will yield about 4.7x 105PBLs.
[00715] In any of the foregoing embodiments, PBMCs may be derived from a whole
blood sample,
by apheresis, from the buffy coat, or from any other method known in the art
for obtaining PBMCs.
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[00716] In some embodiments, PBLs are prepared using the methods described in
U.S. Patent
Application Publication No. US 2020/0347350 Al, the disclosures of which are
incorporated by
reference herein.
2. Methods of Expanding Marrow Infiltrating Lymphocytes
(MILs) from PBMCs
Derived from Bone Marrow
[00717] MIL Method 3. In some embodiments of the invention, the method
comprises obtaining
PBMCs from the bone marrow. On Day 0, the PBMCs are selected for
CD3+/CD33+/CD20+/CD14+
and sorted, and the non-CD3+/CD33+/CD20+/CD14+ cell fraction is sonicated and
a portion of the
sonicated cell fraction is added back to the selected cell fraction.
[00718] In some embodiments of the invention, MIL Method 3 is performed as
follows: On Day 0, a
cryopre served sample of PBMCs is thawed and PBMCs are counted. The cells are
stained with CD3,
CD33, CD20, and CD14 antibodies and sorted using a S3e cell sorted (Bio-Rad).
The cells are sorted
into two fractions ¨ an immune cell fraction (or the MIL fraction)
(CD3+CD33+CD2O+CD14+) and
an AML blast cell fraction (non-CD3+CD33+CD2O+CD14+).
[00719] In some embodiments of the invention, PBMCs are obtained from bone
marrow. In some
embodiments, the PBMCs arc obtained from the bone marrow through apheresis,
aspiration, needle
biopsy, or other similar means known in the art. In some embodiments, the
PBMCs are fresh. In other
embodiments, the PBMCs are cryopresenTed.
[00720] In some embodiments of the invention, MILs are expanded from 10-50 mL
of bone marrow
aspirate. In some embodiments of the invention, 10 mL of bone marrow aspirate
is obtained from the
patient. In other embodiments, 20 mL of bone marrow aspirate is obtained from
the patient. In other
embodiments, 30 mL of bone marrow aspirate is obtained from the patient. In
other embodiments, 40
mL of bone marrow aspirate is obtained from the patient. In other embodiments,
50 mL of bone
marrow aspirate is obtained from the patient.
[00721] In some embodiments of the invention, the number of PBMCs yielded from
about 10-50
mL of bone marrow aspirate is about 5 x107to about 10x107PBMCs. In other
embodiments, the
number of PMBCs yielded is about 7 x 10 PBMCs.
[00722] In some embodiments of the invention, about 5 x107to about
10x107PBMCs, yields about
0.5 x106to about 1.5 x 106 MILs. In some embodiments of the invention, about
lx106MILs is yielded.
[00723] In some embodiments of the invention, 12x 106 PBMC derived from bone
marrow aspirate
yields approximately 1.4x105 MILs.
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[00724] In any of the foregoing embodiments, PBMCs may be derived from a whole
blood sample,
from bone marrow, by apheresis, from the buffy coat, or from any other method
known in the art for
obtaining PBMCs.
[00725] In some embodiments, MILs are prepared using the methods described in
U.S. Patent
Application Publication No. US 2020/0347350 Al, the disclosures of which are
incorporated by
reference herein.
B. STEP B: Priming First Expansion
[00726] In some embodiments, the present methods provide for younger TILs,
which may provide
additional therapeutic benefits over older TILs (i.e., TILs which have further
undergone more rounds
of replication prior to administration to a subject/patient). Features of
young TILs have been described
in the literature, for example Donia, etal., S'cand. .1. Immunol. 2012, 75,
157-167; Dudley, et al., (7/in.
Cancer Res. 2010,16, 6122-6131; Huang, et al., J. Immunother. 2005, 28, 258-
267; Besser, etal.,
Cl/n. Cancer Res 2013, 19, OF1-0F9; Besser, et al õI Immunother. 2009, 32:415-
423; Robbins, et
al., J. Immunol. 2004, 173, 7125-7130; Shen, et al., J. Immunother., 2007, 30,
123-129; Zhou, et al.,
Immunother. 2005, 28, 53-62; and Tran, etal., I Immunother., 2008, 3/, 742-
751, each of which
is incorporated herein by reference.
[00727] After dissection or digestion of tumor fragments and/or tumor
fragments, for example such
as described in Step A of Figure 8 (in particular, e.g., Figure 8A and/or
Figure 8B and/or Figure 8C
and/or Figure 8D), the resulting cells are cultured in serum containing IL-2,
OKT-3, and feeder cells
(e.g., antigen-presenting feeder cells), under conditions that favor the
growth of TILs over tumor and
other cells. In some embodiments, the IL-2, OKT-3, and feeder cells are added
at culture initiation
along with the tumor digest and/or tumor fragments (e.g., at Day 0). In some
embodiments, the tumor
digests and/or tumor fragments are incubated in a container with up to 60
fragments per container and
with 6000 IU/mL of IL-2. In some embodiments, this primary cell population is
cultured for a period
of days, generally from 1 to 8 days, resulting in a bulk TIL population,
generally about 1 x 10s bulk
TIL cells. In some embodiments, this primary cell population is cultured for a
period of days,
generally from Ito 7 days, resulting in a bulk TIL population, generally about
1 x 108 bulk TIL cells.
In some embodiments, priming first expansion occurs for a period of 1 to 8
days, resulting in a bulk
TIL population, generally about 1 x 108 bulk TEL cells. In some embodiments,
priming first expansion
occurs for a period of 1 to 7 days, resulting in a bulk TIL population,
generally about 1 x 108 bulk TIL
cells. In some embodiments, this priming first expansion occurs for a period
of 5 to 8 days, resulting
in a bulk TIL population, generally about 1 x 108 bulk TIL cells. In some
embodiments, this priming
first expansion occurs for a period of 5 to 7 days, resulting in a bulk T1L
population, generally about 1
x 108 bulk TIL cells. In some embodiments, this priming first expansion occurs
for a period of about 6
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to 8 days, resulting in a bulk TIL population, generally about 1 x 10' bulk
TIL cells. In some
embodiments, this priming first expansion occurs for a period of about 6 to 7
days, resulting in a bulk
TIL population, generally about 1 x 10 bulk TTL cells. In some embodiments,
this priming first
expansion occurs for a period of about 7 to 8 days, resulting in a bulk TIL
population, generally about
1 x 108 bulk TIL cells. In some embodiments, this priming first expansion
occurs for a period of about
7 days, resulting in a bulk T1L population, generally about 1 x 108 bulk TIL
cells. In some
embodiments, this priming first expansion occurs for a period of about 8 days,
resulting in a bulk TIL
population, generally about 1 x 108 bulk TIL cells.
[00728] In some embodiments, expansion of TILs may be performed using a
priming first expansion
step (for example such as those described in Step B of Figure 8 (in
particular, e.g., Figure 8A and/or
Figure 8B and/or Figure 8C and/or Figure 8D), which can include processes
referred to as pre-REP or
priming REP and which contains feeder cells from Day 0 and/or from culture
initiation) as described
below and herein, followed by a rapid second expansion (Step D, including
processes referred to as
rapid expansion protocol (REP) steps) as described below under Step D and
herein, followed by
optional cryopreservation, and followed by a second Step D (including
processes referred to as
restimulation REP steps) as described below and herein. The TILs obtained from
this process may be
optionally characterized for phenotypic characteristics and metabolic
parameters as described herein.
In some embodiments, the tumor fragment is between about 1 mm3 and 10 mm3.
[00729] In some embodiments, the first expansion culture medium is referred to
as "CM", an
abbreviation for culture media. In some embodiments, CM for Step B consists of
RPMI 1640 with
GlutaMAX, supplemented with 10% human AB scrum, 25 mM Hcpcs, and 10 mg/mL
gentamicin.
[00730] In some embodiments, there are less than or equal to 240 tumor
fragments. In some
embodiments, there are less than or equal to 240 tumor fragments placed in
less than or equal to 4
containers. In some embodiments, the containers are G-REX100 MCS flasks. In
some embodiments,
less than or equal to 60 tumor fragments are placed in 1 container. In some
embodiments, each
container comprises less than or equal to 500 mL of media per container. In
some embodiments, the
media comprises IL-2. In some embodiments, thc media comprises 6000 IU/mL of
IL-2. In some
embodiments, the media comprises antigen-presenting feeder cells (also
referred to herein as
"antigen-presenting cells"). In some embodiments, the media comprises 2.5 x
108 antigen-presenting
feeder cells per container. In some embodiments, the media comprises OKT-3. In
some embodiments,
the media comprises 30 ng/mL of OKT-3 per container. In some embodiments, the
container is a G-
REX100 MCS flask. In some embodiments, the media comprises 6000 IU/mL of IL-2,
30 ng of OKT-
3, and 2.5 x 108 antigen-presenting feeder cells. In some embodiments, the
media comprises 6000
IU/mL of IL-2, 30 ng/mL of OKT-3, and 2.5 x 10' antigen-presenting feeder
cells per container.
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[00731] After preparation of the tumor fragments, the resulting cells (i.e.,
fragments which is a
primary cell population) are cultured in media containing IL-2, antigen-
presenting feeder cells and
OKT-3 under conditions that favor the growth of TILs over tumor and other
cells and which allow for
TIL priming and accelerated growth from initiation of the culture on Day 0. In
some embodiments,
the tumor digests and/or tumor fragments are incubated in with 6000 IU/mL of
IL-2, as well as
antigen-presenting feeder cells and OKT-3. This primary cell population is
cultured for a period of
days, generally from 1 to 8 days, resulting in a bulk TIL population,
generally about 1 108 bulk TIL
cells. In some embodiments, the growth media during the priming first
expansion comprises IL-2 or a
variant thereof, as well as antigen-presenting feeder cells and OKT-3. In some
embodiments, this
primary cell population is cultured for a period of days, generally from 1 to
7 days, resulting in a bulk
TIL population, generally about i>< 108 bulk TIL cells. In some embodiments,
the growth media during
the priming first expansion comprises IL-2 or a variant thereof, as well as
antigen-presenting feeder
cells and OKT-3. In some embodiments, the 1L-2 is recombinant human 1L-2 (rhiL-
2). In sonic
embodiments the IL-2 stock solution has a specific activity of 20-30x106IU/mg
for a 1 mg vial. In
some embodiments the IL-2 stock solution has a specific activity of 20 x106
IU/mg for a 1 mg vial. In
some embodiments the 1L-2 stock solution has a specific activity of 25 x106
IU/mg for a 1 mg vial. In
some embodiments the IL-2 stock solution has a specific activity of 30 x106
IU/mg for a 1 mg vial. In
some embodiments, the IL- 2 stock solution has a final concentration of 4-
8x106 IU/mg of IL-2. In
some embodiments, the IL- 2 stock solution has a final concentration of 5-
7x106 IU/mg of IL-2. In
some embodiments, the IL- 2 stock solution has a final concentration of
6x106IU/mg of IL-2. In some
embodiments, the IL-2 stock solution is prepare as described in Example C. In
some embodiments,
the priming first expansion culture media comprises about 10,000 IU/mL of IL-
2, about 9,000 IU/mL
of IL-2, about 8,000 IU/mL of IL-2, about 7,000 IU/mL of IL-2, about 6000
IU/mL of IL-2 or about
5,000 IU/mL of IL-2. In some embodiments, the priming first expansion culture
media comprises
about 9,000 IU/mL of iL-2 to about 5,000 IU/mL of iL-2. in some embodiments,
the priming first
expansion culture media comprises about 8,000 IU/mL of IL-2 to about 6,000
IU/mL of IL-2. In some
embodiments, the priming first expansion culture media comprises about 7,000
IU/mL of IL-2 to
about 6,000 IU/mL of 1L-2. In some embodiments, the priming first expansion
culture media
comprises about 6,000 IU/mL of IL-2. In some embodiments, the cell culture
medium further
comprises IL-2. In some embodiments, the priming first expansion cell culture
medium comprises
about 3000 IU/mL of IL-2. In some embodiments, the priming first expansion
cell culture medium
further comprises IL-2. In some embodiments, the priming first expansion cell
culture medium
comprises about 3000 IU/mL of IL-2. In some embodiments, the priming first
expansion cell culture
medium comprises about 1000 IU/mL, about 1500 IU/mL, about 2000 IU/mL, about
2500 IU/mL,
about 3000 IU/mL, about 3500 IU/mL, about 4000 IU/mL, about 4500 IU/mL, about
5000 IU/mL,
about 5500 IU/mL, about 6000 IU/mL, about 6500 IU/mL, about 7000 IU/mL, about
7500 IU/mL, or
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about 8000 IU/mL of IL-2. In some embodiments, the priming first expansion
cell culture medium
comprises between 1000 and 2000 IU/mL, between 2000 and 3000 IU/mL, between
3000 and 4000
IU/mL, between 4000 and 5000 IU/mL, between 5000 and 6000 IU/mL, between 6000
and 7000
IU/mL, between 7000 and 8000 IU/mL, or about 8000 IU/mL of IL-2.
[00732] In some embodiments, priming first expansion culture media comprises
about 500 IU/mL
of IL-15, about 400 IU/mL of IL-15, about 300 IU/mL of IL-15, about 200 IU/mL
of IL-15, about 180
IU/mL of IL-15, about 160 IU/mL of IL-15, about 140 IU/mL of IL-15, about 120
IU/mL of IL-15, or
about 100 Mimi-, of TL-15 In some embodiments, the priming first expansion
culture media
comprises about 500 IU/mL of IL-15 to about 100 IU/mL of IL-15. In some
embodiments, the
priming first expansion culture media comprises about 400 IU/mL of IL-15 to
about 100 IU/mL of IL-
15. In some embodiments, the priming first expansion culture media comprises
about 300 IU/mL of
IL-15 to about 100 IU/mL of IL-15. In some embodiments, the priming first
expansion culture media
comprises about 200 IU/mL of IL-15. In some embodiments, the priming first
expansion cell culture
medium comprises about 180 IU/mL of IL-15. In some embodiments, the priming
first expansion cell
culture medium further comprises IL-15. In some embodiments, the priming first
expansion cell
culture medium comprises about 180 IU/mL of IL-15.
[00733] In some embodiments, priming first expansion culture media comprises
about 20 IU/mL of
1L-21, about 15 IU/mL of TL-21, about 12 IU/mL of TL-21, about 10 IU/mL of 1L-
21, about 5 IU/mL
of IL-21, about 4 IU/mL of IL-21, about 3 IU/mL of IL-21, about 2 IU/mL of IL-
21, about 1 IU/mL
of IL-21, or about 0.5 IU/mL of IL-21. In sonic embodiments, the priming first
expansion culture
media comprises about 20 IU/mL of IL-21 to about 0.5 IU/mL of 1L-21. In some
embodiments, the
priming first expansion culture media comprises about 15 IU/mL of IL-21 to
about 0.5 IU/mL of IL-
21. In some embodiments, the priming first expansion culture media comprises
about 12 IU/mL of IL-
21 to about 0.5 IU/mL of IL-21. In some embodiments, the priming first
expansion culture media
comprises about 10 IU/mL of IL-21 to about 0.5 IU/mL of IL-21. In some
embodiments, the priming
first expansion culture media comprises about 5 IU/mL of IL-21 to about 1
IU/mL of IL-21. In some
embodiments, the priming first expansion culture media comprises about 2 IU/mL
of IL-21. In some
embodiments, the priming first expansion cell culture medium comprises about 1
IU/mL of IL-21. In
some embodiments, the priming first expansion cell culture medium comprises
about 0.5 IU/mL of
1L-21. In some embodiments, the cell culture medium further comprises IL-21.
In some embodiments,
the priming first expansion cell culture medium comprises about 1 IU/mL of IL-
21.
[00734] In some embodiments, the priming first expansion cell culture medium
comprises OKT-3
antibody. In some embodiments, the priming first expansion cell culture medium
comprises about 30
ng/mL of OKT-3 antibody. In some embodiments, the priming first expansion cell
culture medium
comprises about 0.1 ng/mL, about 0.5 ng/mL, about 1 ng/mL, about 2.5 ng/mL,
about 5 ng/mL, about
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7.5 ng/mL, about 10 ng/mL, about 15 ng/mL, about 20 ng/mL, about 25 ng/mL,
about 30 ng/mL,
about 35 ng/mL, about 40 ng/mL, about 50 ng/mL, about 60 ng/mL, about 70
ng/mL, about 80
ng/mL, about 90 ng/mL, about 100 ng/mL, about 200 ng/mL, about 500 ng/mL, and
about 1 vig/mL of
OKT-3 antibody. In some embodiments, the cell culture medium comprises between
0.1 ng/mL and 1
ng/mL, between 1 ng/mL and 5 ng/mL, between 5 ng/mL and 10 ng/mL, between 10
ng/mL and 20
ng/mL, between 20 ng/mL and 30 ng/mL, between 30 ng/mL and 40 ng/mL, between
40 ng/mL and
50 ng/mL, and between 50 ng/mL and 100 ng/mL of OKT-3 antibody. In some
embodiments, the cell
culture medium comprises between 15 ng/mL and 30 ng/mL of OKT-3 antibody. In
some
embodiments, the cell culture medium comprises 30 ng/mL of OKT-3 antibody. In
some
embodiments, the OKT-3 antibody is muromonab. See, Table 1.
[00735] In some embodiments, the priming first expansion cell culture medium
comprises one or
more TNFRSF agonists in a cell culture medium. In some embodiments, the TNFRSF
agonist
comprises a 4-1BB agonist. in some embodiments, the TNFRSF agonist is a 4-I BB
agonist, and the
4-1BB agonist is selected from the group consisting of urelumab, utomilumab,
EU-101, a fusion
protein, and fragments, derivatives, variants, biosimilars, and combinations
thereof In some
embodiments, the TNFRSF agonist is added at a concentration sufficient to
achieve a concentration in
the cell culture medium of between 0.1 g/mL and 100 vtg/mL. In some
embodiments, the TNFRSF
agonist is added at a concentration sufficient to achieve a concentration in
the cell culture medium of
between 20 Kg/mL and 40 ps/mL.
[00736] In some embodiments, in addition to one or more TNFRSF agonists, the
priming first
expansion cell culture medium further comprises 1L-2 at an initial
concentration of about 3000 1U/mL
and OKT-3 antibody at an initial concentration of about 30 ng/mL, and wherein
the one or more
TNFRSF agonists comprises a 4-1BB agonist. In some embodiments, in addition to
one or more
TNFRSF agonists, the priming first expansion cell culture medium further
comprises IL-2 at an initial
concentration of about 6000 IU/mL and OKT-3 antibody at an initial
concentration of about 30
ng/mL, and wherein the one or more TNFRSF agonists comprises a 4-1BB agonist.
[00737] In some embodiments, the priming first expansion culture medium is
referred to as -CM",
an abbreviation for culture media. In some embodiments, it is referred to as
CM1 (culture medium 1).
In some embodiments, CM consists of RPM! 1640 with GlutaMAX, supplemented with
10% human
AB serum, 25 mM Hepes, and 10 mg/mL gentamicin. In some embodiments, the CM is
the CM1
described in the Examples, see, Example A. In some embodiments, the priming
first expansion occurs
in an initial cell culture medium or a first cell culture medium. In some
embodiments, the priming first
expansion culture medium or the initial cell culture medium or the first cell
culture medium comprises
IL-2, OKT-3 and antigen-presenting feeder cells (also referred to herein as
feeder cells).
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[00738] In some embodiments, the culture medium used in the expansion
processes disclosed herein
is a serum-free medium or a defined medium. In some embodiments, the serum-
free or defined
medium comprises a basal cell medium and a serum supplement and/or a serum
replacement. In some
embodiments, the serum-free or defined medium is used to prevent and/or
decrease experimental
variation due in part to the lot-to-lot variation of serum-containing media.
[00739] In some embodiments, the serum-free or defined medium comprises a
basal cell medium
and a serum supplement and/or serum replacement. In some embodiments, the
basal cell medium
includes, but is not limited to CTSTm OpTmizerTm T-cell Expansion Basal Medium
, CTS'
OpTmizerTm T-Cell Expansion SFM, CTSTm AIM-V Medium, CTSTm AIM-V SFM,
LymphoONETM
T-Cell Expansion Xeno-Free Medium, Dulbecco's Modified Eagle's Medium (DMEM),
Minimal
Essential Medium (MEM), Basal Medium Eagle (BME), RPMI 1640, F-10, F-12,
Minimal Essential
Medium (aMEM), Glasgow's Minimal Essential Medium (G-MEM), RPMI growth medium,
and
Iscove's Modified Dulbecco's Medium.
[00740] In some embodiments, the serum supplement or serum replacement
includes, but is not
limited to one or more of CTSTm OpTmizer T-Cell Expansion Serum Supplement,
CTSTm Immune
Cell Serum Replacement, one or more albumins or albumin substitutes, one or
more amino acids, one
or more vitamins, one or more transferrins or transferrin substitutes, one or
more antioxidants, one or
more insulins or insulin substitutes, one or more collagen precursors, one or
more antibiotics, and one
or more trace elements. In some embodiments, the defined medium comprises
albumin and one or
more ingredients selected from the group consisting of glycine, L- histidine,
L-isoleucine, L-
methionine, L-phenylalanine, L-proline, L- hydroxyproline, L-serine, L-
threonine, L-tryptophan, L-
tyrosine, L-valine, thiamine, reduced glutathione, L-ascorbic acid-2-
phosphate, iron saturated
transferrin, insulin, and compounds containing the trace element moieties Ag',
Al", Ba", Cd', Co',
Cr, Ge4+, Se4+, Br, T, mn2+, P. si4+, v5+, mo6+, N=2+,
Rb+, Sn' and Zr4+. In some embodiments, the
defined medium further comprises L-glutamine, sodium bicarbonate and/or 2-
mercaptoethanol.
[00741] In some embodiments, the CTSTmOpTmizerTm T-cell Immune Cell Serum
Replacement is
used with conventional growth media, including but not limited to CTSTm
OpTmizerTm T-cell
Expansion Basal Medium, CTSTm OpTmizerTm T-cell Expansion SFM, CTSTm AIM-V
Medium,
CSTTm AIM-V SFM, LymphoONETM T-Cell Expansion Xeno-Free Medium, Dulbecco's
Modified
Eagle's Medium (DMEM), Minimal Essential Medium (MEM), Basal Medium Eagle
(BME), RPMI
1640, F-10, F-12, Minimal Essential Medium (aMEM), Glasgow's Minimal Essential
Medium (G-
MEM), RPMI growth medium, and Iscove's Modified Dulbecco's Medium.
[00742] In some embodiments, the total serum replacement concentration (vol%)
in the serum-free
or defined medium is from about 1%, 2%, 3%, 4%, 5%, 6%, 7%, 8%, 9%, 10%, 11%,
12%, 13%,
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14%, 15%, 16%, 17%, 18%, 19%, or 20% by volume of the total serum-free or
defined medium. In
some embodiments, the total serum replacement concentration is about 3% of the
total volume of the
serum-free or defined medium. In some embodiments, the total serum replacement
concentration is
about 5% of the total volume of the serum-free or defined medium. In some
embodiments, the total
serum replacement concentration is about 10% of the total volume of the serum-
free or defined
medium.
[00743] In some embodiments, the serum-free or defined medium is CTSTm
OpTmizerTm T-cell
Expansion SFM (ThermoFisher Scientific) Any formulation of CTSTm OpTmizerTm is
usefill in the
present invention. CTSTm OpTmizerTm T-cell Expansion SFM is a combination of
IL CTSTm
OpTmizerTm T-cell Expansion Basal Medium and 26 mL CTSTm OpTmizerTm T-Cell
Expansion
Supplement, which are mixed together prior to use. In some embodiments, the
CTSTm OpTmizerTm T-
cell Expansion SFM is supplemented with about 3% of the CTSTm Immune Cell
Serum Replacement
(SR) (TherrnoFisher Scientific). In some embodiments, the CTS TM OpTmizerTm T-
cell Expansion
SFM is supplemented with about 3% of the CTSTm Immune Cell Serum Replacement
(SR)
(ThermoFisher Scientific), along with 2-mercaptoethanol at 55mM. In some
embodiments, the CTSTm
OpTmizerTm T-cell Expansion SFM is supplemented with about 3% of the CTSTm
Immune Cell
Serum Replacement (SR) (ThermoFisher Scientific) and the final concentration
of 2-mercaptoethanol
in the media is 5511M.
[00744] In some embodiments, the defined medium is CTSTm OpTmizerTm T-cell
Expansion SFM
(ThermoFisher Scientific). Any formulation of CTSTm OpTinizerTm is useful in
the present invention.
CTSTm OpTmizerTm T-cell Expansion SFM is a combination of 11, CTSTm OpTmizerTm
T-cell
Expansion Basal Medium and 26 mL CTSTm OpTmizerTm T-Cell Expansion Supplement,
which are
mixed together prior to use. In some embodiments, the CTSTm OpTmizerTm T-cell
Expansion SFM is
supplemented with about 3% of the CTSTm Immune Cell Serum Replacement (SR)
(ThermoFisher
Scientific), along with 2-mereaptoethanol at 55mM. In some embodiments, the
CTSTmOpTmizerTm T-
cell Expansion SFM is supplemented with about 3% of the CTSTm Immune Cell
Serum Replacement
(SR) (ThermoFisher Scientific), 55mM of 2-mercaptoethanol, and 2mM of L-
glutamine. In some
embodiments, the CTSTmOpTmizerTm T-cell Expansion SFM is supplemented with
about 3% of the
CTSTm Immune Cell Serum Replacement (SR) (ThermoFisher Scientific), 55mM of 2-
mercaptoethanol, and 2mM of L-glutamine, and further comprises about 1000
IU/mL to about 8000
IU/mL of IL-2. In some embodiments, the CTSTmOpTmizerTm T-cell Expansion SFM
is
supplemented with about 3% of the CTSTm Immune Cell Serum Replacement (SR)
(ThermoFisher
Scientific), 55mM of 2-mercaptoethanol, and 2mM of L-glutamine, and further
comprises about 3000
IU/mL of IL-2. In some embodiments, the CTSTmOpTmizerTm T-cell Expansion SFM
is
supplemented with about 3% of the CTSTm Immune Cell Serum Replacement (SR)
(ThermoFisher
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Scientific), 55mM of 2-mercaptoethanol, and 2mM of L-glutamine, and further
comprises about 6000
IU/mL of IL-2. In some embodiments, the CTSTmOpTmizerTm T-cell Expansion SFM
is
supplemented with about 3% of the CTSTm Immune Cell Serum Replacement (SR)
(ThermoFisher
Scientific) and 55mM of 2-mercaptoethanol, and further comprises about 1000
IU/mL to about 8000
IU/mL of IL-2. In some embodiments, the CTSTmOpTmizerTm T-cell Expansion SFM
is
supplemented with about 3% of the CIS IM Immune Cell Serum Replacement (SR)
(ThermoFisher
Scientific) and 55mM of 2-mercaptoethanol, and further comprises about 3000
IU/mL of IL-2. In
some embodiments, the CTSTmOpTmizerTm T-cell Expansion SFM is supplemented
with about 3% of
the CTSTm Immune Cell Serum Replacement (SR) (ThermoFisher Scientific) and
55mM of 2-
mercaptoethanol, and further comprises about 1000 IU/mL to about 6000 IU/mL of
IL-2. In some
embodiments, the CTSTmOpTmizerTm T-cell Expansion SFM is supplemented with
about 3% of the
CTSTm Immune Cell Serum Replacement (SR) (ThermoFisher Scientific) and about
2mM glutamine,
and further comprises about 1000 IU/mL to about 8000 IU/mL of IL-2. In some
embodiments, the
CTSTmOpTmizernm T-cell Expansion SFM is supplemented with about 3% of the
CTSTm Immune
Cell Serum Replacement (SR) (ThernioFisher Scientific) and about 2mM
glutamine, and further
comprises about 3000 IU/mL of IL-2. In some embodiments, the CTSTmOpTmizerTm T-
cell
Expansion SFM is supplemented with about 3% of the CTSTm Immune Cell Serum
Replacement (SR)
(ThermoFisher Scientific) and about 2mM glutamine, and further comprises about
6000 IU/mL of IL-
2. In some embodiments, the CTSTm OpTmizerTm T-cell Expansion SFM is
supplemented with about
3% of the CTSTm Immune Cell Serum Replacement (SR) (ThermoFisher Scientific)
and the final
concentration of 2-mercaptoethanol in the media is 5504.
[00745] In some embodiments, the serum-free medium or defined medium is
supplemented with
glutamine (i.e., GlutaMAXClk) at a concentration of from about 0.1mM to about
10mM, 0.5mM to
about 9mM, 1mM to about 8mM, 2mM to about 7mM, 3mM to about 6mM, or 4mM to
about 5 mM.
In some embodiments, the serum-free medium or defined medium is supplemented
with glutamine
(i.e.. GlutaMAX(t) at a concentration of about 2mM.
[00746] In some embodiments, the serum-free medium or defined medium is
supplemented with 2-
mercaptoethanol at a concentration of from about 5mM to about 150mM, 10mM to
about 140mM,
15mM to about 130mM, 20mM to about 120mM, 25mM to about 110mM, 30mM to about
100mM,
35mM to about 95mM, 40mM to about 90mM, 45mM to about 85mM, 50mM to about
80mM, 55mM
to about 75mM, 60mM to about 70mM, or about 65mM. In some embodiments, the
serum-free
medium or defined medium is supplemented with 2-mercaptoethanol at a
concentration of about
55mM. In some embodiments, the final concentration of 2-mcrcaptocthanol in the
media is 55itM.
[00747] In some embodiments, the defined media described in International PCT
Publication No.
WO/1998/030679, which is herein incorporated by reference, are useful in the
present invention. In
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that publication, serum-free eukaryotic cell culture media are described. The
serum-free, eukaryotic
cell culture medium includes a basal cell culture medium supplemented with a
serum-free supplement
capable of supporting the growth of cells in serum- free culture. The semin-
free eukaryotic cell
culture medium supplement comprises or is obtained by combining one or more
ingredients selected
from the group consisting of one or more albumins or albumin substitutes, one
or more amino acids,
one or more vitamins, one or more transfcrrins or transit:mu substitutes, one
or more antioxidants,
one or more insidins or insulin substitutes, one or more collagen precursors,
one or more trace
elements, and one or more antibiotics. In some embodiments, the defined medium
further comprises
L-glutamine, sodium bicarbonate and/or beta-mercaptoethanol. In some
embodiments, the defined
medium comprises an albumin or an albumin, substitute and. one or more
ingredients selected from
group consisting of one or more amino acids, one or more vitamins, one or more
transferrins or
iransterrin substitutes, one or more antioxidants, one Or more inStilinS or
insulin substitutes., OnC Of
more collagen precursors, and one or more trace elements. In Some embodiments,
the defined medium
comprises albumin and one or more ingredients selected from the group
consisting of glycine,
L-methionine, le-phenylalanine, L-prohne, hydroxyproline, L-
serine,
threonine, L-tryptophan, L-tvrosine, Levaline, thiamine, reduced ghttathione.
L-ascorbic acid-2-
phosphate, iron saturated transferrin, insulin, and compounds containing the
trace element moieties
.Ag', Balf-, Cd2", Co2-', Cr, Ge4-', Se', Br, I. P,
Ni2", Sn.2-' and Zr.
in some embodiments, the basal cell media is selected from the group
consisting of Dulbecco's
Modified Eagle's Medium (DMEM), Minimal Essential Medium (MEM), Basal Medium
Eagle
(BME), RPMI 1640, F-10, F-12, Minimal Essential Medium (ci,MEM), Glasgow's
Minimal Essential
Medium (G-MEM), RPMI growth medium, and iscove's Modified Dulbecco's Medium.
1007481 In some embodiments, the concentration of glycine in the defined
medium is in the range of
from about 5-200 mg/L, the concentration of L- histidine is about 5-250 mg/L,
the concentration of L-
isoleucine is about 5-300 mg/L, the concentration of L-methionine is about 5-
200 mg/L, the
concentration of L-phenylalanine is about 5-400 mg/L, the concentration of L-
proline is about 1-1000
mg/L, the concentration of L- hydroxyproline is about 1-45 mg/L, the
concentration of L-serine is
about 1-250 mg/L, the concentration of L-threonine is about 10-500 mg/L, the
concentration of L-
tryptophan is about 2-110 mg/L, the concentration of L-tyrosine is about 3-175
mg/L, the
concentration of L-valine is about 5-500 mg/L, the concentration of thiamine
is about 1-20 mg/L, the
concentration of reduced glutathione is about 1-20 mg/L, the concentration of
L-ascorbic acid-2-
phosphate is about 1-200 mg/L, the concentration of iron saturated transfen-in
is about 1-50 mg/L, the
concentration of insulin is about 1-100 mg/L, the concentration of sodium
selenite is about 0.000001-
0.0001 mg/L, and the concentration of albumin (e.g., AlbuMAXEC I) is about
5000-50,000 mg/L.
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[00749] In some embodiments, the non-trace element moiety ingredients in the
defined medium are
present in the concentration ranges listed in the column under the heading
"Concentration Range in
1X Medium" in Table 4. In other embodiments, the non-trace element moiety
ingredients in the
defined medium are present in the final concentrations listed in the column
under the heading "A
Preferred Embodiment of the IX Medium" in Table 4. In other embodiments, the
defined medium is a
basal cell medium comprising a serum free supplement. In some of these
embodiments, the serum free
supplement comprises non-trace moiety ingredients of the type and in the
concentrations listed in the
column under the heading "A Preferred Embodiment in Supplement" in Table 4.
[00750] In some embodiments, the osmolarity of the defined medium is between
about 260 and 350
mOsmol. In some embodiments, the osmolarity is between about 280 and 310
mOsmol. In some
embodiments, the defined medium is supplemented with up to about 3.7 g/L, or
about 2.2 g/L sodium
bicarbonate. The defined medium can be further supplemented with L-glutamine
(final concentration
of about 2 mM), one or more antibiotics, non-essential amino acids (NEAA;
final concentration of
about 1001AM), 2-mercaptoethanol (final concentration of about 100 M).
[00751] In some embodiments, the defined media described in Smith, et al.,
Clin Trans'
Immunology, 4(1) 2015 (doi: 10.1038/cti.2014.31) are useful in the present
invention. Briefly, RPMI
or CTSTm OpTmizerTm was used as the basal cell medium, and supplemented with
either 0, 2%, 5%,
or 10% CTSTm Immune Cell Scrum Replacement.
[00752] In some embodiments, the cell medium in the first and/or second gas
permeable container is
unfiltered. The use of unfiltered cell medium may simplify the procedures
necessary to expand the
number of cells. In some embodiments, the cell medium in the first and/or
second gas permeable
container lacks beta-mercaptoethanol (BME or PME; also known as 2-
mercaptoethanol, CAS 60-24-
2).
[00753] In some embodiments, the priming first expansion (including processes
such as for example
those described in Step B of Figure 8 (in particular, e.g, Figure 8A and/or
Figure 8B and/or Figure 8C
and/or Figure 8D), which can include those sometimes referred to as the pre-
REP or priming REP)
process is 1 to 8 days, as discussed in the examples and figures. In some
embodiments, the priming
first expansion (including processes such as for example those described in
Step B of Figure 8 (in
particular, e.g., Figure 8A and/or Figure 8B and/or Figure 8C and/or Figure
8D), which can include
those sometimes referred to as the pre-REP or priming REP) process is 2 to 8
days, as discussed in the
examples and figures. In some embodiments, the priming first expansion
(including processes such as
for example those described in Step B of Figure 8 (in particular, e.g., Figure
8A and/or Figure 8B
and/or Figure 8C and/or Figure 8D), which can include those sometimes referred
to as the pre-REP or
priming REP) process is 3 to 8 days, as discussed in the examples and figures.
In some embodiments,
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the priming first expansion (including processes such as for example those
described in Step B of
Figure 8 (in particular, e.g., Figure 8A and/or Figure 8B and/or Figure 8C
and/or Figure 8D), which
can include those sometimes referred to as the pre-REP or priming REP) process
is 4 to 8 days, as
discussed in the examples and figures. In some embodiments, the priming first
expansion (including
processes such as for example those described in Step B of Figure 8 (in
particular, e.g., Figure 8A
and/or Figure 813 and/or Figure 8C and/or Figure 8D), which can include those
sometimes referred to
as the pre-REP or priming REP) process is 5 to 8 days, as discussed in the
examples and figures. In
some embodiments, the priming first expansion (including processes such as for
example those
described in Step B of Figure 8 (in particular, e.g., Figure 8A and/or Figure
8B and/or Figure 8C
and/or Figure 8D), which can include those sometimes referred to as the pre-
REP or priming REP)
process is 6 to 8 days, as discussed in the examples and figures. In some
embodiments, the priming
first expansion (including processes such as for example those provided in
Step B of Figure 1 (in
particular, e.g. Figure 8A and/or Figure 8B and/or Figure 8C and/or Figure
8D), which can include
those sometimes referred to as the pre-REP or priming REP) process is 7 to 8
days, as discussed in the
examples and figures. In some embodiments, the priming first expansion
(including processes such as
for example those provided in Step B of Figure 8 (in particular, e.g., Figure
8A and/or Figure 8B
and/or Figure 8C and/or Figure 8D), which can include those sometimes referred
to as the pre-REP or
priming REP) process is 8 days, as discussed in the examples and figures. In
some embodiments, the
priming first expansion (including processes such as for example those
described in Step B of Figure
8 (in particular, e.g., Figure 8A and/or Figure 8B and/or Figure 8C and/or
Figure 8D), which can
include those sometimes referred to as the pre-REP or priming REP) process is
1 to 7 days, as
discussed in the examples and figures. In some embodiments, the priming first
expansion (including
processes such as for example those described in Step B of Figure 8 (in
particular, e.g., Figure 8A
and/or Figure 8B and/or Figure 8C and/or Figure 8D), which can include those
sometimes referred to
as the pre-REP or priming REP) process is 2 to 7 days, as discussed in the
examples and figures. In
some embodiments, the priming first expansion (including processes such as for
example those
described in Step B of Figure 8 (in particular, e.g., Figure 8A and/or Figure
8B and/or Figure 8C
and/or Figure 8D), which can include those sometimes referred to as the pre-
REP or priming REP)
process is 3 to 7 days, as discussed in the examples and figures. In some
embodiments, the priming
first expansion (including processes such as for example those described in
Step B of Figure 8 (in
particular, e.g., Figure 8A and/or Figure 8B and/or Figure 8C and/or Figure
8D), which can include
those sometimes referred to as the pre-REP or priming REP) process is 4 to 7
days, as discussed in the
examples and figures. In some embodiments, the priming first expansion
(including processes such as
for example those described in Step B of Figure 8 (in particular, e.g., Figure
8B and/or Figure 8C),
which can include those sometimes referred to as the pre-REP or priming REP)
process is 5 to 7 days,
as discussed in the examples and figures. In some embodiments, the priming
first expansion
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(including processes such as for example those described in Step B of Figure 8
(in particular, e.g.,
Figure 8A and/or Figure 8B and/or Figure 8C and/or Figure 8D), which can
include those sometimes
referred to as the pre-REP or priming REP) process is 6 to 7 days, as
discussed in the examples and
figures. In some embodiments, the priming first expansion (including processes
such as for example
those provided in Step B of Figure 8 (in particular, e.g.. Figure 8A and/or
Figure 8B and/or Figure 8C
and/or Figure 81)), which can include those sometimes referred to as the pre-
REP or priming REP)
process is 7 days, as discussed in the examples and figures.
[00754] In some embodiments, the priming first TIT, expansion can proceed for
1 days to 8 days
from when fragmentation occurs and/or when the first priming expansion step is
initiated. In some
embodiments, the priming first TIL expansion can proceed for 1 days to 7 days
from when
fragmentation occurs and/or when the first priming expansion step is
initiated. In some embodiments,
the priming first TIL expansion can proceed for 2 days to 8 days from when
fragmentation occurs
and/or when the first priming expansion step is initiated. In some
embodiments, the priming first TIL
expansion can proceed for 2 days to 7 days from when fragmentation occurs
and/or when the first
priming expansion step is initiated. In some embodiments, the priming first
TIL expansion can
proceed for 3 days to 8 days from when fragmentation occurs and/or when the
first priming expansion
step is initiated. In some embodiments, the priming first TIL expansion can
proceed for 3 days to 7
days from when fragmentation occurs and/or when the first priming expansion
step is initiated. In
some embodiments, the priming first TIL expansion can proceed for 4 days to 8
days from when
fragmentation occurs and/or when the first priming expansion step is
initiated. In some embodiments,
the priming first TIL expansion can proceed for 4 days to 7 days from when
fragmentation occurs
and/or when the first priming expansion step is initiated. In some
embodiments, the priming first TIL
expansion can proceed for 5 days to 8 days from when fragmentation occurs
and/or when the first
priming expansion step is initiated. In some embodiments, the priming first
TIL expansion can
proceed for 5 days to 7 days from when fragmentation occurs and/or when the
first priming expansion
step is initiated. In some embodiments, the priming first TIL expansion can
proceed for 6 days to 8
days from when fragmentation occurs and/or when the first priming expansion
step is initiated.In
some embodiments, the priming first TIL expansion can proceed for 6 days to 7
days from when
fragmentation occurs and/or when the first priming expansion step is
initiated. In some embodiments,
the priming first TIL expansion can proceed for 7 to 8 days from when
fragmentation occurs and/or
when the first priming expansion step is initiated. In some embodiments, the
priming first TIL
expansion can proceed for 8 days from when fragmentation occurs and/or when
the first priming
expansion step is initiated.In some embodiments, the priming first TIL
expansion can proceed for 7
days from when fragmentation occurs and/or when the first priming expansion
step is initiated.
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[00755] In some embodiments, the priming first expansion of the TILs can
proceed for 1 day, 2
days, 3 days, 4 days, 5 days, 6 days, 7 days, or 8 days. In some embodiments,
the first TIL expansion
can proceed for 1 day to 8 days. In some embodiments, the first TIL expansion
can proceed for 1 day
to 7 days. In some embodiments, the first TIL expansion can proceed for 2 days
to 8 days. In some
embodiments, the first TIL expansion can proceed for 2 days to 7 days. In some
embodiments, the
first TIL expansion can proceed for 3 days to 8 days. In some embodiments, the
first TIL expansion
can proceed for 3 days to 7 days. In some embodiments, the first TIL expansion
can proceed for 4
days to 8 days. In some embodiments, the first TIL expansion can proceed for 4
days to 7 days. In
some embodiments, the first TIL expansion can proceed for 5 days to 8 days. In
some embodiments,
the first TIL expansion can proceed for 5 days to 7 days. In some embodiments,
the first TIL
expansion can proceed for 6 days to 8 days. In some embodiments, the first TIL
expansion can
proceed for 6 days to 7 days. In some embodiments, the first TIL expansion can
proceed for 7 to 8
days. in some embodiments, the first TIL expansion can proceed for 8 days. In
some embodiments,
the first TIL expansion can proceed for 7 days.
[00756] In some embodiments, a combination of IL-2, 1L-7, IL-15, and/or IL-21
are employed as a
combination during the priming first expansion. In some embodiments, IL-2, IL-
7, IL-15, and/or IL-
21 as well as any combinations thereof can be included during the priming
first expansion, including,
for example during Step B processes according to Figure 8 (in particular,
e.g., Figure 8A and/or
Figure 8B and/or Figure 8C and/or Figure 8D), as well as described herein. In
some embodiments, a
combination of IL-2, IL-15, and IL-21 are employed as a combination during the
priming first
expansion. In some embodiments, IL-2, IL-15, and IL-21 as well as any
combinations thereof can be
included during Step B processes according to Figure 8 (in particular, e.g.,
Figure 8A and/or Figure
8B and/or Figure 8C and/or Figure 8D) and as described herein.
[00757] In some embodiments, the priming first expansion, for example, Step B
according to Figure
8 (in particular, e.g., Figure 8A and/or Figure 8B and/or Figure 8C and/or
Figure 8D), is performed in
a closed system bioreactor. In some embodiments, a closed system is employed
for the TIL
expansion, as described herein. In some embodiments, a bioreactor is employed.
In some
embodiments, a bioreactor is employed as the container. In some embodiments,
the bioreactor
employed is for example a G-REX-10 or a G-REX-100. In some embodiments, the
bioreactor
employed is a G-REX-100. In some embodiments, the bioreactor employed is a G-
REX-10.
1. Feeder Cells and Antigen Presenting Cells
[00758] In some embodiments, the priming first expansion procedures described
herein (for
example including expansion such as those described in Step B from Figure 8
(in particular, e.g..
Figure 8A and/or Figure 8B and/or Figure 8C and/or Figure 8D), as well as
those referred to as pre-
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REP or priming REP) does not require feeder cells (also referred to herein as
"antigen-presenting
cells") at the initiation of the TIL expansion, but rather are added during
the priming first expansion.
In some embodiments, the priming first expansion procedures described herein
(for example including
expansion such as those described in Step B from Figure 8 (in particular,
e.g., Figure 8A and/or
Figure 8B and/or Figure 8C and/or Figure 8D), as well as those referred to as
pre-REP or priming
REP) does not require feeder cells (also referred to herein as "antigen-
presenting cells") at the
initiation of the TIL expansion, but rather are added during the priming first
expansion at any time
during days 4-8. In some embodiments, the priming first expansion procedures
described herein (for
example including expansion such as those described in Step B from Figure 8
(in particular, e.g.,
Figure 8A and/or Figure 8B and/or Figure 8C and/or Figure 8D), as well as
those referred to as pre-
REP or priming REP) does not require feeder cells (also referred to herein as -
antigen-presenting
cells") at the initiation of the TIL expansion, but rather are added during
the priming first expansion at
any time during days 4-7. In some embodiments, the priming first expansion
procedures described
herein (for example including expansion such as those described in Step B from
Figure 8 (in
particular, e.g.. Figure 8A and/or Figure 8B and/or Figure 8C and/or Figure
8D), as well as those
referred to as pre-REP or priming REP) does not require feeder cells (also
referred to herein as
"antigen-presenting cells") at the initiation of the TIL expansion, but rather
are added during the
priming first expansion at any time during days 5-8. In some embodiments, the
priming first
expansion procedures described herein (for example including expansion such as
those described in
Step B from Figure 8 (in particular, e.g., Figure 8A and/or Figure 8B and/or
Figure 8C and/or Figure
8D), as well as those referred to as pre-REP or priming REP) does not require
feeder cells (also
referred to herein as "antigen-presenting cells") at the initiation of the TIL
expansion, but rather are
added during the priming first expansion at any time during days 5-7. In some
embodiments, the
priming first expansion procedures described herein (for example including
expansion such as those
described in Step B from Figure 8 (in particular, e.g.. Figure 8A and/or
Figure 8B and/or Figure 8C
and/or Figure 8D), as well as those referred to as pre-REP or priming REP)
does not require feeder
cells (also referred to herein as "antigen-presenting cells") at the
initiation of the TIL expansion, but
rather are added during the priming first expansion at any time during days 6-
8. In some
embodiments, the priming first expansion procedures described herein (for
example including
expansion such as those described in Step B from Figure 8 (in particular,
e.g., Figure 8A and/or
Figure 8B and/or Figure 8C and/or Figure 8D), as well as those referred to as
pre-REP or priming
REP) does not require feeder cells (also referred to herein as "antigen-
presenting cells") at the
initiation of the TIL expansion, but rather are added during the priming first
expansion at any time
during days 6-7. In some embodiments, the priming first expansion procedures
described herein (for
example including expansion such as those described in Step B from Figure 8
(in particular, e.g.,
Figure 8A and/or Figure 8B and/or Figure 8C and/or Figure 8D), as well as
those referred to as pre-
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REP or priming REP) does not require feeder cells (also referred to herein as
"antigen-presenting
cells") at the initiation of the TIL expansion, but rather are added during
the priming first expansion at
any time during day 7 or 8. In some embodiments, the priming first expansion
procedures described
herein (for example including expansion such as those described in Step B from
Figure 8 (in
particular, e.g., Figure 8A and/or Figure 8B and/or Figure 8C and/or Figure
8D), as well as those
referred to as pre-REP or priming REP) docs not require feeder cells (also
referred to herein as
"antigen-presenting cells") at the initiation of the TIL expansion, but rather
are added during the
priming first expansion at any time during day 7. In some embodiments, the
priming first expansion
procedures described herein (for example including expansion such as those
described in Step B from
Figure 8 (in particular, e.g., Figure 8A and/or Figure 8B and/or Figure 8C
and/or Figure 8D), as well
as those referred to as pre-REP or priming REP) does not require feeder cells
(also referred to herein
as "antigen-presenting cells") at the initiation of the TIL expansion, but
rather are added during the
priming first expansion at any time during day 8
[00759] In some embodiments, the priming first expansion procedures described
herein (for
example including expansion such as those described in Step B from Figure 8
(in particular, e.g.,
Figure 8B), as well as those referred to as pre-REP or priming REP) require
feeder cells (also referred
to herein as "antigen-presenting cells-) at the initiation of the TIL
expansion and during the priming
first expansion. In many embodiments, the feeder cells are peripheral blood
mononuclear cells
(PBMCs) obtained from standard whole blood units from allogeneic healthy blood
donors. The
PBMCs are obtained using standard methods such as Ficoll-Paque gradient
separation. In some
embodiments, 2.5 x 108 feeder cells are used during the priming first
expansion. In some
embodiments, 2.5 x 108 feeder cells per container are used during the priming
first expansion. In some
embodiments, 2.5 x 108 feeder cells per GREX-10 are used during the priming
first expansion. In
some embodiments, 2.5 x 108 feeder cells per GREX-100 are used during the
priming first expansion.
[00760] In general, the allogenic PBMCs are inactivated, either via
irradiation or heat treatment, and
used in the REP procedures, as described in the examples, which provides an
exemplary protocol for
evaluating the replication incompetence of irradiate allogeneic PBMCs.
[00761] In some embodiments, PBMCs are considered replication incompetent and
acceptable for
use in the TIL expansion procedures described herein if the total number of
viable cells on day 14 is
less than the initial viable cell number put into culture on day 0 of the
priming first expansion.
[00762] In some embodiments, PBMCs are considered replication incompetent and
acceptable for
use in the TIL expansion procedures described herein if the total number of
viable cells, cultured in
the presence of OKT3 and IL-2, on day 7 have not increased from the initial
viable cell number put
into culture on day 0 of the priming first expansion. In some embodiments, the
PBMCs are cultured in
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the presence of 30 ng/mL OKT3 antibody and 3000 IU/mL IL-2. In some
embodiments, the PBMCs
are cultured in the presence of 30 ng/mL OKT3 antibody and 6000 IU/mL IL-2.
[00763] In some embodiments, PBMCs are considered replication incompetent and
acceptable for
use in the TIL expansion procedures described herein if the total number of
viable cells, cultured in
the presence of OKT3 and IL-2, on day 7 have not increased from the initial
viable cell number put
into culture on day 0 of the priming first expansion. In some embodiments, the
PBMCs are cultured in
the presence of 5-60 ng/mL OKT3 antibody and 1000-6000 IU/mL IL-2. In some
embodiments, the
PRMCs are cultured in the presence of 10-50 ng/mT, OKT3 antibody and 2000-5000
IU/mI, IL-2. In
some embodiments, the PBMCs are cultured in the presence of 20-40 ng/mL OKT3
antibody and
2000-4000 IU/mL IL-2. In some embodiments, the PBMCs are cultured in the
presence of 25-35
ng/mL OKT3 antibody and 2500-3500 IU/mL IL-2. In some embodiments, the PBMCs
are cultured in
the presence of 30 ng/mL OKT3 antibody and 6000 IU/mL IL-2. In some
embodiments, the PBMCs
are cultured in the presence of 15 ng/mL OKT3 antibody and 3000 IU/mL 1L-2. In
some
embodiments, the PBMCs are cultured in the presence of 15 ng/mL OKT3 antibody
and 6000 IU/mL
IL-2.
[00764] In some embodiments, the antigen-presenting feeder cells are PBMCs. In
some
embodiments, the antigen-presenting feeder cells are artificial antigen-
presenting feeder cells. In some
embodiments, the ratio of 'TILs to antigen-presenting feeder cells in the
second expansion is about 1 to
25, about 1 to 50, about 1 to 100, about 1 to 125, about 1 to 150, about 1 to
175, about 1 to 200, about
1 to 225, about 1 to 250, about 1 to 275, about 1 to 300, about 1 to 325,
about 1 to 350, about 1 to 375,
about 1 to 400, or about 1 to 500. In some embodiments, the ratio of TILs to
antigen-presenting feeder
cells in the second expansion is between 1 to 50 and 1 to 300. In some
embodiments, the ratio of TILs
to antigen-presenting feeder cells in the second expansion is between 1 to 100
and 1 to 200.
[00765] In some embodiments, the priming first expansion procedures described
herein require a
ratio of about 2.5 x 108 feeder cells to about 100 x 106 TILs. In some
embodiments, the priming first
expansion procedures described herein require a ratio of about 2.5 x 108
feeder cells to about 50 x 106
TILs. In yet another embodiment, the priming first expansion described herein
require about 2.5 x 108
feeder cells to about 25 x 106 TILs. In yet another embodiment, the priming
first expansion described
herein require about 2.5 x 108 feeder cells. In yet another embodiment, the
priming first expansion
requires one-fourth, one-third, five-twelfths, or one-half of the number of
feeder cells used in the rapid
second expansion.
[00766] In some embodiments, the media in the priming first expansion
comprises IL-2. In some
embodiments, the media in the priming first expansion comprises 6000 IU/mL of
IL-2. In some
embodiments, the media in the priming first expansion comprises antigen-
presenting feeder cells. In
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some embodiments, the media in the priming first expansion comprises 2.5 x 108
antigen-presenting
feeder cells per container. In some embodiments, the media in the priming
first expansion comprises
OKT-3. In some embodiments, the media comprises 30 ng of OKT-3 per container.
In some
embodiments, the container is a GREX100 MCS flask. In some embodiments, the
media comprises
6000 IU/mL of IL-2, 30 ng/mL of OKT-3, and 2.5 x 108 antigen-presenting feeder
cells. In some
embodiments, the media comprises 6000 1U/mL of IL-2, 30 ng/mL of OKT-3, and
2.5 x 108 antigen-
presenting feeder cells per container. In some embodiments, the media
comprises 500 mL of culture
medium and 15 pg of OKT-3 per 2.5 x 108 antigen-presenting feeder cells per
container. In some
embodiments, the media comprises 500 mL of culture medium and 15 mg of OKT-3
per container. In
some embodiments, the container is a GREX100 MCS flask. In some embodiments,
the media
comprises 500 mL of culture medium. 6000 IU/mL of IL-2, 30 ng/mL of OKT-3, and
2.5 x 108
antigen-presenting feeder cells. In some embodiments, the media comprises 500
mL of culture
medium, 6000 IU/mL of IL-2, 15 lag of OKT-3, and 2.5 x 108 antigen-presenting
feeder cells per
container. In some embodiments, the media comprises 500 mL of culture medium
and 15 jig of OKT-
3 per 2.5 x 108 antigen-presenting feeder cells per container.
[00767] In some embodiments, the priming first expansion procedures described
herein require an
excess of feeder cells over TILs during the second expansion. In many
embodiments, the feeder cells
are peripheral blood mononuclear cells (PBMCs) obtained from standard whole
blood units from
allogeneic healthy blood donors. The PBMCs are obtained using standard methods
such as Ficoll-
Paque gradient separation. In some embodiments, artificial antigen-presenting
(aAPC) cells are used
in place of PBMCs.
[00768] In general, the allogenic PBMCs are inactivated, either via
irradiation or heat treatment, and
used in the TIL expansion procedures described herein, including the exemplary
procedures described
in the figures and examples.
[00769] In some embodiments, artificial antigen presenting cells are used in
the priming first
expansion as a replacement for, or in combination with, PBMCs.
2. Cytokines
[00770] The expansion methods described herein generally use culture media
with high doses of a
cytokine, in particular IL-2, as is known in the art.
[00771] Alternatively, using combinations of cytokines for the priming first
expansion of TILs is
additionally possible, with combinations of two or more of IL-2, IL-15 and IL-
21 as is generally
outlined in International Publication No. WO 2015/189356 and WO 2015/189357,
hereby expressly
incorporated by reference in their entirety. Thus, possible combinations
include 1L-2 and 1L-15, 1L-2
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and IL-21, IL-15 and IL-21, and IL-2, IL-15 and IL-21, with the latter finding
particular use in many
embodiments. The use of combinations of cytokines specifically favors the
generation of
lymphocytes, and in particular T-cells as described therein. See, Table 2.
[00772] In some embodiments, Step B may also include the addition of OKT-3
antibody or
muromonab to the culture media, as described elsewhere herein. In some
embodiments, Step B may
also include the addition of a 4-1BB agonist to the culture media, as
described elsewhere herein. In
some embodiments, Step B may also include the addition of an OX-40 agonist to
the culture media, as
described elsewhere herein. In addition, additives such as peroxisome
proliferator-activated receptor
gamma coactivator I-alpha agonists, including proliferator-activated receptor
(PPAR)-gamma agonists
such as a thiazolidinedione compound, may be used in the culture media during
Step B, as described
in U.S. Patent Application Publication No. US 2019/0307796 Al, the disclosure
of which is
incorporated by reference herein.
C. STEP C: Priming First Expansion to Rapid Second Expansion
Transition
[00773] In some cases, the bulk TIL population obtained from the priming first
expansion (which
can include expansions sometimes referred to as pre-REP), including, for
example the TIL population
obtained from for example, Step B as indicated in Figure 8 (in particular,
e.g., Figure 8A and/or
Figure 8B and/or Figure 8C and/or Figure 8D), can be subjected to a rapid
second expansion (which
can include expansions sometimes referred to as Rapid Expansion Protocol
(REP)) and then
cryopreserved as discussed below. Similarly, in the case where genetically
modified TILs will be used
in therapy, the expanded TIL population from the priming first expansion or
the expanded TIL
population from the rapid second expansion can be subjected to genetic
modifications for suitable
treatments prior to the expansion step or after the priming first expansion
and prior to the rapid second
expansion.
[00774] In some embodiments, the TILs obtained from the priming first
expansion (for example,
from Step B as indicated in Figure 8 (in particular, e.g., Figure 8A and/or
Figure 8B and/or Figure 8C
and/or Figure 8D)) are stored until phenotyped for selection. In some
embodiments, the TILs obtained
from the priming first expansion (for example, from Step B as indicated in
Figure 8 (in particular,
e.g. Figure 8A and/or Figure 8B and/or Figure 8C and/or Figure 8D)) are not
stored and proceed
directly to the rapid second expansion. In some embodiments, the TILs obtained
from the priming
first expansion are not cryopreserved after the priming first expansion and
prior to the rapid second
expansion. In some embodiments, the transition from the priming first
expansion to the second
expansion occurs at about 2 days, 3 days, 4, days, 5 days, 6 days, 7 days, or
8 days from when tumor
fragmentation occurs and/or when the first priming expansion step is
initiated. In some embodiments,
the transition from the priming first expansion to the rapid second expansion
occurs at about 3 days to
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7 days from when fragmentation occurs and/or when the first priming expansion
step is initiated. In
some embodiments, the transition from the priming first expansion to the rapid
second expansion
occurs at about 3 days to 8 days from when fragmentation occurs and/or when
the first priming
expansion step is initiated. In some embodiments, the transition from the
priming first expansion to
the second expansion occurs at about 4 days to 7 days from when fragmentation
occurs and/or when
the first priming expansion step is initiated. In some embodiments, the
transition from the priming
first expansion to the second expansion occurs at about 4 days to 8 days from
when fragmentation
occurs and/or when the first priming expansion step is initiated. In some
embodiments, the transition
from the priming first expansion to the second expansion occurs at about 5
days to 7 days from when
fragmentation occurs and/or when the first priming expansion step is
initiated. In some embodiments,
the transition from the priming first expansion to the second expansion occurs
at about 5 days to 8
days from when fragmentation occurs and/or when the first priming expansion
step is initiated. In
some embodiments, the transition from the priming first expansion to the
second expansion occurs at
about 6 days to 7 days from when fragmentation occurs and/or when the first
priming expansion step
is initiated. In some embodiments, the transition from the priming first
expansion to the second
expansion occurs at about 6 days to 8 days from when fragmentation occurs
and/or when the first
priming expansion step is initiated. In some embodiments, the transition from
the priming first
expansion to the second expansion occurs at about 7 days to 8 days from when
fragmentation occurs
and/or when the first priming expansion step is initiated. In some
embodiments, the transition from
the priming first expansion to the second expansion occurs at about 7 days
from when fragmentation
occurs and/or when the first priming expansion step is initiated. In some
embodiments, the transition
from the priming first expansion to the second expansion occurs at about 8
days from when
fragmentation occurs and/or when the first priming expansion step is
initiated.
[00775] In some embodiments, the transition from the priming first expansion
to the rapid second
expansion occurs at 1 day, 2 days, 3 days, 4 days, 5 days, 6 days, 7 days, or
8 days from when
fragmentation occurs and/or when the first priming expansion step is
initiated. In some embodiments,
the transition from the priming first expansion to the rapid second expansion
occurs 1 day to 7 days
from when fragmentation occurs and/or when the first priming expansion step is
initiated. In some
embodiments, the transition from the priming first expansion to the rapid
second expansion occurs 1
day to 8 days from when fragmentation occurs and/or when the first priming
expansion step is
initiated. In some embodiments, the transition from the priming first
expansion to the second
expansion occurs 2 days to 7 days from when fragmentation occurs and/or when
the first priming
expansion step is initiated. In some embodiments, the transition from the
priming first expansion to
the second expansion occurs 2 days to 8 days from when fragmentation occurs
and/or when the first
priming expansion step is initiated. In some embodiments, the transition from
the priming first
expansion to the second expansion occurs 3 days to 7 days from when
fragmentation occurs and/or
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when the first priming expansion step is initiated. In some embodiments, the
transition from the
priming first expansion to the second expansion occurs 3 days to 8 days from
when fragmentation
occurs and/or when the first priming expansion step is initiated. In some
embodiments, the transition
from the priming first expansion to the rapid second expansion occurs 4 days
to 7 days from when
fragmentation occurs and/or when the first priming expansion step is
initiated. In some embodiments,
the transition from the priming first expansion to the rapid second expansion
occurs 4 days to 8 days
from when fragmentation occurs and/or when the first priming expansion step is
initiated. In some
embodiments, the transition from the priming first expansion to the rapid
second expansion occurs 5
days to 7 days from when fragmentation occurs and/or when the first priming
expansion step is
initiated. In some embodiments, the transition from the priming first
expansion to the rapid second
expansion occurs 5 days to 8 days from when fragmentation occurs and/or when
the first priming
expansion step is initiated. In some embodiments, the transition from the
priming first expansion to
the rapid second expansion occurs 6 days to 7 days from when fragmentation
occurs and/or when the
first priming expansion step is initiated. . In some embodiments, the
transition from the priming first
expansion to the rapid second expansion occurs 6 days to 8 days from when
fragmentation occurs
and/or when the first priming expansion step is initiated. In some
embodiments, the transition from
the priming first expansion to the rapid second expansion occurs 7 days to 8
days from when
fragmentation occurs and/or when the first priming expansion step is
initiated. In some embodiments,
the transition from the priming first expansion to the rapid second expansion
occurs 7 days from when
fragmentation occurs and/or when the first priming expansion step is
initiated. In some embodiments,
the transition from the priming first expansion to the rapid second expansion
occurs 8 days from when
fragmentation occurs and/or when the first priming expansion step is initiated
[00776] In some embodiments, the TILs are not stored after the primary first
expansion and prior to
the rapid second expansion, and the TILs proceed directly to the rapid second
expansion (for example,
in some embodiments, there is no storage during the transition from Step B to
Step D as shown in
Figure 8 (in particular, e.g., Figure 8A and/or Figure 8B and/or Figure 8C
and/or Figure 8D)). In some
embodiments, the transition occurs in closed system, as described herein. In
some embodiments, the
TILs from the priming first expansion, the second population of TILs, proceeds
directly into the rapid
second expansion with no transition period.
[00777] In some embodiments, the transition from the priming first expansion
to the rapid second
expansion, for example, Step C according to Figure 8 (in particular, e.g.,
Figure 8A and/or Figure 8B
and/or Figure 8C and/or Figure 8D), is performed in a closed system
bioreactor. In some
embodiments, a closed system is employed for the T1L expansion, as described
herein. In some
embodiments, a single bioreactor is employed. In some embodiments, the single
bioreactor employed
is for example a GREX-10 or a GREX-100. In some embodiments, the closed system
bioreactor is a
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single bioreactor. In some embodiments, the transition from the priming first
expansion to the rapid
second expansion involves a scale-up in container size. In some embodiments,
the priming first
expansion is performed in a smaller container than the rapid second expansion.
In some embodiments,
the priming first expansion is performed in a GREX-100 and the rapid second
expansion is performed
in a GREX-500.
D. STEP D: Rapid Second Expansion
[00778] In some embodiments, the TIL cell population is further expanded in
number after harvest
and the priming first expansion, after Step A and Step B, and the transition
referred to as Step C, as
indicated in Figure 8 (in particular, e.g., Figure 8A and/or Figure 8B and/or
Figure 8C and/or Figure
8D)). This further expansion is referred to herein as the rapid second
expansion or a rapid expansion,
which can include expansion processes generally referred to in the art as a
rapid expansion process
(Rapid Expansion Protocol or REP; as well as processes as indicated in Step D
of Figure 8 (in
particular, e.g., Figure 8A and/or Figure 8B and/or Figure 8C and/or Figure
8D). The rapid second
expansion is generally accomplished using a culture media comprising a number
of components,
including feeder cells, a cytokine source, and an anti-CD3 antibody, in a gas-
permeable container. In
some embodiments, 1 day, 2 days, 3 days, or 4 days after initiation of the
rapid second expansion (i.e.,
at days 8, 9, 10, or 11 of the overall Gen 3 process), the TILs are
transferred to a larger volume
container.
[00779] In some embodiments, the rapid second expansion (which can include
expansions
sometimes referred to as REP; as well as processes as indicated in Step D of
Figure 8 (in particular,
e.g., Figure 8A and/or Figure 8B and/or Figure 8C and/or Figure 8D)) of TIL
can be performed using
any TIL flasks or containers known by those of skill in the art. In some
embodiments, the second TIL
expansion can proceed for 1 day, 2 days, 3 days, 4, days, 5 days, 6 days, 7
days, 8 days, 9 days or 10
days after initiation of the rapid second expansion. In some embodiments, the
second TIL expansion
can proceed for about 1 days to about 9 days after initiation of the rapid
second expansion. In some
embodiments, the second TIL expansion can proceed for about 1 days to about 10
days after initiation
of the rapid second expansion. In some embodiments, the second TIL expansion
can proceed for
about 2 days to about 9 days after initiation of the rapid second expansion.
In some embodiments, the
second TIL expansion can proceed for about 2 days to about 10 days after
initiation of the rapid
second expansion. in some embodiments, the second TIL expansion can proceed
for about 3 days to
about 9 days after initiation of the rapid second expansion. In some
embodiments, the second TIL
expansion can proceed for about 3 days to about 10 days after initiation of
the rapid second
expansion. In some embodiments, the second TIL expansion can proceed for about
4 days to about 9
days after initiation of the rapid second expansion. In some embodiments, the
second TIL expansion
can proceed for about 4 days to about 10 days after initiation of the rapid
second expansion. In some
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embodiments, the second TIL expansion can proceed for about 5 days to about 9
days after initiation
of the rapid second expansion. In some embodiments, the second TIL expansion
can proceed for
about 5 days to about 10 days after initiation of the rapid second expansion.
In some embodiments,
the second TIL expansion can proceed for about 6 days to about 9 days after
initiation of the rapid
second expansion. In some embodiments, the second TIL expansion can proceed
for about 6 days to
about 10 days after initiation of the rapid second expansion. In some
embodiments, the second TIL
expansion can proceed for about 7 days to about 9 days after initiation of the
rapid second expansion.
In some embodiments, the second TIL expansion can proceed for about 7 days to
about 10 days after
initiation of the rapid second expansion. In some embodiments, the second TIL
expansion can
proceed for about 8 days to about 9 days after initiation of the rapid second
expansion. In some
embodiments, the second TIL expansion can proceed for about 8 days to about 10
days after initiation
of the rapid second expansion. In some embodiments, the second TIL expansion
can proceed for
about 9 days to about 10 days after initiation of the rapid second expansion.
In some embodiments,
the second TIL expansion can proceed for about 1 day after initiation of the
rapid second expansion.
In some embodiments, the second TIL expansion can proceed for about 2 days
after initiation of the
rapid second expansion. In some embodiments, the second TIL expansion can
proceed for about 3
days after initiation of the rapid second expansion. In some embodiments, the
second TIL expansion
can proceed for about 4 days after initiation of the rapid second expansion.
In some embodiments, the
second TIL expansion can proceed for about 5 days after initiation of the
rapid second expansion. In
some embodiments, the second TIL expansion can proceed for about 6 days after
initiation of the
rapid second expansion. In some embodiments, the second TIL expansion can
proceed for about 7
days after initiation of the rapid second expansion. In some embodiments, the
second TIL expansion
can proceed for about 8 days after initiation of the rapid second expansion.
In some embodiments, the
second TIL expansion can proceed for about 9 days after initiation of the
rapid second expansion. In
some embodiments, the second TIL expansion can proceed for about 10 days after
initiation of the
rapid second expansion.
[00780] In some embodiments, the ra.pid second expansion can be performed in a
gas permeable
container using the methods of the present disclosure (including, for example,
expansions referred to
as REP; as well as processes as indicated in Step D of Figure 8 (in
particular, e.g., Figure 8A and/or
Figure 8B and/or Figure 8C and/or Figure 8D). In some embodiments, the TILs
are expanded in the
rapid second expansion in the presence of IL-2, OKT-3, and feeder cells (also
referred herein as
"antigen-presenting cells"). In some embodiments, the Tits are expanded in the
rapid second
expansion in the presence of IL-2, OKT-3, and feeder cells, wherein the feeder
cells are added to a
final concentration that is twice, 2.4 times, 2.5 times, 3 times, 3.5 times or
4 times the concentration of
feeder cells present in the priming first expansion. For example, TILs can be
rapidly expanded using
non-specific T-cell receptor stimulation in the presence of interleukin-2 (IL-
2) or interleukin-15 (IL-
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15). The non-specific T-cell receptor stimulus can include, for example, an
anti-CD3 antibody, such
as about 30 ng/mL of OKT3, a mouse monoclonal anti-CD3 antibody (commercially
available from
Ortho-McNeil, Raritan, NJ or Miltenyi Biotech, Auburn, CA) or UHCT-1
(commercially available
from BioLegend, San Diego, CA, USA). TILs can be expanded to induce further
stimulation of the
TILs in vitro by including one or more antigens during the second expansion,
including antigenic
portions thereof, such as epitope(s), of the cancer, which can be optionally
expressed from a vector,
such as a human leukocyte antigen A2 (HLA-A2) binding peptide, e.g., 0.3 t.tM
MART-1 :26-35 (27
L) or gpl 00:209-217 (210M), optionally in the presence of a T-cell growth
factor, such as 300 IU/mL
IL-2 or IL-15. Other suitable antigens may include, e.g., NY-ESO-1, TRP-1, TRP-
2, tyrosinase cancer
antigen, MAGE-A3, SSX-2, and VEGFR2, or antigenic portions thereof. TIL may
also be rapidly
expanded by re-stimulation with the same antigen(s) of the cancer pulsed onto
HLA-A2-expressing
antigen-presenting cells. Alternatively, the TILs can be further re-stimulated
with, e.g., example,
irradiated, autologous lymphocytes or with irradiated HLA-A2+ allogeneic
lymphocytes and IL-2. In
some embodiments, the re-stimulation occurs as part of the second expansion.
In some embodiments,
the second expansion occurs in the presence of irradiated, autologous
lymphocytes or with irradiated
HLA-A2+ allogeneic lymphocytes and IL-2.
[00781] In some embodiments, the cell culture medium further comprises IL-2.
In some
embodiments, the cell culture medium comprises about 3000 IU/mL of IL-2. In
some embodiments,
the cell culture medium comprises about 1000 IU/mL, about 1500 IU/mL, about
2000 IU/mL, about
2500 IU/mL, about 3000 IU/mL, about 3500 TU/mL, about 4000 IU/mL, about 4500
IU/mL, about
5000 IU/mL, about 5500 IU/mL, about 6000 IU/mL, about 6500 IU/mL, about 7000
IU/mL, about
7500 IU/mL, or about 8000 IU/mL of IL-2. In some embodiments, the cell culture
medium comprises
between 1000 and 2000 IU/mL, between 2000 and 3000 IU/mL, between 3000 and
4000 IU/mL,
between 4000 and 5000 IU/mL, between 5000 and 6000 IU/mL, between 6000 and
7000 IU/mL,
between 7000 and 8000 IU/mL, or between 8000 IU/mL of IL-2.
[00782] In some embodiments, the cell culture medium comprises OKT-3 antibody.
In some
embodiments, the cell culture medium comprises about 30 ng/mL of OKT-3
antibody. In some
embodiments, the cell culture medium comprises about 0.1 ng/mL, about 0.5
ng/mL, about 1 ng/mL,
about 2.5 ng/mL, about 5 ng/mL, about 7.5 ng/mL, about 10 ng/mL, about 15
ng/mL, about 20
ng/mL, about 25 ng/mL, about 30 ng/mL, about 35 ng/mL, about 40 ng/mL, about
50 ng/mL, about
60 ng/mL, about 70 ng/mL, about 80 ng/mL, about 90 ng/mL, about 100 ng/mL,
about 200 ng/mL,
about 500 ng/mL, and about 1 i_ig/mL of OKT-3 antibody. In some embodiments,
the cell culture
medium comprises between 0.1 ng/mL and 1 ng/mL, between 1 ng/mL and 5 ng/mL,
between 5
ng/mL and 10 ng/mL, between 10 ng/mL and 20 ng/mL, between 20 ng/mL and 30
ng/mL, between
30 ng/mL and 40 ng/mL, between 40 ng/mL and 50 ng/mL, and between 50 ng/mL and
100 ng/mL of
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OKT-3 antibody. In some embodiments, the cell culture medium comprises between
15 ng/mL and 30
ng/mL of OKT-3 antibody. In some embodiments, the cell culture medium
comprises between 30
ng/mL and 60 ng/mL of OKT-3 antibody. In sonic embodiments, the cell culture
medium comprises
about 30 ng/mL OKT-3. In some embodiments, the cell culture medium comprises
about 60 ng/mL
OKT-3. In some embodiments, the OKT-3 antibody is muromonab.
[00783] In some embodiments, the media in the rapid second expansion comprises
IL-2. In some
embodiments, the media comprises 6000 IU/mL of IL-2. In some embodiments, the
media in the rapid
second expansion comprises antigen-presenting feeder cells. In some
embodiments, the media in the
rapid second expansion comprises 7.5 x 10' antigen-presenting feeder cells per
container. In some
embodiments, the media in the rapid second expansion comprises OKT-3. In some
embodiments, the
in the rapid second expansion media comprises 500 mL of culture medium and 30
Kg of OKT-3 per
container. In some embodiments, the container is a GREX100 MCS flask. In some
embodiments, the
in the rapid second expansion media comprises 6000 IU/mL of 1L-2, 60 ng/mL of
OKT-3, and 7.5 x
108 antigen-presenting feeder cells. In some embodiments, the media comprises
500 mL of culture
medium and 6000 1U/mL of 1L-2, 301,ig of OKT-3, and 7.5 x 108 antigen-
presenting feeder cells per
container.
[00784] In some embodiments, the media in the rapid second expansion comprises
IL-2. In some
embodiments, the media comprises 6000 TU/mL of 1L-2. In some embodiments, the
media in the rapid
second expansion comprises antigen-presenting feeder cells. In some
embodiments, the media
comprises between 5 x 10' and 7.5 x 108 antigen-presenting feeder cells per
container. In some
embodiments, the media in the rapid second expansion comprises OKT-3. In some
embodiments, the
media in the rapid second expansion comprises 500 mL of culture medium and 30
lig of OKT-3 per
container. In some embodiments, the container is a GREX100 MCS flask. In some
embodiments, the
media in the rapid second expansion comprises 6000 IU/mL of IL-2, 60 ng/mL of
OKT-3, and
between 5 108 and 7.5 x 108 antigen-presenting feeder cells. In some
embodiments, the media in the
rapid second expansion comprises 500 mL of culture medium and 6000 IU/mL of IL-
2, 30 lig of
OKT-3, and between 5 x 10' and 7.5 x 108 antigen-presenting feeder cells per
container.
[00785] In some embodiments, the cell culture medium comprises one or more
'TNFRSF agonists in
a cell culture medium. In some embodiments, the TNFRSF agonist comprises a 4-
1BB agonist. In
some embodiments, the TNFRSF agonist is a 4-1BB agonist, and the 4-1BB agonist
is selected from
the group consisting of urelumab, utomilumab, EU-101, a fusion protein, and
fragments, derivatives,
variants, biosimilars, and combinations thereof. In some embodiments, the
TNFRSF agonist is added
at a concentration sufficient to achieve a concentration in the cell culture
medium of between 0.1
ti.g/mL and 1001..t.g/mL. In some embodiments, the TNFRSF agonist is added at
a concentration
sufficient to achieve a concentration in the cell culture medium of between 20
ps/mL and 40 is/mL.
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[00786] In some embodiments, in addition to one or more TNFRSF agonists, the
cell culture
medium further comprises IL-2 at an initial concentration of about 3000 IU/mL
and OKT-3 antibody
at an initial concentration of about 30 ng/mL, and wherein the one or more
TNFRSF agonists
comprises a 4-1BB agonist.
[00787] In some embodiments, a combination of IL-2, IL-7, IL-15, and/or IL-21
are employed as a
combination during the second expansion. In some embodiments, IL-2, IL-7, IL-
15, and/or IL-21 as
well as any combinations thereof can be included during the second expansion,
including, for example
during a Step D processes according to Figure 8 (in particular, e.g., Figure
8A and/or Figure 8B
and/or Figure 8C and/or Figure 8D), as well as described herein. In some
embodiments, a
combination of IL-2, IL-15, and IL-21 are employed as a combination during the
second expansion.
In some embodiments, 1L-2, IL-15, and 1L-21 as well as any combinations
thereof can be included
during Step D processes according to Figure 8 (in particular, e.g., Figure 8A
and/or Figure 8B and/or
Figure 8C and/or Figure 8D) and as described herein.
[00788] In some embodiments, the second expansion can be conducted in a
supplemented cell
culture medium comprising TL-2, OKT-3, antigen-presenting feeder cells, and
optionally a TNFRSF
agonist. In some embodiments, the second expansion occurs in a supplemented
cell culture medium.
In some embodiments, the supplemented cell culture medium comprises IL-2, OKT-
3, and antigen-
presenting feeder cells. In some embodiments, the second cell culture medium
comprises IL-2. OKT-
3, and antigen-presenting cells (APCs; also referred to as antigen-presenting
feeder cells). In some
embodiments, the second expansion occurs in a cell culture medium comprising
IL-2, OKT-3, and
antigen-presenting feeder cells (i.e., antigen presenting cells).
[00789] In some embodiments, the second expansion culture media comprises
about 500 IU/mL of
IL-15, about 400 IU/mL of IL-15, about 300 IU/mL of IL-15, about 200 IU/mL of
IL-15, about 180
IU/mL of IL-15, about 160 IU/mL of IL-15, about 140 IU/mL of IL-15, about 120
IU/mL of IL-15, or
about 100 IU/mL of IL-15. In some embodiments, the second expansion culture
media comprises
about 500 IU/mL of IL-15 to about 100 IU/mL of IL-15. In some embodiments, the
second expansion
culture media comprises about 400 IU/mL of IL-15 to about 100 IU/mL of IL-15.
In some
embodiments, the second expansion culture media comprises about 300 IU/mL of
IL-15 to about 100
IU/mL of IL-15. In some embodiments, the second expansion culture media
comprises about 200
IU/mL of 1L-15. In some embodiments, the cell culture medium comprises about
180 IU/mL of IL-15.
In some embodiments, the cell culture medium further comprises IL-15. In some
embodiments, the
cell culture medium comprises about 180 IU/mL of IL-15.
[00790] In some embodiments, the second expansion culture media comprises
about 20 IU/mL of
IL-21, about 15 IU/mL of IL-21, about 12 IU/mL of IL-21, about 10 IU/mL of IL-
21, about 5 IU/mL
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of IL-21, about 4 IU/mL of IL-21, about 3 IU/mL of IL-21, about 2 IU/mL of IL-
21, about 1 IU/mL
of IL-21, or about 0.5 IU/mL of IL-21. In some embodiments, the second
expansion culture media
comprises about 20 IU/mL of TL-21 to about 0.5 TU/mL of IL-21. In some
embodiments, the second
expansion culture media comprises about 15 IU/mL of IL-21 to about 0.5 IU/mL
of IL-21. In some
embodiments, the second expansion culture media comprises about 12 IU/mL of IL-
21 to about 0.5
IU/mL of 1L-21. In some embodiments, the second expansion culture media
comprises about 10
IU/mL of IL-21 to about 0.5 IU/mL of IL-21. In some embodiments, the second
expansion culture
media comprises about 5 IU/mL of IL-21 to about 1 IU/mL of IL-21. In some
embodiments, the
second expansion culture media comprises about 2 IU/mL of IL-21. In some
embodiments, the cell
culture medium comprises about 1 IU/mL of IL-21. In some embodiments, the cell
culture medium
comprises about 0.5 IU/mL of IL-21. In some embodiments, the cell culture
medium further
comprises IL-21. In some embodiments, the cell culture medium comprises about
1 IU/mL of IL-21.
[00791] In some embodiments, the antigen-presenting feeder cells (APCs) are
PBMCs. In some
embodiments, the ratio of TILs to PBMCs and/or antigen-presenting cells in the
rapid expansion
and/or the second expansion is about 1 to 10, about 1 to 15, about 1 to 20,
about 1 to 25, about 1 to 30,
about 1 to 35, about 1 to 40, about 1 to 45, about 1 to 50, about 1 to 75,
about 1 to 100, about 1 to 125,
about 1 to 150, about 1 to 175, about 1 to 200, about 1 to 225, about 1 to
250, about 1 to 275, about 1
to 300, about 1 to 325, about 1 to 350, about 1 to 375, about 1 to 400, or
about 1 to 500. In some
embodiments, the ratio of TILs to PBMCs in the rapid expansion and/or the
second expansion is
between 1 to 50 and 1 to 300. in sonic embodiments, the ratio of TILs to PBMCs
in the rapid
expansion and/or the second expansion is between 1 to 100 and Ito 200.
[00792] In some embodiments, REP and/or the rapid second expansion is
performed in flasks with
the bulk TILs being mixed with a 100- or 200-fold excess of inactivated feeder
cells, wherein the
feeder cell concentration is at least 1.1 times (1.1X), 1.2X, 1.3X, 1.4X,
1.5X, 1.6X, 1.7X, 1.8X, 1.8X,
2X, 2.1X2.2X, 2.3X, 2.4X, 2.5X, 2.6X, 2.7X, 2.8X, 2.9X, 3.0X, 3.1X, 3.2X,
3.3X, 3.4X, 3.5X, 3.6X,
3.7X, 3.8X, 3.9X or 4.0X the feeder cell concentration in the priming first
expansion, 30 ng/mL
OKT3 anti-CD3 antibody and 6000 IU/mL IL-2 in 150 mL media. Media replacement
is done
(generally 2/3 media replacement via aspiration of 2/3 of spent media and
replacement with an equal
volume of fresh media) until the cells are transferred to an alternative
growth chamber. Alternative
growth chambers include G-REX flasks and gas permeable containers as more
fully discussed below.
[00793] In some embodiments, the rapid second expansion (which can include
processes referred to
as the REP process) is 7 to 9 days, as discussed in the examples and figures.
In some embodiments,
the second expansion is 7 days. In some embodiments, the second expansion is 8
days. In some
embodiments, the second expansion is 9 days.
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[00794] In some embodiments, the second expansion (which can include
expansions referred to as
REP, as well as those referred to in Step D of Figure 8 (in particular, e.g.,
Figure 8A and/or Figure 8B
and/or Figure 8C and/or Figure 8D) may be performed in 500 mL capacity gas
permeable flasks with
100 cm gas-permeable silicon bottoms (G-REX 100, commercially available from
Wilson Wolf
Manufacturing Corporation, New Brighton, MN, USA), 5 x 106 or 10 x 106 TIL may
be cultured with
PBMCs in 400 mL of 50/50 medium, supplemented with 5% human AB serum, 3000 IU
per mL of
IL-2 and 30 ng per mL of anti-CD3 (OKT3). The G-REX 100 flasks may be
incubated at 37 C in 5%
CO2. On day 5, 250 mL of supernatant may be removed and placed into centrifuge
bottles and
centrifuged at 1500 rpm (491 x g) for 10 minutes. The TIL pellets may be re-
suspended with 150 mL
of fresh medium with 5% human AB serum, 6000 IU per mL of IL-2, and added back
to the original
GREX-100 flasks. When TIL are expanded serially in GREX-100 flasks, on day 10
or lithe TILs
can be moved to a larger flask, such as a GREX-500. The cells may be harvested
on day 14 of culture.
The cells may be harvested on day 15 of culture. The cells may be harvested on
day 16 of culture. In
some embodiments, media replacement is done until the cells are transferred to
an alternative growth
chamber. In some embodiments, 2/3 of the media is replaced by aspiration of
spent media and
replacement with an equal volume of fresh media. In some embodiments,
alternative growth chambers
include GREX flasks and gas permeable containers as more fully discussed
below.
[007951 In some embodiments, the culture medium used in the expansion
processes disclosed herein
is a serum-free medium or a defined medium. In some embodiments, the serum-
free or defined
medium comprises a basal cell medium and a serum supplement and/or a serum
replacement. In some
embodiments, the serum-free or defined medium is used to prevent and/or
decrease experimental
variation due in part to the lot-to-lot variation of serum-containing media.
[007961 In some embodiments, the serum-free or defined medium comprises a
basal cell medium
and a serum supplement and/or serum replacement. In some embodiments, the
basal cell medium
includes, bui; is not limited to CTSTm OpTmizerTm T-cell Expansion Basal
Medium , CTSIm
OpTmizeirm T-Coli Expansion STM, CTS:im AIM-V Medium, CTS" AIM-V STM,
LymphoONEim
T-Ceil Expansion Xeno-Free Medium, Dulbecco's Modified Eagle's Medium (DMEM),
Minimal
Essential Medium (MEM), Basal Medium Eagle (B1'.'viE)õ RPM! 1640, F-10, F-1.2,
Minimal Essential
Medium (aMEM), Glasgow's Minimal Essential Medium (G-MEM)õ RPMI growth mediumõ
and
Iseove's Modified Dulbeceo's Medium.
[00797] In some embodiments, the serum supplement or serum replacement
includes, but is not
limited to one or more of CTSTm OpTmizer T-Cell Expansion Serum Supplement,
CTSTm Immune
Cell Serum Replacement, one or more albumins or albumin substitutes, one or
more amino acids, one
or more vitamins, one or more transferrins or transferrin substitutes, one or
more antioxidants, one or
more insulins or insulin substitutes, one or more collagen precursors, one or
more antibiotics, and one
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or more trace elements. In some embodiments, the defined medium comprises
albumin and one or
more ingredients selected from the group consisting of glycine, L- histidine,
L-isoleucine, L-
methionine, L-phenylalanine, L-proline, L- hydroxyproline, L-serine, L-
threonine, L-tryptophan, L-
tyrosine, L-valine, thiamine, reduced glutathione, L-ascorbic acid-2-
phosphate, iron saturated
transferrin, insulin, and compounds containing the trace element moieties Ag '
, A13', Ba2', Cd2' , Co2',
Cr, Get', Se', Br, T, Mn2 , P. Si4, v5+, mcp% Ni2-% Rb , Sn2 and Zr4 . In
some embodiments, the
defined medium further comprises L-glutamine, sodium bicarbonate and/or 2-
mercaptoethanol.
[00798] In some embodiments, the CTSTmOpTmizerTm T-cell Immune Cell Senim
Replacement is
used with conventional growth media, including but not limited to CTSTm
OpTmizerTm T-cell
Expansion Basal Medium, CTSTm OpTmizerTm T-cell Expansion SFM, CTSTm AIM-V
Medium,
CSTTm AIM-V SFM, LymphoONETM T-Cell Expansion Xeno-Free Medium, Dulbecco's
Modified
Eagle's Medium (DMEM), Minimal Essential Medium (MEM), Basal Medium Eagle
(BME), RPMI
1640, F-10, F-12, Minimal Essential Medium (aMEM), Glasgow's Minimal Essential
Medium (G-
MEM), RPMI growth medium, and Iscove's Modified Dulbecco's Medium.
[00799] In some embodiments, the total serum replacement concentration (vol%)
in the serum-free
or defined medium is from about 1%, 2%, 3%, 4%, 5%, 6%, 7%, 8%, 9%, 10%, 11%,
12%, 13%,
14%, 15%, 16%, 17%, 18%, 19%, or 20% by volume of the total serum-free or
defined medium. In
some embodiments, the total serum replacement concentration is about 3% of the
total volume of the
serum-free or defined medium. In some embodiments, the total serum replacement
concentration is
about 5% of the total volume of the serum-free or defined medium. In some
embodiments, the total
scrum replacement concentration is about 10% of the total volume of the serum-
free or defined
medium.
[00800] In some embodiments, the serum-free or defined medium is CTSTm
OpTmizerTm T-cell
Expansion SFM (ThermoFisher Scientific). Any formulation of CTSTm OpTmizerTm
is useful in the
present invention. CTSTm OpTmizerTm T-cell Expansion SFM is a combination of
1L CTSTm
OpTmizerTm T-cell Expansion Basal Medium and 26 mL CTSTm OpTmizerTm T-Cell
Expansion
Supplement, which are mixed together prior to use. In some embodiments, the
CTSTm OpTmizerTm T-
cell Expansion SFM is supplemented with about 3% of the CTSTm Immune Cell
Serum Replacement
(SR) (ThermoFisher Scientific), along with 2-mercaptoethanol at 55mM.
[00801] In some embodiments, the defined medium is CTSTm OpTmizerTm T-cell
Expansion SFM
(ThermoFisher Scientific). Any formulation of CTSTm OpTmizerTm is useful in
the present invention.
CTSTm OpTmizerTm T-cell Expansion SFM is a combination of 1L CTSTm OpTmizerTm
T-cell
Expansion Basal Medium and 26 mL CTSTm OpTmizerTm T-Cell Expansion Supplement,
which are
mixed together prior to use. In some embodiments, the CTSTm OpTmizerTm T-cell
Expansion SFM is
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supplemented with about 3% of the CTSTm Immune Cell Serum Replacement (SR)
(ThermoFisher
Scientific), along with 2-mercaptoethanol at 55mM. In some embodiments, the
CTSTmOpTmizerTm T-
eel] Expansion SFM is supplemented with about 3% of the CTSTm Immune Cell
Serum Replacement
(SR) (ThermoFisher Scientific), 55mM of 2-mercaptoethanol, and 2mM of L-
glutamine. In some
embodiments, the CTSTmOpTmizerTm T-cell Expansion SFM is supplemented with
about 3% of the
CIS lm Immune Cell Scrum Replacement (SR) (ThermoFisher Scientific), 55mM of 2-
mercaptoethanol, and 2mM of L-glutamine, and further comprises about 1000
IU/mL to about 8000
IU/mL of IL-2. In some embodiments, the CTSTmOpTmizerTm T-cell Expansion SFM
is
supplemented with about 3% of the CTSTm Immune Cell Serum Replacement (SR)
(ThermoFisher
Scientific), 55mM of 2-mercaptoethanol, and 2mM of L-glutamine, and further
comprises about 3000
IU/mL of IL-2. In some embodiments, the CTSTmOpTmizerTm T-cell Expansion SFM
is
supplemented with about 3% of the CTSTm Immune Cell Serum Replacement (SR)
(ThermoFisher
Scientific), 55mM of 2-mercaptoethanol, and 2mM of L-glutaminc, and further
comprises about 6000
IU/mL of IL-2. In some embodiments, the CTSTmOpTmizerTm T-cell Expansion SFM
is
supplemented with about 3% of the CTSTm Immune Cell Serum Replacement (SR)
(ThermoFisher
Scientific) and 55mM of 2-mercaptoethanol, and further comprises about 1000
IU/mL to about 8000
IU/mL of IL-2. In some embodiments, the CTSTmOpTmizerTm T-cell Expansion SFM
is
supplemented with about 3% of the CTSTm Immune Cell Serum Replacement (SR)
(ThermoFisher
Scientific) and 55mM of 2-mercaptoethanol, and further comprises about 3000
IU/mL of IL-2. In
some embodiments, the CTSTmOpTmizerTm T-cell Expansion SFM is supplemented
with about 3% of
the CTSTm Immune Cell Serum Replacement (SR) (ThermoFisher Scientific) and
55mM of 2-
mercaptoethanol, and further comprises about 1000 IU/mL to about 6000 IU/mL of
IL-2. In some
embodiments, the CTSTmOpTmizerTm T-cell Expansion SFM is supplemented with
about 3% of the
CTSTm Immune Cell Serum Replacement (SR) (ThermoFisher Scientific) and about
2mM glutamine,
and further comprises about 1000 IU/mL to about 8000 IU/mL of IL-2. In some
embodiments, the
CTSTmOpTmizerTm T-cell Expansion SFM is supplemented with about 3% of the
CTSTm Immune
Cell Serum Replacement (SR) (ThermoFisher Scientific) and about 2mM glutamine,
and further
comprises about 3000 IU/mL of IL-2. In some embodiments, the CTSTmOpTmizerTm T-
cell
Expansion SFM is supplemented with about 3% of the CTSTm Immune Cell Serum
Replacement (SR)
(ThermoFisher Scientific) and about 2mM glutamine, and further comprises about
6000 IU/mL of IL-
2.
[00802] In some embodiments, the serum-free medium or defined medium is
supplemented with
glutamine (i.e., GlutaMAX(R)) at a concentration of from about 0.1mM to about
10mM, 0.5mM to
about 9mM, 1mM to about 8mM, 2mM to about 7mM, 3mM to about 6mM, or 4mM to
about 5 mM.
In some embodiments, the serum-free medium or defined medium is supplemented
with glutamine
(i.e., GlutaMAX0) at a concentration of about 2mM.
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[00803] In some embodiments, the serum-free medium or defined medium is
supplemented with 2-
mercaptoethanol at a concentration of from about 5mM to about 150mM, 10mM to
about 140mM,
15mM to about 130mM, 20mM to about 120mM, 25mM to about 110mM, 30mM to about
100mM,
35mM to about 95mM, 40mM to about 90mM, 45mM to about 85mM, 50mM to about
80mM, 55mM
to about 75mM, 60mM to about 70mM, or about 65mM. In some embodiments, the
serum-free
medium or defined medium is supplemented with 2-mercaptoethanol at a
concentration of about
55mM.
[00804] In some embodiments, the defined media described in International
Patent Application
Publication No. WO/1998/030679 and U.S. Patent Application Publication No. US
2002/0076747 Al,
which is herein incorporated by reference, are useful in the present
invention. In that publication,
serum-free eukaryotic cell culture media are described. The scrum-free,
eukaryotic cell culture
medium includes a basal cell culture medium supplemented with a serum-free
supplement capable of
supporting the growth of cells in serum- free culture. The serum-free
eukaryotic cell culture medium
supplement comprises or is obtained by combining one or more ingredients
selected from the group
consisting of one or more albumins or albumin substitutes, one or more amino
acids, one or more
vitamins, one or more transferrins or transferrin substitutes, one or more
antioxidants, one or more
insulins or insulin substitutes, one or more collagen precursors, one or more
trace elements, and one
or more antibiotics. In some embodiments, the defined medium further comprises
L-glutamine,
sodium bicarbonate and/or beta-mercaptoethanol. In some embodiments, the
defined medium
comprises an albumin or an albumin substitute and one or more ingredients
selected from group
consisting of one or more amino acids, one or more vitamins, one or more
transferrins or transferrin
substitutes, one or more antioxidants, one or more insulins or insulin
substitutes, one or more collagen
precursors, and one or more trace elements. In some embodiments, the defined
medium comprises
albumin and one or more ingredients selected from the group consisting of
glycine, L- histidine, L-
isoleucine, L-methionine, L-phenylalanine, L-proline, L- hydroxyproline, L-
serine, L-threonine, L-
tryptophan, L-tyrosine, L-valine, thiamine, reduced glutathione, L-ascorbic
acid-2-phosphate, iron
saturated transferrin, insulin, and compounds containing the trace element
moieties Ag+, Al', Ba2+,
Cd', Co', Cr', Ge", Se", Br, T, mn2+, P. si4+, v5+, mo6+, Ni2+, w
Sn' and Zr". In some
embodiments, the basal cell media is selected from the group consisting of
Dulbecco's Modified
Eagle's Medium (DMEM), Minimal Essential Medium (MEM), Basal Medium Eagle
(BME), RPMI
1640, F-10, F-12, Minimal Essential Medium (aMEM), Glasgow's Minimal Essential
Medium (G-
MEM), RPMI growth medium, and Iscove's Modified Dulbecco's Medium.
[00805] In some embodiments, the concentration of glycine in the defined
medium is in the range of
from about 5-200 mg/L, the concentration of L- histidine is about 5-250 mg/L,
the concentration of L-
isoleucine is about 5-300 mg/L, the concentration of L-methionine is about 5-
200 mg/L, the
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concentration of L-phenylalanine is about 5-400 mg/L, the concentration of L-
proline is about 1-1000
mg/L, the concentration of L- hydroxyproline is about 1-45 mg/L, the
concentration of L-serine is
about 1-250 mg/L, the concentration of L-threonine is about 10-500 mg/L, the
concentration of L-
tryptophan is about 2-110 mg/L, the concentration of L-tyrosine is about 3-175
mg/L, the
concentration of L-valine is about 5-500 mg/L, the concentration of thiamine
is about 1-20 mg/L, the
concentration of reduced glutathione is about 1-20 mg/L, the concentration of
L-ascorbic acid-2-
phosphate is about 1-200 mg/L, the concentration of iron saturated transferrin
is about 1-50 mg/L, the
concentration of insulin is about 1-100 mg/L, the concentration of sodium
selenite is about 0.000001-
0.0001 mg/L, and the concentration of albumin (e.g., AlbuMAX0 I) is about 5000-
50,000 mg/L.
[00806] In some embodiments, the non-trace element moiety ingredients in the
defined medium are
present in the concentration ranges listed in the column under the heading
"Concentration Range in
1X Medium" in Table 4. In other embodiments, the non-trace element moiety
ingredients in the
defined medium are present in the final concentrations listed in the column
under the heading "A
Preferred Embodiment of the 1X Medium" in Table 4. In other embodiments, the
defined medium is a
basal cell medium comprising a serum free supplement. In some of these
embodiments, the serum free
supplement comprises non-trace moiety ingredients of the type and in the
concentrations listed in the
column under the heading "A Preferred Embodiment in Supplement- in Table 4.
[00807] In some embodiments, the osmolarity of the defined medium is between
about 260 and 350
mOsmol. In some embodiments, the osmolarity is between about 280 and 310
mOsmol. In some
embodiments, the defined medium is supplemented with up to about 3.7 g/L, or
about 2.2 g/L sodium
bicarbonate. The defined medium can be further supplemented with L-glutaminc
(final concentration
of about 2 mM), one or more antibiotics, non-essential amino acids (NEAA;
final concentration of
about 100 ptM), 2-mercaptoethanol (final concentration of about 100 ilM).
[00808] In some embodiments, the defined media described in Smith, et at.,
Clin Transl
Immunology, 4(1) 2015 (doi: 10.1038/cti.2014.31) are useful in the present
invention. Briefly, RPMI
or CTSTm OpTmizerTm was used as the basal cell medium, and supplemented with
either 0, 2%, 5%,
or 10% CTSTm Immune Cell Scrum Replacement.
[00809] In some embodiments, the cell medium in the first and/or second gas
permeable container is
unfiltered. The use of unfiltered cell medium may simplify the procedures
necessary to expand the
number of cells. In some embodiments, the cell medium in the first and/or
second gas permeable
container lacks beta-mercaptoethanol (BME or PME; also known as 2-
mercaptoethanol, CAS 60-24-
2).
[00810] In some embodiments, the rapid second expansion (including expansions
referred to as
REP) is performed and further comprises a step wherein TILs are selected for
superior tumor
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reactivity. Any selection method known in the art may be used. For example,
the methods described
in U.S. Patent Application Publication No. 2016/0010058 Al, the disclosures of
which are
incorporated herein by reference, may be used for selection of TILs for
superior tumor reactivity.
[00811] Optionally, a cell viability assay can be performed after the rapid
second expansion
(including expansions referred to as the REP expansion), using standard assays
known in the art. For
example, a trypan blue exclusion assay can be done on a sample of the bulk
TILs, which selectively
labels dead cells and allows a viability assessment. In some embodiments, TIL
samples can be
counted and viability determined using a Cellometer K2 automated cell counter
(Nexcelom
Bioscience, Lawrence, MA). In some embodiments, viability is determined
according to the standard
Cellometer K2 Image Cytometer Automatic Cell Counter protocol.
[00812] The diverse antigen receptors of T and B lymphocytes are produced by
somatic
recombination of a limited, but large number of gene segments. These gene
segments: V (variable), D
(diversity), J (joining), and C (constant), determine the binding specificity
and downstream
applications of immunoglobulins and T-cell receptors (TCRs). The present
invention provides a
method for generating TILs which exhibit and increase the T-cell repertoire
diversity. In some
embodiments, the TILs obtained by the present method exhibit an increase in
the T-cell repertoire
diversity. In some embodiments, the TILs obtained in the second expansion
exhibit an increase in the
T-cell repertoire diversity. In some embodiments, the increase in diversity is
an increase in the
immunoglobulin diversity and/or the T-cell receptor diversity. In some
embodiments, the diversity is
in the immunoglobulin is in the immunoglobulin heavy chain. In some
embodiments, the diversity is
in the immunoglobulin is in the immunoglobulin light chain. In some
embodiments, the diversity is in
the T-cell receptor. In some embodiments, the diversity is in one of the T-
cell receptors selected from
the group consisting of alpha, beta, gamma, and delta receptors. In some
embodiments, there is an
increase in the expression of T-cell receptor (TCR) alpha and/or beta. In some
embodiments, there is
an increase in the expression of T-cell receptor (TCR) alpha. In some
embodiments, there is an
increase in the expression of T-cell receptor (TCR) beta. In some embodiments,
there is an increase in
the expression of TCRab (i.e., TCRa/r3).
[00813] In some embodiments, the rapid second expansion culture medium (e.g.,
sometimes
referred to as CM2 or the second cell culture medium), comprises IL-2, OKT-3,
as well as the
antigen-presenting feeder cells (APCs), as discussed in more detail below. In
some embodiments,
the rapid second expansion culture medium (e.g., sometimes referred to as CM2
or the second cell
culture medium), comprises 6000 IU/mL IL-2, 30 ug/flask OKT-3, as well as 7.5
x 108 antigen-
presenting feeder cells (APCs), as discussed in more detail below. In some
embodiments, the rapid
second expansion culture medium (e.g., sometimes referred to as CM2 or the
second cell culture
medium), comprises IL-2, OKT-3, as well as the antigen-presenting feeder cells
(APCs), as
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discussed in more detail below. In some embodiments, the rapid second
expansion culture medium
(e.g., sometimes referred to as CM2 or the second cell culture medium),
comprises 6000 IU/mL
1L-2, 30 ug/flask OKT-3, as well as 5 > l0 antigen-presenting feeder cells
(APCs), as discussed in
more detail below.
[00814] In some embodiments, the rapid second expansion, for example, Step D
according to Figure
8 (in particular, e.g., Figure 8A and/or Figure 8B and/or Figure 8C and/or
Figure 8D), is performed in
a closed system bioreactor. In some embodiments, a closed system is employed
for the TIL
expansion, as described herein. In sonic embodiments, a bioreactor is
employed. In some
embodiments, a bioreactor is employed as the container. In some embodiments,
the bioreactor
employed is for example a G-REX-100 or a G-REX-500. In some embodiments, the
bioreactor
employed is a G-REX-100. In some embodiments, the bioreactor employed is a G-
REX-500.
[00815] In some embodiments, the step of rapid second expansion
is split into a plurality of
steps to achieve a scaling up of the culture by: (a) performing the rapid
second expansion by culturing
TILs in a small scale culture in a first container, e.g., a G-REX-100 MCS
container, for a period of
about 3 to 7 days, and then (b) effecting the transfer of the TILs in the
small scale culture to a second
container larger than the first container, e.g., a G-REX-500-MCS container,
and culturing the TILs
from the small scale culture in a larger scale culture in the second container
for a period of about 4 to
7 days.
[00816] In some embodiments, the step of rapid second expansion
is split into a plurality of
steps to achieve a scaling out of the culture by: (a) performing the rapid
second expansion by
culturing TILs in a first small scale culture in a first container, e.g., a G-
REX-100 MCS container, for
a period of about 3 to 7 days, and then (b) effecting the transfer and
apportioning of the TILs from the
first small scale culture into and amongst at least 2, 3, 4, 5, 6, 7, 8, 9,
10, 11, 12, 13, 14, 15, 16, 17, 18,
19, or 20 second containers that are equal in size to the first container,
wherein in each second
container the portion of the TILs from first small scale culture transferred
to such second container is
cultured in a second small scale culture for a period of about 4 to 7 days.
[00817] In some embodiments, the first small scale TIL culture is
apportioned into a plurality
of about 2 to 5 subpopulations of TILs.
[00818] In some embodiments, the step of rapid second expansion
is split into a plurality of
steps to achieve a scaling out and scaling up of the culture by: (a)
performing the rapid second
expansion by culturing TILs in a small scale culture in a first container,
e.g., a G-REX-100 MCS
container, for a period of about 3 to 7 days, and then (b) effecting the
transfer and apportioning of the
TILs from the small scale culture into and amongst at least 2, 3, 4, 5, 6, 7,
8, 9, 10, 11, 12, 13, 14, 15,
16, 17, 18, 19, or 20 second containers that are larger in size than the first
container, e.g., G-REX-
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500MCS containers, wherein in each second container the portion of the TILs
from the small scale
culture transferred to such second container is cultured in a larger scale
culture for a period of about 4
to 7 days.
[00819] In some embodiments, the step of rapid second expansion
is split into a plurality of
steps to achieve a scaling out and scaling up of the culture by: (a)
performing the rapid or second
expansion by culturing TILs in a small scale culture in a first container,
e.g., a G-REX-100 MCS
container, for a period of about 5 days, and then (b) effecting the transfer
and apportioning of the TILs
from the small scale culture into and amongst 2, 3 or 4 second containers that
are larger in size than
the first container, e.g., G-REX-500 MCS containers, wherein in each second
container the portion of
the TILs from the small scale culture transferred to such second container is
cultured in a larger scale
culture for a period of about 6 days.
[00820] In some embodiments, upon the splitting of the rapid
second expansion, each second
container comprises at least 108 TILs. In some embodiments, upon the splitting
of the rapid or second
expansion, each second container comprises at least 108 TILs, at least 109
TILs, or at least 1010 TILs.
In one exemplary embodiment, each second container comprises at least 1010
TILs.
[00821] In some embodiments, the first small scale TIL culture is
apportioned into a plurality
of subpopulations. In some embodiments, the first small scale TIL culture is
apportioned into a
plurality of about 2 to 5 subpopulations. In some embodiments, the first small
scale TIL culture is
apportioned into a plurality of about 2, 3, 4, or 5 subpopulations.
[00822] In some embodiments, after the completion of the rapid
second expansion, the
plurality of subpopulations comprises a therapeutically effective amount of
TILs. In some
embodiments, after the completion of the rapid or second expansion, one or
more subpopulations of
TILs are pooled together to produce a therapeutically effective amount of
TILs. In some
embodiments, after the completion of the rapid expansion, each subpopulation
of TILs comprises a
therapeutically effective amount of TILs.
[00823] In some embodiments, the rapid second expansion is
performed for a period of about
3 to 7 days before being split into a plurality of steps. In some embodiments,
the splitting of the rapid
second expansion occurs at about day 3, day 4, day 5, day 6, or day 7 after
the initiation of the rapid
or second expansion.
[00824] In some embodiments, the splitting of the rapid second
expansion occurs at about day
7, day 8, day 9, day 10, day 11, day 12, day 13, day 14, day 15, or day 16 day
17, or day 18 after the
initiation of the first expansion (i.e., pre-REP expansion). In one exemplary
embodiment, the splitting
of the rapid or second expansion occurs at about day 16 after the initiation
of the first expansion.
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[00825] In some embodiments, the rapid second expansion is
further performed for a period of
about 7 to 11 days after the splitting. In some embodiments, the rapid second
expansion is further
performed for a period of about 5 days, 6 days, 7 days, 8 days, 9 days, 10
days, or 11 days after the
splitting.
[00826] In some embodiments, the cell culture medium used for the
rapid second expansion
before the splitting comprises the same components as the cell culture medium
used for the rapid
second expansion after the splitting. In some embodiments, the cell culture
medium used for the rapid
second expansion before the splitting comprises different components from the
cell culture medium
used for the rapid second expansion after the splitting.
[00827] In some embodiments, the cell culture medium used for the
rapid second expansion
before the splitting comprises IL-2, optionally OKT-3 and further optionally
APCs. In some
embodiments, the cell culture medium used for the rapid second expansion
before the splitting
comprises IL-2, OKT-3, and further optionally APCs. In some embodiments, the
cell culture medium
used for the rapid second expansion before the splitting comprises IL-2, OKT-3
and APCs.
[00828] In some embodiments, the cell culture medium used for the
rapid second expansion
before the splitting is generated by supplementing the cell culture medium in
the first expansion with
fresh culture medium comprising IL-2, optionally OKT-3 and further optionally
APCs. In some
embodiments, the cell culture medium used for the rapid second expansion
before the splitting is
generated by supplementing the cell culture medium in the first expansion with
fresh culture medium
comprising IL-2, OKT-3 and APCs. In some embodiments, the cell culture medium
used for the rapid
second expansion before the splitting is generated by replacing the cell
culture medium in the first
expansion with fresh cell culture medium comprising IL-2, optionally OKT-3 and
further optionally
APCs. In some embodiments, the cell culture medium used for the rapid second
expansion before the
splitting is generated by replacing the cell culture medium in the first
expansion with fresh cell culture
medium comprising IL-2, OKT-3 and APCs.
[00829] In some embodiments, the cell culture medium used for the
rapid second expansion
after the splitting comprises IL-2, and optionally OKT-3. In some embodiments,
the cell culture
medium used for the rapid second expansion after the splitting comprises IL-2,
and OKT-3. In some
embodiments, the cell culture medium used for the rapid second expansion after
the splitting is
generated by replacing the cell culture medium used for the rapid second
expansion before the
splitting with fresh culture medium comprising IL-2 and optionally OKT-3. In
some embodiments,
the cell culture medium used for the rapid second expansion after the
splitting is generated by
replacing the cell culture medium used for the rapid second expansion before
the splitting with fresh
culture medium comprising IL-2 and OKT-3.
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1. Feeder Cells and Antigen Presenting Cells
[00830] In some embodiments, the rapid second expansion procedures described
herein (for
example including expansion such as those described in Step D from Figure 8
(in particular, e.g.,
Figure 8A and/or Figure 8B and/or Figure 8C and/or Figure 8D), as well as
those referred to as REP)
require an excess of feeder cells during REP TIL expansion and/or during the
rapid second expansion.
In many embodiments, the feeder cells are peripheral blood mononuclear cells
(PBMCs) obtained
from standard whole blood units from healthy blood donors. The PBMCs are
obtained using standard
methods such as Ficoll-Paque gradient separation.
[00831] In general, the allogenic PBMCs are inactivated, either via
irradiation or heat treatment, and
used in the REP procedures, as described in the examples, which provides an
exemplary protocol for
evaluating the replication incompetence of irradiate allogcncic PBMCs.
[00832] In some embodiments, PBMCs are considered replication incompetent and
acceptable for
use in the TIL expansion procedures described herein if the total number of
viable cells on day 7 or 14
is less than the initial viable cell number put into culture on day 0 of the
REP and/or day 0 of the
second expansion (i.e., the start day of the second expansion).
[00833] hi sonic embodiments, PBMCs are considered replication incompetent and
acceptable for
use in the TIL expansion procedures described herein if the total number of
viable cells, cultured in
the presence of OKT3 and IL-2, on day 7 and day 14 has not increased from the
initial viable cell
number put into culture on day 0 of the REP and/or day 0 of the second
expansion (i.e., the start day
of the second expansion). In some embodiments, the PBMCs are cultured in the
presence of 30 ng/mL
OKT3 antibody and 3000 IU/mL 1L-2. In some embodiments, the PBMCs are cultured
in the presence
of 60 ng/mL OKT3 antibody and 6000 IU/mL IL-2. In some embodiments, the PBMCs
are cultured in
the presence of 60 ng/mL OKT3 antibody and 3000 IU/mL IL-2. In some
embodiments, the PBMCs
are cultured in the presence of 30 ng/mL OKT3 antibody and 6000 IU/mL IL-2.
[00834] In some embodiments, PBMCs are considered replication incompetent and
acceptable for
use in the TIL expansion procedures described herein if the total number of
viable cells, cultured in
the presence of OKT3 and IL-2, on day 7 and day 14 has not increased from the
initial viable cell
number put into culture on day 0 of the REP and/or day 0 of the second
expansion (i.e., the start day
of the second expansion). In some embodiments, the PBMCs are cultured in the
presence of 30-60
ng/mL OKT3 antibody and 1000-6000 IU/mL IL-2. In some embodiments, the PBMCs
are cultured in
the presence of 30-60 ng/mL OKT3 antibody and 2000-5000 IU/mL IL-2. In some
embodiments, the
PBMCs are cultured in the presence of 30-60 ng/mL OKT3 antibody and 2000-4000
IU/mL IL-2. In
some embodiments, the PBMCs are cultured in the presence of 30-60 ng/mL OKT3
antibody and
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2500-3500 IU/mL IL-2. In some embodiments, the PBMCs are cultured in the
presence of 30-60
ng/mL OKT3 antibody and 6000 IU/mL IL-2.
[00835] hi some embodiments, the antigen-presenting feeder cells are PBMCs. In
some
embodiments, the antigen-presenting feeder cells are artificial antigen-
presenting feeder cells. In some
embodiments, the ratio of TILs to antigen-presenting feeder cells in the
second expansion is about 1 to
10, about 1 to 25, about 1 to 50, about 1 to 100, about 1 to 125, about 1 to
150, about 1 to 175, about 1
to 200, about 1 to 225, about 1 to 250, about 1 to 275, about 1 to 300, about
1 to 325, about 1 to 350,
about 1 to 375, about 1 to 400, or about 1 to 500. In some embodiments, the
ratio of TII,s to antigen-
presenting feeder cells in the second expansion is between 1 to 50 and 1 to
300. In some
embodiments, the ratio of TILs to antigen-presenting feeder cells in the
second expansion is between
1 to 100 and 1 to 200.
[00836] In some embodiments, the second expansion procedures described herein
require a ratio of
about 5 x 108 feeder cells to about 100 x 106 TILs. In some embodiments, the
second expansion
procedures described herein require a ratio of about 7.5 x 108 feeder cells to
about 100 >< 106 TILs. In
some embodiments, the second expansion procedures described herein require a
ratio of about 5 x 108
feeder cells to about 50 >< 106 TILs. In some embodiments, the second
expansion procedures described
herein require a ratio of about 7.5 x 108 feeder cells to about 50 x 106 TILs.
In yet another
embodiment, the second expansion procedures described herein require about 5
108 feeder cells to
about 25 x 106 TILs. In yet another embodiment, the second expansion
procedures described herein
require about 7.5 x 108 feeder cells to about 25 x 106 TILs. In yet another
embodiment, the rapid
second expansion requires twice the number of feeder cells as the rapid second
expansion. In yet
another embodiment, when the priming first expansion described herein requires
about 2.5 x 108
feeder cells, the rapid second expansion requires about 5 >< 108 feeder cells.
In yet another
embodiment, when the priming first expansion described herein requires about
2.5x 108 feeder cells,
the rapid second expansion requires about 7.5 x 108 feeder cells. In yet
another embodiment, the rapid
second expansion requires two times (2.0X), 2.5X, 3.0X, 3.5X or 4.0X the
number of feeder cells as
the priming first expansion.
[00837] In some embodiments, the rapid second expansion procedures described
herein require an
excess of feeder cells during the rapid second expansion. In many embodiments,
the feeder cells are
peripheral blood mononuclear cells (PBMCs) obtained from standard whole blood
units from
allogeneic healthy blood donors. The PBMCs are obtained using standard methods
such as Ficoll-
Paque gradient separation. In some embodiments, artificial antigen-presenting
(aAPC) cells are used
in place of PBMCs. In some embodiments, the PBMCs are added to the rapid
second expansion at
twice the concentration of PBMCs that were added to the priming first
expansion.
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[00838] In general, the allogenic PBMCs are inactivated, either via
irradiation or heat treatment, and
used in the TIL expansion procedures described herein, including the exemplary
procedures described
in the figures and examples.
[00839] In some embodiments, artificial antigen presenting cells are used in
the rapid second
expansion as a replacement for, or in combination with, PBMCs.
2. Cytokincs
[00840] The rapid second expansion methods described herein generally use
culture media with
high doses of a cytokine, in particular IL-2, as is known in the art.
[00841] Alternatively, using combinations of cytokines for the rapid second
expansion of TILs is
additionally possible, with combinations of two or more of IL-2, IL-15 and IL-
21 as is generally
outlined in WO 2015/189356 and WO 2015/189357, hereby expressly incorporated
by reference in
their entirety. Thus, possible combinations include IL-2 and IL-15, IL-2 and
IL-21, IL-15 and IL-21,
and IL-2, IL-15 and IL-21, with the latter finding particular use in many
embodiments. The use of
combinations of cytokines specifically favors the generation of lymphocytes,
and in particular T-cells
as described therein.
[00842] In some embodiments, Step D (from in particular, e.g., Figure 8A
and/or Figure 8B and/or
Figure 8C and/or Figure 8D) may also include the addition of OKT-3 antibody or
muromonab to the
culture media, as described elsewhere herein. In some embodiments, Step D may
also include the
addition of a 4-1BB agonist to the culture media, as described elsewhere
herein. In some
embodiments, Step D (from, in particular, e.g., Figure 8A and/or Figure 8B
and/or Figure 8C and/or
Figure SD) may also include the addition of an OX-40 agonist to the culture
media, as described
elsewhere herein. In addition, additives such as peroxisome proliferator-
activated receptor gamma
coactivator I-alpha agonists, including proliferator-activated receptor (PPAR)-
gamma agonists such as
a thiazolidincdionc compound, may be used in the culture media during Step D
(from, in particular,
e.g., Figure 8A and/or Figure 8B and/or Figure 8C and/or Figure 8D), as
described in U.S. Patent
Application Publication No. US 2019/0307796 Al, the disclosure of which is
incorporated by
reference herein.
E. STEP E: Harvest TILs
1008431 After the rapid second expansion step, cells can be harvested. In some
embodiments the
TILs are harvested after one, two, three, four or more expansion steps, for
example as provided in
Figure 8 (in particular, e.g., Figure 8A and/or Figure 8B and/or Figure 8C
and/or Figure 8D). In some
embodiments the TILs are harvested after two expansion steps, for example as
provided in Figure 8
(in particular, e.g., Figure 8A and/or Figure 8B and/or Figure 8C and/or
Figure 8D). in some
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embodiments the TILs are harvested after two expansion steps, one priming
first expansion and one
rapid second expansion, for example as provided in Figure 8 (in particular,
e.g., Figure 8A and/or
Figure 8B and/or Figure 8C and/or Figure 8D).
[00844] TILs can bc harvested in any appropriate and sterile manner,
including, for example by
centrifugation. Methods for TIL harvesting are well known in the art and any
such known methods
can be employed with the present process. In some embodiments, TILs are
harvested using an
automated system.
[00845] Cell harvesters and/or cell processing systems are commercially
available from a variety of
sources, including, for example, Fresenius Kabi, Tomtec Life Science, Perkin
Elmer, and Inotech
Biosystems International, Inc. Any cell based harvester can be employed with
the present methods. In
some embodiments, the cell harvester and/or cell processing system is a
membrane-based cell
harvester. In some embodiments, cell harvesting is via a cell processing
system, such as the LOVO
system (manufactured by Fresenius Kabi). The term "LOVO cell processing
system" also refers to
any instrument or device manufactured by any vendor that can pump a solution
comprising cells
through a membrane or filter such as a spinning membrane or spinning filter in
a sterile and/or closed
system environment, allowing for continuous flow and cell processing to remove
supernatant or cell
culture media without pelletization. In some embodiments, the cell harvester
and/or cell processing
system can perform cell separation, washing, fluid-exchange, concentration,
and/or other cell
processing steps in a closed, sterile system.
[00846] In some embodiments, the rapid second expansion, for example, Step D
according to Figure
8 (in particular, e.g., Figure 8A and/or Figure 8B and/or Figure 8C and/or
Figure 8D), is performed in
a closed system bioreactor. In some embodiments, a closed system is employed
for the TIL
expansion, as described herein. In some embodiments, a bioreactor is employed.
In some
embodiments, a bioreactor is employed as the container. In some embodiments,
the bioreactor
employed is for example a G-REX-100 or a G-REX-500. In some embodiments, the
bioreactor
employed is a G-REX-100. In some embodiments, the bioreactor employed is a G-
REX-500.
[00847] In some embodiments, Step E according to Figure 8 (in particular,
e.g., Figure 8A and/or
Figure 8B and/or Figure 8C and/or Figure 8D), is performed according to the
processes described
herein. In some embodiments, the closed system is accessed via syringes under
sterile conditions in
order to maintain the sterility and closed nature of the system. In some
embodiments, a closed system
as described herein is employed.
[00848] In some embodiments, TILs are harvested according to the methods
described in herein. In
some embodiments, TILs between days 14 and 16 are harvested using the methods
as described
herein. In some embodiments, TILs are harvested at 14 days using the methods
as described herein. In
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some embodiments, TILs are harvested at 15 days using the methods as described
herein. In some
embodiments, TILs are harvested at 16 days using the methods as described
herein.
F. STEP F: Final Formulation and Transfer to Infusion Container
[00849] After Steps A through E as provided in an exemplary order in Figure 8
(in particular, e.g.,
Figure 8A and/or Figure 8B and/or Figure 8C and/or Figure 8D) and as outlined
in detailed above and
herein are complete, cells are transferred to a container for use in
administration to a patient, such as
an infusion bag or sterile vial. In some embodiments, once a therapeutically
sufficient number of TILs
are obtained using the expansion methods described above, they are transferred
to a container for use
in administration to a patient.
[00850] In some embodiments, TILs expanded using the methods of the present
disclosure are
administered to a patient as a pharmaceutical composition. In some
embodiments, the pharmaceutical
composition is a suspension of TILs in a sterile buffer. TILs expanded as
disclosed herein may be
administered by any suitable route as known in the art. In some embodiments,
the TILs are
administered as a single intra-arterial or intravenous infusion, which
preferably lasts approximately 30
to 60 minutes. Other suitable routes of administration include
intraperitoneal, intrathecal, and
intralymphatic.
V. Further Gen 2, Gen 3, and Other TIL Manufacturing Process
Embodiments
A. PBMC Feeder Cell Ratios
1008511 In some embodiments, the culture media used in expansion methods
described herein (see
for example, Figure 8 (in particular, e.g., Figure 8A and/or Figure 8B and/or
Figure 8C and/or Figure
8D)) include an anti-CD3 antibody e.g. OKT-3. An anti-CD3 antibody in
combination with IL-2
induces T cell activation and cell division in the TIL population. This effect
can be seen with full
length antibodies as well as Fab and F(ab')2 fragments, with the former being
generally preferred;
see, e.g., Tsoukas etal., I Inirnunol. 1985, 135, 1719, hereby incorporated by
reference in its entirety.
[00852] In some embodiments, the number of PBMC feeder layers is calculated as
follows:
A. Volume of a T-cell (10 gm diameter): V= (4/3) rre =523.6 gm"
B. Column of G-REX 100 (M) with a 40 gm (4 cells) height: V= (4/3) rre = 4><
1012 gm'
C. Number cell required to fill column B: 4x 1012 jim3 / 523.6 gm' = 7.6x108
gm' * 0.64 = 4.86< 108
D. Number cells that can be optimally activated in 4D space: 4.86x 108/ 24 =
20.25x 106
E. Number of feeders and TIL extrapolated to G-REX 500: TIL: 100< 106 and
Feeder: 2.5 x109
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In this calculation, an approximation of the number of mononuclear cells
required to provide an
icosahedral geometry for activation of TIL in a cylinder with a 100 cm2 base
is used. The calculation
derives the experimental result of ¨5 x10s for threshold activation of T-cells
which closely mirrors
NCI experimental data, as described in Jin, etal., I Immunother. 2012, 35, 283-
292. In (C), the
multiplier (0.64) is the random packing density for equivalent spheres as
calculated by Jaeger and
Nagel, Science, 1992, 255, 1523-3. In (D), the divisor 24 is the number of
equivalent spheres that
could contact a similar object in 4 -dimensional space or "the Newton number"
as described in Musin,
Russ. Math. Surv., 2003, 58, 794-795.
[00853] In other embodiments, the number of antigen-presenting feeder cells
exogenously supplied
during the priming first expansion is approximately one-half the number of
antigen-presenting feeder
cells exogenously supplied during the rapid second expansion. In certain
embodiments, the method
comprises performing the priming first expansion in a cell culture medium
which comprises
approximately 50% fewer antigen presenting cells as compared to the cell
culture medium of the rapid
second expansion.
[00854] In other embodiments, the number of antigen-presenting feeder cells
(APCs) exogenously
supplied during the rapid second expansion is greater than the number of APCs
exogenously supplied
during the priming first expansion.
[00855] In other embodiments, the ratio of the number of APCs exogenously
supplied during the
rapid second expansion to the number of APCs exogenously supplied during the
priming first
expansion is selected from a range of from at or about 1.1:1 to at or about
20:1.
[00856] In other embodiments, the ratio of the number of APCs exogenously
supplied during the
rapid second expansion to the number of APCs exogenously supplied during the
priming first
expansion is selected from a range of from at or about 1.1:1 to at or about
10:1.
[00857] In other embodiments, the ratio of the number of APCs exogenously
supplied during the
rapid second expansion to the number of APCs exogenously supplied during the
priming first
expansion is selected from a range of from at or about 1.1:1 to at or about
9:1.
[00858] In other embodiments, the ratio of the number of APCs exogenously
supplied during the
rapid second expansion to the number of APCs exogenously supplied during the
priming first
expansion is selected from a range of from at or about 1.1:1 to at or about
8:1.
[00859] In other embodiments, the ratio of the number of APCs exogenously
supplied during the
rapid second expansion to the number of APCs exogenously supplied during the
priming first
expansion is selected from a range of from at or about 1.1:1 to at or about
7:1.
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[00860] In other embodiments, the ratio of the number of APCs exogenously
supplied during the
rapid second expansion to the number of APCs exogenously supplied during the
priming first
expansion is selected from a range of from at or about 1.1:1 to at or about
6:1.
[00861] In other embodiments, the ratio of the number of APCs exogenously
supplied during the
rapid second expansion to the number of APCs exogenously supplied during the
priming first
expansion is selected from a range of from at or about 1.1:1 to at or about
5:1.
[00862] In other embodiments, the ratio of the number of APCs exogenously
supplied during the
rapid second expansion to the number of APCs exogenously supplied during the
priming first
expansion is selected from a range of from at or about 1.1:1 to at or about
4:1.
[00863] In other embodiments, the ratio of the number of APCs exogenously
supplied during the
rapid second expansion to the number of APCs exogenously supplied during the
priming first
expansion) is selected from a range of from at or about 1.1:1 to at or about
3:1.
[00864] In other embodiments, the ratio of the number of APCs exogenously
supplied during the
rapid second expansion to the number of APCs exogenously supplied during the
priming first
expansion is selected from a range of from at or about 1.1:1 to at or about
2.9:1.
[00865] In other embodiments, the ratio of the number of APCs exogenously
supplied during the
rapid second expansion to the number of APCs exogenously supplied during the
priming first
expansion is selected from a range of from at or about 1.1:1 to at or about
2.8:1.
[00866] In other embodiments, the ratio of the number of APCs exogenously
supplied during the
rapid second expansion to the number of APCs exogenously supplied during the
priming first
expansion is selected from a range of from at or about 1.1: Ito at or about
2.7:1.
[00867] In other embodiments, the ratio of the number of APCs exogenously
supplied during the
rapid second expansion to the number of APCs exogenously supplied during the
priming first
expansion is selected from a range of from at or about 1.1:1 to at or about
2.6:1.
[00868] In other embodiments, the ratio of the number of APCs exogenously
supplied during the
rapid second expansion to the number of APCs exogenously supplied during the
priming first
expansion is selected from a range of from at or about 1.1:1 to at or about
2.5:1.
[00869] In other embodiments, the ratio of the number of APCs exogenously
supplied during the
rapid second expansion to the number of APCs exogenously supplied during the
priming first
expansion is selected from a range of from at or about 1.1:1 to at or about
2.4:1.
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[00870] In other embodiments, the ratio of the number of APCs exogenously
supplied during the
rapid second expansion to the number of APCs exogenously supplied during the
priming first
expansion is selected from a range of from at or about 1.1:1 to at or about
2.3:1.
[00871] In other embodiments, the ratio of the number of APCs exogenously
supplied during the
rapid second expansion to the number of APCs exogenously supplied during the
priming first
expansion is selected from a range of from at or about 1.1:1 to at or about
2.2:1.
[00872] In other embodiments, the ratio of the number of APCs exogenously
supplied during the
rapid second expansion to the number of APCs exogenously supplied during the
priming first
expansion is selected from a range of from at or about 1.1:1 to at or about
2.1:1.
[00873] In other embodiments, the ratio of the number of APCs exogenously
supplied during the
rapid second expansion to the number of APCs exogenously supplied during the
priming first
expansion is selected from a range of from at or about 1.1:1 to at or about
2:1.
[00874] In other embodiments, the ratio of the number of APCs exogenously
supplied during the
rapid second expansion to the number of APCs exogenously supplied during the
priming first
expansion is selected from a range of from at or about 2:1 to at or about
10:1.
[00875] In other embodiments, the ratio of the number of APCs exogenously
supplied during the
rapid second expansion to the number of APCs exogenously supplied during the
priming first
expansion is selected from a range of from at or about 2:1 to at or about 5:1.
[00876] In other embodiments, the ratio of the number of APCs exogenously
supplied during the
rapid second expansion to the number of APCs exogenously supplied during the
priming first
expansion is selected from a range of from at or about 2:1 to at or about 4:1.
[00877] In other embodiments, the ratio of the number of APCs exogenously
supplied during the
rapid second expansion to the number of APCs exogenously supplied during the
priming first
expansion is selected from a range of from at or about 2:1 to at or about 3:1.
[00878] In other embodiments, the ratio of the number of APCs exogenously
supplied during the
rapid second expansion to the number of APCs exogenously supplied during the
priming first
expansion is selected from a range of from at or about 2:1 to at or about
2.9:1.
[00879] In other embodiments, the ratio of the number of APCs exogenously
supplied during the
rapid second expansion to the number of APCs exogenously supplied during the
priming first
expansion is selected from a range of from at or about 2:1 to at or about
2.8:1.
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[00880] In other embodiments, the ratio of the number of APCs exogenously
supplied during the
rapid second expansion to the number of APCs exogenously supplied during the
priming first
expansion is selected from a range of from at or about 2:1 to at or about
2.7:1.
[00881] In other embodiments, the ratio of the number of APCs exogenously
supplied during the
rapid second expansion to the number of APCs exogenously supplied during the
priming first
expansion is selected from a range of from at or about 2:1 to at or about
2.6:1.
[00882] In other embodiments, the ratio of the number of APCs exogenously
supplied during the
rapid second expansion to the number of APCs exogenously supplied during the
priming first
expansion is selected from a range of from at or about 2:1 to at or about
2.5:1.
[00883] In other embodiments, the ratio of the number of APCs exogenously
supplied during the
rapid second expansion to the number of APCs exogenously supplied during the
priming first
expansion is selected from a range of from at or about 2:1 to at or about
2.4:1.
[00884] In other embodiments, the ratio of the number of APCs exogenously
supplied during the
rapid second expansion to the number of APCs exogenously supplied during the
priming first
expansion is selected from a range of from at or about 2:1 to at or about
2.3:1.
[00885] In other embodiments, the ratio of the number of APCs exogenously
supplied during the
rapid second expansion to the number of APCs exogenously supplied during the
priming first
expansion is selected from a range of from at or about about 2:1 to at or
about 2.2:1.
[00886] In other embodiments, the ratio of the number of APCs exogenously
supplied during the
rapid second expansion to the number of APCs exogenously supplied during the
priming first
expansion is selected from a range of from at or about 2:1 to at or about
2.1:1.
[00887] In other embodiments, the ratio of the number of APCs exogenously
supplied during the
rapid second expansion to the number of APCs exogenously supplied during the
priming first
expansion is at or about 2:1.
[00888] In other embodiments, the ratio of the number of APCs exogenously
supplied during the
rapid second expansion to the number of APCs exogenously supplied during the
priming first
expansion is at or about 1.1:1, 1.2:1, 1.3:1, 1.4:1, 1.5:1, 1.6:1, 1.7:1,
1.8:1, 1.9:1, 2:1, 2.1:1, 2.2:1,
2.3:1, 2.4:1, 2.5:1, 2.6:1, 2.7:1, 2.8:1, 2.9:1, 3:1, 3.1:1, 3.2:1, 3.3:1,
3.4:1, 3.5:1, 3.6:1, 3.7:1, 3.8:1,
3.9:1, 4:1, 4.1:1, 4.2:1, 4.3:1, 4.4:1, 4.5:1, 4.6:1, 4.7:1, 4.8:1, 4.9:1, or
5:1.
[00889] In other embodiments, the number of APCs exogenously supplied during
the priming first
expansion is at or about 1><1O, 1.1x10, 1.2>1O, 1.3 x10', 1.4 x 10', 1.5 x 10,
1.6>1O, 1.7>1O,
1.8x108, 1.9x10, 2>1O, 2.1x 10', 2.2x108, 2.3x 10, 2.4><108, 2.5><108,
2.6><108, 2.7><108, 2.8><108,
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2.9x108, 3x108, 3.1x 108, 3.2x 108, 3.3 x108, 3.4x 108 or 3.5 x 108 APCs, and
the number of APCs
exogenously supplied during the rapid second expansion is at or about 3.5x
108, 3.6x 108, 3.7x 108,
3.8x108, 3.9x108, 4x108, 4.1x108, 4.2x108, 4.3x108, 4.4x108, 4.5x108, 4.6x108,
4.7x108, 4.8x108,
4.9x108, 5x108, 5.1x108, 5.2x108, 53x108 5.4x108, 55x108 5.6x108, 5.7x108,
5.8x108, 5.9x108,
6x108, 6.1x108, 6.2x108, 6.3x 108, 6.4x108, 6.5x108, 6.6x108, 6.7x108,
6.8x108, 6.9x108, 7x108,
7.1x108, 7.2x108, 7.3x 108, 7.4x 108, 7.5x108, 7.6x108, 7.7x108, 7.8x108,
7.9x108, 8x108, 8.1x108,
8.2><108, 8.3x108, 8.4x 108, 8.5 x 108, 8.6 x 108, 8.7 x 108, 8.8x108,
8.9x108, 9x108, 9.1><108, 9.2><108,
9.3x108, 9.4x108, 9.5x108, 9.6x 108, 9.7x 108, 9.8x 108, 9.9x108 or 1>(109
APCs.
[00890] In other embodiments, the number of APCs exogenously supplied during
the priming first
expansion is selected from the range of at or about 1.5 x108 APCs to at or
about 3x 108 APCs, and the
number of APCs exogenously supplied during the rapid second expansion is
selected from the range
of at or about 4x 108 APCs to at or about 7.5 x108 APCs.
[00891] In other embodiments, the number of APCs exogenously supplied during
the priming first
expansion is selected from the range of at or about 2x 108 APCs to at or about
2.5 x 108 APCs, and the
number of APCs exogenously supplied during the rapid second expansion is
selected from the range
of at or about 4.5 x108 APCs to at or about 5.5 x108 APCs.
[00892] In other embodiments, the number of APCs exogenously supplied during
the priming first
expansion is at or about 2.5 x108 APCs, and the number of APCs exogenously
supplied during the
rapid second expansion is at or about 5>< 108 APCs.
[00893] In other embodiments, the number of APCs (including, for example,
PBMCs) added at day
0 of the priming first expansion is approximately one-half of the number of
PBMCs added at day 7 of
the priming first expansion (e.g., day 7 of the method). In certain
embodiments, the method comprises
adding antigen presenting cells at day 0 of the priming first expansion to the
first population of TILs
and adding antigen presenting cells at day 7 to the second population of TILs,
wherein the number of
antigen presenting cells added at day 0 is approximately 50% of the number of
antigen presenting
cells added at day 7 of the priming first expansion (e.g., day 7 of the
method).
[00894] In other embodiments, the number of APCs (including, for example,
PBMCs) exogenously
supplied at day 7 of the rapid second expansion is greater than the number of
PBMCs exogenously
supplied at day 0 of the priming first expansion.
[00895] In other embodiments, the APCs exogenously supplied in the priming
first expansion are
seeded in the culture flask at a density selected from a range of at or about
1.0 x 106 APCs/cm2 to at or
about 4.5 x106 APCs/cm2.
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[00896] In other embodiments, the APCs exogenously supplied in the priming
first expansion are
seeded in the culture flask at a density selected from a range of at or about
l.5>< 106 APCs/cm2 to at or
about 3.5x 106 APCs/cm2.
[00897] In other embodiments, the APCs exogenously supplied in the priming
first expansion are
seeded in the culture flask at a density selected from a range of at or about
2 x106 APCs/cm2 to at or
about 3)< 106 APCs/cm2.
[00898] In other embodiments, the APCs exogenously supplied in the priming
first expansion are
seeded in the culture flask at a density of at or about 2 x 106 APCs/cm2.
[00899] In other embodiments, the APCs exogenously supplied in the priming
first expansion are
seeded in the culture flask at a density of at or about 1.0 x 106, 1.1x106,
1.2 x 106, 1.3x 106, 1.4x106,
1.5x106, 1.6x106, 1.7x106, 1.8x106, 1.9x106, 2x106, 2.1x106, 2.2x106, 2.3x106,
2.4x106, 2.5x106,
2.6x106, 2.7x106, 2.8x 106, 2.9x 106, 3 x106, 3.1x106, 3.2x106, 3.3><106,
3.4x106, 3.5x106, 3.6x106,
3.7x106, 3.8x106, 3.9x 106, 4x 106, 4.1 x 106, 4.2x106, 4.3><106, 4.4><106 or
4.5><106 APCs/cm2.
[00900] In other embodiments, the APCs exogenously supplied in the rapid
second expansion are
seeded in the culture flask at a density selected from a range of at or about
2.5 x106 APCs/cm2 to at or
about 7.5 x106 APCs/cm2.
[00901] In other embodiments, the APCs exogenously supplied in the rapid
second expansion are
seeded in the culture flask at a density selected from a range of at or about
3.5>< 106 APCs/cm2 to about
6.0 x106 APCs/cm2.
[00902] In other embodiments, the APCs exogenously supplied in the rapid
second expansion are
seeded in the culture flask at a density selected from a range of at or about
4.0> 106 APCs/cm2 to about
5.5 x106 APCs/cm2.
[00903] In other embodiments, the APCs exogenously supplied in the rapid
second expansion are
seeded in the culture flask at a density selected from a range of at or about
4.0>< 106 APCs/cm2.
[00904] In other embodiments, the APCs exogenously supplied in the rapid
second expansion are
seeded in the culture flask at a density of at or about 2.5>< 106 APCs/cm2,
2.6 x106 APCs/cm2, 2.7x 106
APCs/cm2, 2.8x106, 2.9x106, 3x106, 3.1x106, 3.2x106, 3.3x106, 3.4x106,
3.5x106, 3.6x106, 3.7x106,
3.8x106, 3.9x106, 4x106, 4.1x106, 4.2x106, 4.3x106, 4.4x106, 4.5x106, 4.6x106,
4.7x106, 4.8x106,
4.9x106, 5x106, 5.1x 106, 5.2<106, 5.3x106, 5.4x106, 5.5<106, 5.6><106,
5.7x106, 5.8x106, 5.9><106,
6x106, 6.1x106, 6.2 x 106, 6.3 x 106, 6.4 x 106, 6.5 x 106, 6.6x106, 6.7x106,
6.8x106, 6.9><106, 7x106
7.1x106, 7.2x106, 7.3x 106, 7.4x 106 or 75x106 APCs/cm2.
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[00905] In other embodiments, the APCs exogenously supplied in the priming
first expansion are
seeded in the culture flask at a density of at or about 1.0>106, 1.1>106, 1.2
x 106, 1.3 x 106, 1.4 x 10',
1.5>106, 1.6x106, 1.7x 106, 1.8x 106, 1.9x 106, 2x106, 2.1x106, 2.2 x106,
2.3x106, 2.4x106, 2.5 x106,
2.6>106,2.7>106, 2.8x 106, 2.9x 106, 3x 106, 3.1x106, 3.2><106, 3.3><106, 3.4x
106, 3.5 x 106, 3.6><106,
3.7 >106, 3.8 >106, 3.9x 106, 4>< 106, 4.1x 106, 4.2x106, 4.3 >106, 4.4 x106
or 4.5>106 APCs/cm2 and the
APCs exogenously supplied in the rapid second expansion are seeded in the
culture flask at a density
of at or about 2,5x106 APCs/cm2, 2.6x 106 APCs/cm2, 2.7>106 APCs/cm2, 2.8>106,
2.9>106, 3>106,
3.1x106, 3.2x106, 3.3x 106, 3.4x 106, 3.5x 106, 3.6x 106, 3.7x106, 3.8x106,
3.9x106, 4x106, 4.1x106,
4.2x106, 4.3x106, 4.4x 106, 4.5x 106, 4.6x 106, 4.7x 106, 4.8x106, 4.9 x106,
5x 106, 5.1x 106, 5.2 >106,
5.3x106, 5.4x106, 5.5x 106, 5.6x 106, 5.7x 106, 5.8x 106, 5.9x106, 6x106,
6.1x106, 6.2x106, 6.3>106,
6.4x106, 6.5 x106, 6.6x 106, 6.7>10, 6.8x 106, 6.9x 106, 7x106, 7.1 x106,
7.2x106, 7.3 x 106, 7.4 >106 or
7.5>106 APCs/cm2.
[00906] In other embodiments, the APCs exogenously supplied in the priming
first expansion are
seeded in the culture flask at a density selected from a range of at or about
1.0 x 106 APCs/cm2 to at or
about 4.5 x106 APCs/cm2, and the APCs exogenously supplied in the rapid second
expansion are
seeded in the culture flask at a density selected from a range of at or about
2.5>106 APCs/cm2 to at or
about 7.5 >106 APCs/cm2.
[00907] In other embodiments, the APCs exogenously supplied in the priming
first expansion arc
seeded in the culture flask at a density selected from a range of at or about
1.5 x 106 APCs/cm2 to at or
about 3.5>106 APCs/cm2, and the APCs exogenously supplied in the rapid second
expansion are
seeded in the culture flask at a density selected from a range of at or about
3.5 x 10' APCs/cm2 to at or
about 6 x 106 APCs/cm2.
[00908] In other embodiments, the APCs exogenously supplied in the priming
first expansion are
seeded in the culture flask at a density selected from a range of at or about
2 >10' APCs/cm2 to at or
about 3> 106 APCs/cm2, and the APCs exogenously supplied in the rapid second
expansion are seeded
in the culture flask at a density selected from a range of at or about 4 >106
APCs/cm2 to at or about
5.5>106 APCs/cm2.
[00909] In other embodiments, the APCs exogenously supplied in the priming
first expansion are
seeded in the culture flask at a density at or about 2 x106 APCs/cm2 and the
APCs exogenously
supplied in the rapid second expansion are seeded in the culture flask at a
density of at or about 4 x 10'
APCs/cm2.
[00910] In other embodiments, the ratio of the number of APCs (including, for
example, PBMCs)
exogenously supplied at day 7 of the rapid second expansion to the number of
PBMCs exogenously
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supplied at day 0 of the priming first expansion is selected from a range of
from at or about 1.1:1 to at
or about 20:1.
[00911] In other embodiments, the ratio of the number of APCs (including, for
example, PBMCs)
exogenously supplied at day 7 of the rapid second expansion to the number of
PBMCs exogenously
supplied at day 0 of the priming first expansion is selected from a range of
from at or about 1.1:1 to at
or about 10:1.
[00912] In other embodiments, the ratio of the number of APCs (including, for
example. PBMCs)
exogenously supplied at day 7 of the rapid second expansion to the number of
PBMCs exogenously
supplied at day 0 of the priming first expansion is selected from a range of
from at or about 1.1:1 to at
or about 9:1.
[00913] In other embodiments, the ratio of the number of APCs (including, for
example, PBMCs)
exogenously supplied at day 7 of the rapid second expansion to the number of
APCs (including, for
example, PBMCs) exogenously supplied at day 0 of the priming first expansion
is selected from a
range of from at or about 1.1:1 to at or about 8:1.
[00914] In other embodiments, the ratio of the number of APCs (including, for
example, PBMCs)
exogenously supplied at day 7 of the rapid second expansion to the number of
APCs (including, for
example, PBMCs) exogenously supplied at day 0 of the priming first expansion
is selected from a
range of from at or about 1.1:1 to at or about 7:1.
[00915] In other embodiments, the ratio of the number of APCs (including, for
example, PBMCs)
exogenously supplied at day 7 of the rapid second expansion to the number of
APCs (including, for
example, PBMCs) exogenously supplied at day 0 of the priming first expansion
is selected from a
range of from at or about 1.1:110 at or about 6:1.
[00916] In other embodiments, the ratio of the number of APCs (including, for
example, PBMCs)
exogenously supplied at day 7 of the rapid second expansion to the number of
APCs (including, for
example, PBMCs) exogenously supplied at day 0 of the priming first expansion
is selected from a
range of from at or about 1.1:1 to at or about 5:1.
[00917] In other embodiments, the ratio of the number of APCs (including, for
example, PBMCs)
exogenously supplied at day 7 of the rapid second expansion to the number of
APCs (including, for
example, PBMCs) exogenously supplied at day 0 of the priming first expansion
is selected from a
range of from at or about 1.1:1 to at or about 4:1.
[00918] In other embodiments, the ratio of the number of APCs (including, for
example, PBMCs)
exogenously supplied at day 7 of the rapid second expansion to the number of
APCs (including, for
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example, PBMCs) exogenously supplied at day 0 of the priming first expansion
is selected from a
range of from at or about 1.1:1 to at or about 3:1.
[00919] In other embodiments, the ratio of the number of APCs (including, for
example, PBMCs)
exogenously supplied at day 7 of the rapid second expansion to the number of
APCs (including, for
example, PBMCs) exogenously supplied at day 0 of the priming first expansion
is selected from a
range of from at or about 1.1:1 to at or about 2.9:1.
[00920] In other embodiments, the ratio of the number of APCs (including, for
example, PBMCs)
exogenously supplied at day 7 of the rapid second expansion to the number of
APCs (including, for
example, PBMCs) exogenously supplied at day 0 of the priming first expansion
is selected from a
range of from at or about 1.1:1 to at or about 2.8:1.
[00921] In other embodiments, the ratio of the number of APCs (including, for
example, PBMCs)
exogenously supplied at day 7 of the rapid second expansion to the number of
APCs (including, for
example, PBMCs) exogenously supplied at day 0 of the priming first expansion
is selected from a
range of from at or about 1.1:1 to at or about 2.7:1.
[00922] In other embodiments, the ratio of the number of APCs (including, for
example, PBMCs)
exogenously supplied at day 7 of the rapid second expansion to the number of
APCs (including, for
example, PBMCs) exogenously supplied at day 0 of the priming first expansion
is selected from a
range of from at or about 1.1:1 to at or about 2.6:1.
[00923] In other embodiments, the ratio of the number of APCs (including, for
example, PBMCs)
exogenously supplied at day 7 of the rapid second expansion to the number of
APCs (including, for
example, PBMCs) exogenously supplied at day 0 of the priming first expansion
is selected from a
range of from at or about 1.1:1 to at or about 2.5:1.
[00924] In other embodiments, the ratio of the number of APCs (including, for
example, PBMCs)
exogenously supplied at day 7 of the rapid second expansion to the number of
APCs (including, for
example, PBMCs) exogenously supplied at day 0 of the priming first expansion
is selected from a
range of from at or about 1.1:1 to at or about 2.4:1.
[00925] In other embodiments, the ratio of the number of APCs (including, for
example, PBMCs)
exogenously supplied at day 7 of the rapid second expansion to the number of
APCs (including, for
example, PBMCs) exogenously supplied at day 0 of the priming first expansion
is selected from a
range of from at or about 1.1:1 to at or about 2.3:1.
[00926] In other embodiments, the ratio of the number of APCs (including, for
example, PBMCs)
exogenously supplied at day 7 of the rapid second expansion to the number of
APCs (including, for
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example, PBMCs) exogenously supplied at day 0 of the priming first expansion
is selected from a
range of from at or about 1.1:1 to at or about 2.2:1.
[00927] In other embodiments, the ratio of the number of APCs (including, for
example, PBMCs)
exogenously supplied at day 7 of the rapid second expansion to the number of
APCs (including, for
example, PBMCs) exogenously supplied at day 0 of the priming first expansion
is selected from a
range of from at or about 1.1:1 to at or about 2.1:1.
[00928] In other embodiments, the ratio of the number of APCs (including, for
example, PBMCs)
exogenously supplied at day 7 of the rapid second expansion to the number of
APCs (including, for
example, PBMCs) exogenously supplied at day 0 of the priming first expansion
is selected from a
range of from at or about 1.1:1 to at or about 2:1.
[00929] In other embodiments, the ratio of the number of APCs (including, for
example, PBMCs)
exogenously supplied at day 7 of the rapid second expansion to the number of
APCs (including, for
example, PBMCs) exogenously supplied at day 0 of the priming first expansion
is selected from a
range of from at or about 2:1 to at or about 10:1.
[00930] In other embodiments, the ratio of the number of APCs (including, for
example, PBMCs)
exogenously supplied at day 7 of the rapid second expansion to the number of
APCs (including, for
example, PBMCs) exogenously supplied at day 0 of the priming first expansion
is selected from a
range of from at or about 2:1 to at or about 5:1.
[00931] In other embodiments, the ratio of the number of APCs (including, for
example, PBMCs)
exogenously supplied at day 7 of the rapid second expansion to the number of
APCs (including, for
example, PBMCs) exogenously supplied at day 0 of the priming first expansion
is selected from a
range of from at or about 2:1 to at or about 4:1.
[00932] In other embodiments, the ratio of the number of APCs (including, for
example, PBMCs)
exogenously supplied at day 7 of the rapid second expansion to the number of
APCs (including, for
example, PBMCs) exogenously supplied at day 0 of the priming first expansion
is selected from a
range of from at or about 2:1 to at or about 3:1.
[00933] In other embodiments, the ratio of the number of APCs (including, for
example, PBMCs)
exogenously supplied at day 7 of the rapid second expansion to the number of
APCs (including, for
example, PBMCs) exogenously supplied at day 0 of the priming first expansion
is selected from a
range of from at or about 2:1 to at or about 2.9:1.
[00934] In other embodiments, the ratio of the number of APCs (including, for
example, PBMCs)
exogenously supplied at day 7 of the rapid second expansion to the number of
APCs (including, for
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example, PBMCs) exogenously supplied at day 0 of the priming first expansion
is selected from a
range of from at or about 2:1 to at or about 2.8:1.
[00935] In other embodiments, the ratio of the number of APCs (including, for
example, PBMCs)
exogenously supplied at day 7 of the rapid second expansion to the number of
APCs (including, for
example, PBMCs) exogenously supplied at day 0 of the priming first expansion
is selected from a
range of from at or about 2:1 to at or about 2.7:1.
[00936] In other embodiments, the ratio of the number of APCs (including, for
example, PBMCs)
exogenously supplied at day 7 of the rapid second expansion to the number of
APCs (including, for
example, PBMCs) exogenously supplied at day 0 of the priming first expansion
is selected from a
range of from at or about 2:1 to at or about 2.6:1.
[00937] In other embodiments, the ratio of the number of APCs (including, for
example, PBMCs)
exogenously supplied at day 7 of the rapid second expansion to the number of
APCs (including, for
example, PBMCs) exogenously supplied at day 0 of the priming first expansion
is selected from a
range of from at or about 2:1 to at or about 2.5:1.
[00938] In other embodiments, the ratio of the number of APCs (including, for
example, PBMCs)
exogenously supplied at day 7 of the rapid second expansion to the number of
APCs (including, for
example, PBMCs) exogenously supplied at day 0 of the priming first expansion
is selected from a
range of from at or about 2:1 to at or about 2.4:1.
[00939] In other embodiments, the ratio of the number of APCs (including, for
example, PBMCs)
exogenously supplied at day 7 of the rapid second expansion to the number of
APCs (including, for
example, PBMCs) exogenously supplied at day 0 of the priming first expansion
is selected from a
range of from at or about 2:1 to at or about 2.3:1.
[00940] In other embodiments, the ratio of the number of APCs (including, for
example, PBMCs)
exogenously supplied at day 7 of the rapid second expansion to the number of
APCs (including, for
example, PBMCs) exogenously supplied at day 0 of the priming first expansion
is selected from a
range of from at or about about 2:1 to at or about 2.2:1.
[00941] In other embodiments, the ratio of the number of APCs (including, for
example, PBMCs)
exogenously supplied at day 7 of the rapid second expansion to the number of
APCs (including, for
example, PBMCs) exogenously supplied at day 0 of the priming first expansion
is selected from a
range of from at or about 2:1 to at or about 2.1:1.
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[00942] In other embodiments, the ratio of the number of APCs (including, for
example, PBMCs)
exogenously supplied at day 7 of the rapid second expansion to the number of
APCs (including, for
example, PBMCs) exogenously supplied at day 0 of the priming first expansion
is at or about 2:1.
[00943] In other embodiments, the ratio of the number of APCs (including, for
example, PBMCs)
exogenously supplied at day 7 of the rapid second expansion to the number of
APCs (including, for
example, PBMCs) exogenously supplied at day 0 of the priming first expansion
is at or about 1.1:1,
1.2:1, 1.3:1, 1.4:1, 1.5:1, 1.6:1, 1.7:1, 1.8:1, 1.9:1, 2:1, 2.1:1, 2.2:1,
2.3:1, 2.4:1, 2.5:1, 2.6:1, 2.7:1,
2.8:1, 2.9:1, 3:1, 3.1:1, 3.2:1, 3.3:1, 3.4:1, 3.5:1, 3.6:1, 3.7:1, 3.8:1,
3.9:1, 4:1, 4.1:1, 4.2:1, 4.3:1,
4.4:1, 4.5:1, 4.6:1, 4.7:1, 4.8:1, 4.9:1, or 5:1.
[00944] In other embodiments, the number of APCs (including, for example,
PBMCs) exogenously
supplied at day 0 of the priming first expansion is at or about lx108,
1.1x108, 1.2x 108, 1.3 x108,
1.4x108, 1.5x108, 1.6x108, 1.7x 108, 1.8x108, 1.9x108, 2x108, 2.1x108,
2.2x108, 2.3x108, 2.4x108,
2.5x108, 2.6x108, 2.7x 108, 2.8x 108, 2.9x 108, 3x108, 3.1x108, 3.2x108,
3.3x108, 3.4x108 or 3.5x108
APCs (including, for example, PBMCs), and the number of APCs (including, for
example, PBMCs)
exogenously supplied at day 7 of the rapid second expansion is at or about 3.5
x108, 3.6 x108, 3.7x 108,
3.8x108, 3.9x108, 4x108, 4.1x 108, 4.2x 108, 4.3x108, 4.4x108, 4.5x108,
4.6x108, 4.7x108, 4.8x108,
4.9x108, 5x108, 5.1x108, 5.2x108, 5.3x108, 5.4x108, 5.5x108, 5.6x108, 5.7x108,
5.8x108, 5.9x108,
6x108, 6.1x108, 6.2x108, 6.3x108, 6.4x10, 6.5x108, 6.6x108, 6.7x108, 6.8x108,
6.9x108, 7x108,
7.1x108, 7.2x108, 7.3x108, 74x108 7.5x108, 7.6x108, 7.7x108, 7.8x108, 7.9x108,
8x108, 8.1x108,
8.2x108, 8.3x108, 8.4x108, 8.5x108, 8.6x108, 8.7x108, 8.8x108, 8.9x108, 9x108,
9.1x108, 9.2x108,
9.3 x108, 9.4x108, 9.5x 108, 9.6x 108, 9.7x 108, 9.8x 108, 9.9x 108 or 1x109
APCs (including, for
example, PBMCs).
[00945] In other embodiments, the number of APCs (including, for example,
PBMCs) exogenously
supplied at day 0 of the priming first expansion is selected from the range of
at or about lx 108 APCs
(including, for example, PBMCs) to at or about 3.5< 108 APCs (including, for
example, PBMCs), and
the number of APCs (including, for example, PBMCs) exogenously supplied at day
7 of the rapid
second expansion is selected from the range of at or about 3=5x 108 APCs
(including, for example,
PBMCs) to at or about lx 109 APCs (including, for example, PBMCs).
[00946] In other embodiments, the number of APCs (including, for example,
PBMCs) exogenously
supplied at day 0 of the priming first expansion is selected from the range of
at or about 1.5 x108 APCs
to at or about 3x 10 APCs (including, for example, PBMCs), and the number of
APCs (including, for
example, PBMCs) exogenously supplied at day 7 of the rapid second expansion is
selected from the
range of at or about 4x108 APCs (including, for example, PBMCs) to at or about
7.5 x108 APCs
(including, for example, PBMCs).
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[00947] In other embodiments, the number of APCs (including, for example,
PBMCs) exogenously
supplied at day 0 of the priming first expansion is selected from the range of
at or about 2x 10 APCs
(including, for example, PBMCs) to at or about 2.5x 108APCs (including, for
example, PBMCs), and
the number of APCs (including, for example, PBMCs) exogenously supplied at day
7 of the rapid
second expansion is selected from the range of at or about 4=5x 10' APCs
(including, for example,
PBMCs) to at or about 5.5 x108 APCs (including, for example, PBMCs).
[00948] In other embodiments, the number of APCs (including, for example,
PBMCs) exogenously
supplied at day 0 of the priming first expansion is at or about 2.5 x108 APCs
(including, for example,
PBMCs) and the number of APCs (including, for example, PBMCs) exogenously
supplied at day 7 of
the rapid second expansion is at or about 5 x108 APCs (including, for example,
PBMCs)
[00949] In other embodiments, the number of layers of APCs (including, for
example, PBMCs)
added at day 0 of the priming first expansion is approximately one-half of the
number of layers of
APCs (including, for example, PBMCs) added at day 7 of the rapid second
expansion. In certain
embodiments, the method comprises adding antigen presenting cell layers at day
0 of the priming first
expansion to the first population of TILs and adding antigen presenting cell
layers at day 7 to the
second population of TILs, wherein the number of antigen presenting cell layer
added at day 0 is
approximately 50% of the number of antigen presenting cell layers added at day
7.
[00950] In other embodiments, the number of layers of APCs (including, for
example, PBMCs)
exogenously supplied at day 7 of the rapid second expansion is greater than
the number of layers of
APCs (including, for example, PBMCs) exogenously supplied at day 0 of the
priming first expansion.
[00951] In other embodiments, day 0 of the priming first expansion occurs in
the presence of
layered APCs (including, for example, PBMCs) with an average thickness of at
or about 2 cell layers
and day 7 of the rapid second expansion occurs in the presence of layered APCs
(including, for
example, PBMCs) with an average thickness of at or about 4 cell layers.
[00952] In other embodiments, day 0 of the priming first expansion occurs in
the presence of
layered APCs (including, for example, PBMCs) with an average thickness of at
or about one cell layer
and day 7 of the rapid second expansion occurs in the presence of layered APCs
(including, for
example, PBMCs) with an average thickness of at or about 3 cell layers.
[00953] In other embodiments, day 0 of the priming first expansion occurs in
the presence of
layered APCs (including, for example, PBMCs) with an average thickness of at
or about 1.5 cell
layers to at or about 2.5 cell layers and day 7 of the rapid second expansion
occurs in the presence of
layered APCs (including, for example, PBMCs) with an average thickness of at
or about 3 cell layers.
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[00954] In other embodiments, day 0 of the priming first expansion occurs in
the presence of
layered APCs (including, for example, PBMCs) with an average thickness of at
or about one cell layer
and day 7 of the rapid second expansion occurs in the presence of layered APCs
(including, for
example, PBMCs) with an average thickness of at or about 2 cell layers.
[00955] In other embodiments, day 0 of the priming first expansion occurs in
the presence of
layered APCs (including, for example, PBMCs) with an average thickness of of
at or about 1, 1.1, 1.2,
1.3, 1.4, 1.5, 1.6, 1.7, 1.8, 1.9, 2,2.1, 2.2, 2.3, 2.4, 2.5, 2.6, 2.7, 2.8,
2.9 or 3 cell layers and day 7 of
the rapid second expansion occurs in the presence of layered APCs (including,
for example, PBMCs)
with an average thickness of at or about 3.1, 3.2, 3.3, 3.4, 3.5, 3.6, 3.7,
3.8, 3.9,4, 4.1, 4.2, 4.3, 4.4,
4.5, 4.6, 4.7, 4.8, 4.9, 5, 5.1, 5.2, 5.3, 5.4, 5.5, 5.6, 5.7, 5.8, 5.9, 6,
6.1, 6.2, 6.3, 6.4, 6.5, 6.6, 6.7, 6.8,
6.9, 7, 7.1, 7.2, 7.3, 7.4, 7.5, 7.6, 7.7, 7.8, 7.9 or 8 cell layers.
[00956] In other embodiments, day 0 of the priming first expansion occurs in
the presence of
layered APCs (including, for example, PBMCs) with an average thickness of at
or about 1 cell layer
to at or about 2 cell layers and day 7 of the rapid second expansion occurs in
the presence of layered
APCs (including, for example, PBMCs) with an average thickness of at or about
3 cell layers to at or
about 10 cell layers.
[00957] In other embodiments, day 0 of the priming first expansion occurs in
the presence of
layered APCs (including, for example, PBMCs) with an average thickness of at
or about 2 cell layers
to at or about 3 cell layers and day 7 of the rapid second expansion occurs in
the presence of layered
APCs (including, for example, PBMCs) with an average thickness of at or about
4 cell layers to at or
about 8 cell layers.
[00958] In other embodiments, day 0 of the priming first expansion occurs in
the presence of
layered APCs (including, for example, PBMCs) with an average thickness of at
or about 2 cell layers
and day 7 of the rapid second expansion occurs in the presence of layered APCs
(including, for
example, PBMCs) with an average thickness of at or about 4 cell layers to at
or about 8 cell layers.
[00959] In other embodiments, day 0 of the priming first expansion occurs in
the presence of
layered APCs (including, for example, PBMCs) with an average thickness of at
or about 1, 2 or 3 cell
layers and day 7 of the rapid second expansion occurs in the presence of
layered APCs (including, for
example, PBMCs) with an average thickness of at or about 3, 4, 5, 6, 7, 8, 9
or 10 cell layers.
[00960] In other embodiments, day 0 of the priming first expansion occurs in
the presence of
layered APCs (including, for example, PBMCs) with a first average thickness
equal to a first number
of layers of APCs (including, for example, PBMCs) and day 7 of the rapid
second expansion occurs in
the presence of layered APCs (including, for example, PBMCs) with a second
average thickness equal
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to a second number of layers of APCs (including, for example. PBMCs), wherein
the ratio of the first
number of layers of APCs (including, for example, PBMCs) to the second number
of layers of APCs
(including, for example, PBMCs) is selected from the range of at or about
1:1.1 to at or about 1:10.
[00961] In other embodiments, day 0 of the priming first expansion occurs in
the presence of
layered APCs (including, for example, PBMCs) with a first average thickness
equal to a first number
of layers of APCs (including, for example, PBMCs) and day 7 of the rapid
second expansion occurs in
the presence of layered APCs (including, for example, PBMCs) with a second
average thickness equal
to a second number of layers of APCs (including, for example. PBMCs), wherein
the ratio of the first
number of layers of APCs (including, for example, PBMCs) to the second number
of layers of APCs
(including, for example, PBMCs) is selected from the range of at or about
1:1.1 to at or about 1:8.
[00962] In other embodiments, day 0 of the priming first expansion occurs in
the presence of
layered APCs (including, for example, PBMCs) with a first average thickness
equal to a first number
of layers of APCs (including, for example, PBMCs) and day 7 of the rapid
second expansion occurs in
the presence of layered APCs (including, for example, PBMCs) with a second
average thickness equal
to a second number of layers of APCs (including, for example, PBMCs), wherein
the ratio of the first
number of layers of APCs (including, for example, PBMCs) to the second number
of layers of APCs
(including, for example, PBMCs) is selected from the range of at or about
1:1.1 to at or about 1:7.
[00963] In other embodiments, day 0 of the priming first expansion occurs in
the presence of
layered APCs (including, for example, PBMCs) with a first average thickness
equal to a first number
of layers of APCs (including, for example, PBMCs) and day 7 of the rapid
second expansion occurs in
the presence of layered APCs (including, for example, PBMCs) with a second
average thickness equal
to a second number of layers of APCs (including, for example, PBMCs), wherein
the ratio of the first
number of layers of APCs (including, for example, PBMCs) to the second number
of layers of APCs
(including, for example, PBMCs) is selected from the range of at or about
1:1.1 to at or about 1:6.
[00964] In other embodiments, day 0 of the priming first expansion occurs in
the presence of
layered APCs (including, for example, PBMCs) with a first average thickness
equal to a first number
of layers of APCs (including, for example, PBMCs) and day 7 of the rapid
second expansion occurs in
the presence of layered APCs (including, for example, PBMCs) with a second
average thickness equal
to a second number of layers of APCs (including, for example, PBMCs), wherein
the ratio of the first
number of layers of APCs (including, for example, PBMCs) to the second number
of layers of APCs
(including, for example, PBMCs) is selected from the range of at or about
1:1.1 to at or about 1:5.
[00965] In other embodiments, day 0 of the priming first expansion occurs in
the presence of
layered APCs (including, for example, PBMCs) with a first average thickness
equal to a first number
of layers of APCs (including, for example, PBMCs) and day 7 of the rapid
second expansion occurs in
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the presence of layered APCs (including, for example, PBMCs) with a second
average thickness equal
to a second number of layers of APCs (including, for example. PBMCs), wherein
the ratio of the first
number of layers of APCs (including, for example, PBMCs) to the second number
of layers of APCs
(including, for example, PBMCs) is selected from the range of at or about
1:1.1 to at or about 1:4.
[00966] In other embodiments, day 0 of the priming first expansion occurs in
the presence of
layered APCs (including, for example, PBMCs) with a first average thickness
equal to a first number
of layers of APCs (including, for example, PBMCs) and day 7 of the rapid
second expansion occurs in
the presence of layered APCs (including, for example, PBMCs) with a second
average thickness equal
to a second number of layers of APCs (including, for example, PBMCs), wherein
the ratio of the first
number of layers of APCs (including, for example, PBMCs) to the second number
of layers of APCs
(including, for example, PBMCs) is selected from the range of at or about
1:1.1 to at or about 1:3.
[00967] In other embodiments, day 0 of the priming first expansion occurs in
the presence of
layered APCs (including, for example, PBMCs) with a first average thickness
equal to a first number
of layers of APCs (including, for example, PBMCs) and day 7 of the rapid
second expansion occurs in
the presence of layered APCs (including, for example, PBMCs) with a second
average thickness equal
to a second number of layers of APCs (including, for example. PBMCs), wherein
the ratio of the first
number of layers of APCs (including, for example, PBMCs) to the second number
of layers of APCs
(including, for example, PBMCs) is selected from the range of at or about
1:1.1 to at or about 1:2.
[00968] In other embodiments, day 0 of the priming first expansion occurs in
the presence of
layered APCs (including, for example, PBMCs) with a first average thickness
equal to a first number
of layers of APCs (including, for example, PBMCs) and day 7 of the rapid
second expansion occurs in
the presence of layered APCs (including, for example, PBMCs) with a second
average thickness equal
to a second number of layers of APCs (including, for example, PBMCs), wherein
the ratio of the first
number of layers of APCs (including, for example, PBMCs) to the second number
of layers of APCs
(including, for example, PBMCs) is selected from the range of at or about
1:1.2 to at or about 1:8.
[00969] In other embodiments, day 0 of the priming first expansion occurs in
the presence of
layered APCs (including, for example, PBMCs) with a first average thickness
equal to a first number
of layers of APCs (including, for example, PBMCs) and day 7 of the rapid
second expansion occurs in
the presence of layered APCs (including, for example, PBMCs) with a second
average thickness equal
to a second number of layers of APCs (including, for example. PBMCs), wherein
the ratio of the first
number of layers of APCs (including, for example, PBMCs) to the second number
of layers of APCs
(including, for example, PBMCs) is selected from the range of at or about
1:1.3 to at or about 1:7.
[00970] In other embodiments, day 0 of the priming first expansion occurs in
the presence of
layered APCs (including, for example, PBMCs) with a first average thickness
equal to a first number
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of layers of APCs (including, for example, PBMCs) and day 7 of the rapid
second expansion occurs in
the presence of layered APCs (including, for example, PBMCs) with a second
average thickness equal
to a second number of layers of APCs (including, for example, PBMCs), wherein
the ratio of the first
number of layers of APCs (including, for example, PBMCs) to the second number
of layers of APCs
(including, for example, PBMCs) is selected from the range of at or about
1:1.4 to at or about 1:6.
[00971] In other embodiments, day 0 of the priming first expansion occurs in
the presence of
layered APCs (including, for example, PBMCs) with a first average thickness
equal to a first number
of layers of APCs (including, for example, PRMCs) and day 7 of the rapid
second expansion occurs in
the presence of layered APCs (including, for example, PBMCs) with a second
average thickness equal
to a second number of layers of APCs (including, for example. PBMCs), wherein
the ratio of the first
number of layers of APCs (including, for example, PBMCs) to the second number
of layers of APCs
(including, for example, PBMCs) is selected from the range of at or about
1:1.5 to at or about 1:5.
[00972] In other embodiments, day 0 of the priming first expansion occurs in
the presence of
layered APCs (including, for example, PBMCs) with a first average thickness
equal to a first number
of layers of APCs (including, for example, PBMCs) and day 7 of the rapid
second expansion occurs in
the presence of layered APCs (including, for example, PBMCs) with a second
average thickness equal
to a second number of layers of APCs (including, for example. PBMCs), wherein
the ratio of the first
number of layers of APCs (including, for example, PBMCs) to the second number
of layers of APCs
(including, for example, PBMCs) is selected from the range of at or about
1:1.6 to at or about 1:4.
[00973] In other embodiments, day 0 of the priming first expansion occurs in
the presence of
layered APCs (including, for example, PBMCs) with a first average thickness
equal to a first number
of layers of APCs (including, for example, PBMCs) and day 7 of the rapid
second expansion occurs in
the presence of layered APCs (including, for example, PBMCs) with a second
average thickness equal
to a second number of layers of APCs (including, for example. PBMCs), wherein
the ratio of the first
number of layers of APCs (including, for example, PBMCs) to the second number
of layers of APCs
(including, for example, PBMCs) is selected from the range of at or about
1:1.7 to at or about 1:3.5.
[00974] In other embodiments, day 0 of the priming first expansion occurs in
the presence of
layered APCs (including, for example, PBMCs) with a first average thickness
equal to a first number
of layers of APCs (including, for example, PBMCs) and day 7 of the rapid
second expansion occurs in
the presence of layered APCs (including, for example, PBMCs) with a second
average thickness equal
to a second number of layers of APCs (including, for example, PBMCs), wherein
the ratio of the first
number of layers of APCs (including, for example, PBMCs) to the second number
of layers of APCs
(including, for example, PBMCs) is selected from the range of at or about
1:1.g to at or about 1:3.
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[00975] In other embodiments, day 0 of the priming first expansion occurs in
the presence of
layered APCs (including, for example, PBMCs) with a first average thickness
equal to a first number
of layers of APCs (including, for example, PBMCs) and day 7 of the rapid
second expansion occurs in
the presence of layered APCs (including, for example, PBMCs) with a second
average thickness equal
to a second number of layers of APCs (including, for example, PBMCs), wherein
the ratio of the first
number of layers of APCs (including, for example, PBMCs) to the second number
of layers of APCs
(including, for example, PBMCs) is selected from the range of at or about
1:1.9 to at or about 1:2.5.
[00976] In other embodiments, day 0 of the priming first expansion occurs in
the presence of
layered APCs (including, for example, PBMCs) with a first average thickness
equal to a first number
of layers of APCs (including, for example, PBMCs) and day 7 of the rapid
second expansion occurs in
the presence of layered APCs (including, for example, PBMCs) with a second
average thickness equal
to a second number of layers of APCs (including, for example, PBMCs), wherein
the ratio of the first
number of layers of APCs (including, for example, PBMCs) to the second number
of layers of APCs
(including, for example, PBMCs) is at or about 1: 2.
[00977] In other embodiments, day 0 of the priming first expansion occurs in
the presence of
layered APCs (including, for example, PBMCs) with a first average thickness
equal to a first number
of layers of APCs (including, for example, PBMCs) and day 7 of the rapid
second expansion occurs in
the presence of layered APCs (including, for example, PBMCs) with a second
average thickness equal
to a second number of layers of APCs (including, for example, PBMCs), wherein
the ratio of the first
number of layers of APCs (including, for example, PBMCs) to the second number
of layers of APCs
(including, for example, PBMCs) is selected from at or about 1:1.1, 1:1.2,
1:1.3, 1:1.4, 1:1.5, 1:1.6,
1:1.7, 1:1.8, 1:1.9, 1:2, 1:2.1, 1:2.2, 1:2.3, 1:2.4, 1:2.5, 1:2.6, 1:2.7,
1:2.8, 1:2.9, 1:3, 1:3.1, 1:3.2,
1:3.3, 1:3.4, 1:3.5, 1:3.6, 1:3.7, 1:3.8, 1:3.9, 1:4, 1:4.1, 1:4.2, 1:4.3,
1:4.4, 1:4.5, 1:4.6, 1:4.7, 1:4.8,
1:4.9, 1:5, 1:5.1, 1:5.2, 1:5.3, 1:5.4, 1:5.5, 1:5.6, 1:5.7, 1:5.8, 1:5.9,
1:6, 1:6.1, 1:6.2, 1:6.3, 1:6.4,
1:6.5, 1:6.6, 1:6.7, 1:6.8, 1:6.9, 1:7, 1:7.1, 1:7.2, 1:7.3, 1:7.4, 1:7.5,
1:7.6, 1:7.7, 1:7.8, 1:7.9, 1:8,
1:8.1, 1:8.2, 1:8.3, 1:8.4, 1:8.5, 1:8.6, 1:8.7, 1:8.8, 1:8.9, 1:9, 1:9.1,
1:9.2, 1:9.3, 1:9.4, 1:9.5, 1:9.6,
1:9.7, 1:9.8, 1:9.9 or 1:10.
[00978] In some embodiments, the number of APCs in the priming first expansion
is selected from
the range of about 1.0x106 APCs/cm2 to about 4.5 x106 APCs/cm2, and the number
of APCs in the
rapid second expansion is selected from the range of about 2.5 x106 APCs/cm2
to about 7.5 x106
APCs/cm2.
[00979] In some embodiments, the number of APCs in the priming first expansion
is selected from
the range of about 1.5 x106 APCs/cm2 to about 3.5 x106 APCs/cm2, and the
number of APCs in the
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rapid second expansion is selected from the range of about 3.5 x106 APCs/cm2
to about 6.0x106
APCs/cm2.
[00980] In some embodiments, the number of APCs in the priming first expansion
is selected from
the range of about 2.0x106APCs/cm2 to about 3.0 x106 APCs/cm2, and the number
of APCs in the
rapid second expansion is selected from the range of about 4.0x106 APCs/cm2 to
about 5.5 x106
APCs/cm2.
B. Optional Cell Medium Components
1. Anti-CD3 Antibodies
[00759] In some embodiments, the culture media used in expansion methods
described herein
(including those referred to as REP, see for example, Figures 1 and 8 (in
particular, e.g., Figure 8B))
include an anti-CD3 antibody. An anti-CD3 antibody in combination with IL-2
induces T cell
activation and cell division in the TIL population. This effect can be seen
with full length antibodies
as well as Fab and F(abl2 fragments, with the former being generally
preferred; sec, e.g., Isoukas et
at., I Immunol. 1985, 135, 1719, hereby incorporated by reference in its
entirety.
[00760] As will be appreciated by those in the art, there are a number of
suitable anti-human CD3
antibodies that find use in the invention, including anti-human CD3 polyclonal
and monoclonal
antibodies from various mammals, including, but not limited to, murine, human,
primate, rat, and
canine antibodies. In particular embodiments, the OKT3 anti-CD3 antibody
muromonab is used
(commercially available from Ortho-McNeil, Raritan, NJ or Miltenyi Biotech,
Auburn, CA). See,
Table 1.
[00761] As will be appreciated by those in the art, there are a number of
suitable anti-human CD3
antibodies that find use in the invention, including anti-human CD3 polyclonal
and monoclonal
antibodies from various mammals, including, but not limited to, murine, human,
primate, rat, and
canine antibodies. In some embodiments, the OKT3 anti-CD3 antibody muromonab
is used
(commercially available from Ortho-McNeil, Raritan, NJ or Miltenyi Biotech,
Auburn, CA).
2. 4-1BB (CD137) Agonists
[00762] In some embodiments, the cell culture medium of the priming first
expansion and/or the
rapid second expansion comprises a TNFRSF agonist. In some embodiments, the
TNFRSF agonist is
a 4-1BB (CD137) agonist. The 4-1BB agonist may be any 4-1BB binding molecule
known in the art.
The 4-1BB binding molecule may be a monoclonal antibody or fusion protein
capable of binding to
human or mammalian 4-1BB. The 4-1BB agonists or 4-1BB binding molecules may
comprise an
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immunoglobulin heavy chain of any isotype (e.g., IgG, IgE, IgM, IgD, IgA, and
IgY), class (e.g.,
IgGl, IgG2, IgG3, IgG4, IgAl and IgA2) or subclass of immunoglobulin molecule.
The 4-1BB
agonist or 4-1BB binding molecule may have both a heavy and a light chain. As
used herein, the term
binding molecule also includes antibodies (including full length antibodies),
monoclonal antibodies
(including full length monoclonal antibodies), polyclonal antibodies,
multispecific antibodies (e.g.,
bispecific antibodies), human, humanized or chimeric antibodies, and antibody
fragments, e.g., Fab
fragments, F(ab') fragments, fragments produced by a Fab expression library,
epitope-binding
fragments of any of the above, and engineered forms of antibodies, e.g., scFy
molecules, that bind to
4-1BB. In some embodiments, the 4-1BB agonist is an antigen binding protein
that is a fully human
antibody. In some embodiments, the 4-1BB agonist is an antigen binding protein
that is a humanized
antibody. In some embodiments, 4-1BB agonists for use in the presently
disclosed methods and
compositions include anti-4-1BB antibodies, human anti-4-1BB antibodies, mouse
anti-4-1BB
antibodies, mammalian anti-4-1BB antibodies, monoclonal anti-4-1BB antibodies,
polyclonal anti-4-
1BB antibodies, chimeric anti-4-1BB antibodies, anti-4-1BB adnectins, anti-4-
1BB domain
antibodies, single chain anti-4-1 BB fragments, heavy chain anti-4-1BB
fragments, light chain anti-4-
1BB fragments, anti-4-1BB fusion proteins, and fragments, derivatives,
conjugates, variants, or
biosimilars thereof. Agonistic anti-4-1BB antibodies are known to induce
strong immune responses.
Lee, etal., PLOS One 2013, 8, e69677. In some embodiments, the 4-1BB agonist
is an agonistic, anti-
4-1BB humanized or fully human monoclonal antibody (i.e., an antibody derived
from a single cell
line). In some embodiments, the 4-1BB agonist is EU-101 (Eutilex Co. Ltd.),
utomilumab, or
urelumab, or a fragment, derivative, conjugate, variant, or biosimilar
thereof. In some embodiments,
the 4-1BB agonist is utomilumab or urelumab, or a fragment, derivative,
conjugate, variant, or
biosimilar thereof.
1007631 In some embodiments, the 4-1BB agonist or 4-1BB binding molecule may
also be a fusion
protein. In some embodiments, a multimeric 4-1BB agonist, such as a trimeric
or hexameric 4-1BB
agonist (with three or six ligand binding domains), may induce superior
receptor (4-1BBL) clustering
and internal cellular signaling complex formation compared to an agonistic
monoclonal antibody,
which typically possesses two ligand binding domains. Trimeric (trivalent) or
hexameric (or
hexavalent) or greater fusion proteins comprising three TNFRSF binding domains
and IgGl-Fc and
optionally further linking two or more of these fusion proteins are described,
e.g., in Gieffers, etal.,
Mol. Cancer Therapeutics 2013, 12, 2735-47.
[00764] Agonistic 4-1BB antibodies and fusion proteins are known to induce
strong immune
responses. In some embodiments, the 4-1BB agonist is a monoclonal antibody or
fusion protein that
binds specifically to 4-1BB antigen in a manner sufficient to reduce toxicity.
In some embodiments,
the 4-1BB agonist is an agonistic 4-1BB monoclonal antibody or fusion protein
that abrogates
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antibody-dependent cellular toxicity (ADCC), for example NK cell cytotoxicity.
In some
embodiments, the 4-1BB agonist is an agonistic 4-1BB monoclonal antibody or
fusion protein that
abrogates antibody-dependent cell phagocytosis (ADCP). In some embodiments,
the 4-1BB agonist is
an agonistic 4-1BB monoclonal antibody or fusion protein that abrogates
complement-dependent
cytotoxicity (CDC). In some embodiments, the 4-1BB agonist is an agonistic 4-
1BB monoclonal
antibody or fusion protein which abrogates Fe region functionality.
[00765] In some embodiments, the 4-1BB agonists are characterized by binding
to human 4-1BB
(SEQ ID NO:9) with high affinity and agonistic activity. In some embodiments,
the 4-1BB agonist is
a binding molecule that binds to human 4-1BB (SEQ ID NO:40). In some
embodiments, the 4-1BB
agonist is a binding molecule that binds to murinc 4-1BB (SEQ ID NO:41). The
amino acid sequences
of 4-1BB antigen to which a 4-1BB agonist or binding molecule binds are
summarized in Table 5.
TABLE 5. Amino acid sequences of 4-1BB antigens.
Identifier Sequence (One-Letter Amino Acid
Symbols)
SEQ ID NO:40 MGNSCYNIVA TLLLVLNFER TRSLQDPCSN CPAGTFCDNN
RNQICSPCPP NSFSSAGGQR 60
human 4 12E,
Tumor necrosis TCDICRQCKG VERTRKECSS TSNAECDCTP GFHCLGAGCS
NCEQDCKQGQ ELTKKGCKDC 120
factor receptor
CFGTFNDQKR GICRPWTNCS LDGKSVLVNG TKERDVVCGP SPADLSPGAS SVTPPAPARE
180
superfami1y,
member 9 (Homo
PGHSPQIISF FLALTSTALL FLLFYLTLRF SVVRRGRKKL LYIFY.QPFMR PVQTTQEEDG
240
sapiens)
CSCRFPEEEE GGCEL
255
SEQ ID 50:11 MGNNCYNVVV IVLLLVGCEK VGAVQNSCDN CQPGTFCRKY
NPVCKSCPPS TFSSIGGQPN 60
murine 4 1E5,
CNICRVCAGY FREKKFCSST HNAECECIEG FHCLGPQCTR CEKDCRPGQE LTKQGCKTCS
120
Tumor nec:Lulö
factor receptor LGTFNDQNGT GVCRPWTNCS LDGRSVLKTG TTEKDVVCGP
PVVSFSPSTT ISVTPEGGPG 160
superfamily,
GHSLQVLTLF LALTSALLLA LIFITLLFSV LKWIRKKFPH IFKQPFKKTT GAAQEEDACS
240
member 9 (Mus
musculus)
CRCPQEEEGG GGGYEL
256
[00766] In some embodiments, the compositions, processes and methods described
include a 4-1BB
agonist that binds human or murine 4-1BB with a KD of about 100 pM or lower,
binds human or
murine 4-1BB with a Kn of about 90 pM or lower, binds human or murine 4-1BB
with a Kn of about
80 pM or lower, binds human or murine 4-1BB with a K0 of about 70 pM or lower,
binds human or
murine 4-1BB with a K0 of about 60 pM or lower, binds human or murine 4-1BB
with a KD of about
50 pM or lower, binds human or murine 4-1BB with a Kip of about 40 pM or
lower, or binds human or
murine 4-1BB with a KD of about 30 pM or lower.
[00767] In some embodiments, the compositions, processes and methods described
include a 4-1BB
agonist that binds to human or murine 4-1BB with a kassoc of about 7.5 x 105
1/NI- s or faster, binds to
human or murine 4-1BB with a Icas50, of about 7.5 x 1051/M.s or faster, binds
to human or murine 4-
1BB with a kassoc of about 8 x 1051/M.s or faster, binds to human or murine 4-
1BB with a kassoc of
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about 8.5 x 105 1/M- s or faster, binds to human or murine 4-1BB with a kassoc
of about 9>< 105 1/M- s
or faster, binds to human or murine 4-1BB with a kasso, of about 9.5 x 105
1/M= s or faster, or binds to
human or murine 4-1BB with a kassoc of about 1 x 106 1/Ms or faster.
[00768] In some embodiments, the compositions, processes and methods described
include a 4-1BB
agonist that binds to human or murine 4-1BB with a kdissoc of about 2 x 10-5
1/s or slower, binds to
human or murine 4-1BB with a kaissoc of about 2.1 x 10-5 1/s or slower, binds
to human or murine 4-
1BB with a kaissoc of about 2.2 x 10-5 1/s or slower, binds to human or murine
4-1BB with a kaissoc of
about 2.3 x 10-5 1/s or slower, binds to human or murine 4-1BB with a kaissoc
of about 2.4 x 10-5 1/s or
slower, binds to human or murine 4-1BB with a kdissoc of about 2.5 x 10-5 1/s
or slower, binds to
human or murinc 4-1BB with a kcjissoc of about 2.6 x 10-5 1/s or slower or
binds to human or murine 4-
1BB with a kdi. of about 2.7< 10 1/s or slower, binds to human or murine 4-1BB
with a kdissoc of
about 2.8 x 10' 1/s or slower, binds to human or murine 4-1BB with a kaissoc
of about 2.9 x 10-5 1/s or
slower, or binds to human or murine 4-1BB with a kossoc of about 3 x 10-5 1/s
or slower.
[00769] In some embodiments, the compositions, processes and methods described
include a 4-1BB
agonist that binds to human or murine 4-1BB with an IC50 of about 10 nM or
lower, binds to human
or murine 4-1BB with an IC50 of about 9 nM or lower, binds to human or murine
4-1BB with an IC50
of about 8 nM or lower, binds to human or murine 4-1BB with an IC50 of about 7
nM or lower, binds
to human or murine 4- I BB with an IC50 of about 6 nM or lower, binds to human
or murine 4-1BB
with an 1050 of about 5 nM or lower, binds to human or murine 4-1BB with an
IC50 of about 4 nM or
lower, binds to human or murine 4-1BB with an IC50 of about 3 nM or lower,
binds to human or
murine 4-1BB with an IC50 of about 2 nM or lower, or binds to human or murine
4-1BB with an IC50
of about 1 nM or lower.
[00770] In some embodiments, the 4-1BB agonist is utomilumab, also known as PF-
05082566 or
MOR-7480, or a fragment, derivative, variant, or biosimilar thereof Utomilumab
is available from
Pfizer, Inc. Utomilumab is an immunoglobulin G2-lambda, anti-Homo sapiens
TNFRSF9 (tumor
necrosis factor receptor (TNFR) superfamily member 9, 4-1BB, T cell antigen
ILA, CD137)1, Homo
sapiens (fully human) monoclonal antibody. The amino acid sequences of
utomilumab are set forth in
Table 6. ITtomiluniab comprises glyeosylation sites at Am-159 and Asn292;
heavy chain intrachain
disulfide bridges at positions 22-96 (VII-VL), 143-199 (CHI-CL), 256-316
(C112) and 362-420 (CH3);
light chain intrachain disulfide bridges at positions 22'-87' (VH-VL) and 136'-
195' (CH1-CL);
interchain heavy chain-heavy chain disulfide bridges at IgG2A isoform
positions 218-218, 219-219,
222-222, and 225-225, at IgG2A/B isofonn positions 218-130, 219-219, 222-222,
and 225-225, and at
IgG2B isoform positions 219-130 (2), 222-222, and 225-225; and interchain
heavy chain-light chain
disulfide bridges at IgG2A isoform positions 130-213' (2), IgG2A/B isoform
positions 218-213' and
130-213', and at IgG2B isoform positions 218-213' (2). The preparation and
properties of
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utomilumab and its variants and fragments are described in U.S. Patent Nos.
8,821,867; 8,337,850;
and 9,468,678, and International Patent Application Publication No. WO
2012/032433 Al, the
disclosures of each of which are incorporated by reference herein. Preclinical
characteristics of
utomilumab are described in Fisher, et at., Cancer Immuno & Immunother.
2012, 61, 1721-33.
Current clinical trials of utomilumab in a variety of hematological and solid
tumor indications include
U.S. National Institutes of Health chnicaltrials.gov identifiers NCT02444793,
NCT01307267,
NCT02315066, and NCT02554812.
1007711 In some embodiments, a 4-1BB agonist comprises a heavy chain given by
SF() IT) NO-42
and a light chain given by SEQ ID NO:43. In some embodiments, a 4-1BB agonist
comprises heavy
and light chains having the sequences shown in SEQ ID NO:42 and SEQ ID NO:43,
respectively, or
antigen binding fragments, Fab fragments, single-chain variable fragments
(scFv), variants, or
conjugates thereof. In some embodiments, a 4-1BB agonist comprises heavy and
light chains that are
each at least 99% identical to the sequences shown in SEQ ID NO:42 and SEQ ID
NO:43,
respectively. In some embodiments, a 4-1BB agonist comprises heavy and light
chains that are each at
least 98% identical to the sequences shown in SEQ ID NO:42 and SEQ ID NO:43,
respectively. In
some embodiments, a 4-1BB agonist comprises heavy and light chains that are
each at least 97%
identical to the sequences shown in SEQ ID NO:42 and SEQ ID NO:43,
respectively. In some
embodiments, a 4-1BB agonist comprises heavy and light chains that are each at
least 96% identical
to the sequences shown in SEQ ID NO:42 and SEQ ID NO:43, respectively. In some
embodiments, a
4-1BB agonist comprises heavy and light chains that are each at least 95%
identical to the sequences
shown in SEQ ID NO:42 and SEQ ID NO:43, respectively.
1007721 In some embodiments, the 4-1BB agonist comprises the heavy and light
chain CDRs or
variable regions (VRs) of utomilumab. In some embodiments, the 4-1BB agonist
heavy chain variable
region (VH) comprises the sequence shown in SEQ ID NO:44, and the 4-1BB
agonist light chain
variable region (VL) comprises the sequence shown in SEQ ID NO:45, and
conservative amino acid
substitutions thereof. In some embodiments, a 4-1BB agonist comprises VH and
VL regions that are
each at least 99% identical to the sequences shown in SEQ ID NO:44 and SEQ ID
NO:45,
respectively. In some embodiments, a 4-1BB agonist comprises VII and VL
regions that are each at
least 98% identical to the sequences shown in SEQ ID NO:44 and SEQ ID NO:45,
respectively. In
some embodiments, a 4-1BB agonist comprises Vu and VL regions that are each at
least 97% identical
to the sequences shown in SEQ ID NO:44 and SEQ ID NO:45, respectively. In some
embodiments, a
4-1BB agonist comprises VII and VL regions that are each at least 96%
identical to the sequences
shown in SEQ ID NO:44 and SEQ ID NO:45, respectively. In some embodiments, a 4-
1BB agonist
comprises VH and VL regions that are each at least 95% identical to the
sequences shown in SEQ ID
NO:44 and SEQ ID NO:45, respectively. In some embodiments, a 4-1BB agonist
comprises an scFy
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antibody comprising VH and VL regions that are each at least 99% identical to
the sequences shown in
SEQ ID NO:44 and SEQ ID NO:45.
[00773] In some embodiments, a 4-1BB agonist comprises heavy chain CDR1, CDR2
and CDR3
domains having the sequences set forth in SEQ ID NO:46, SEQ ID NO:47, and SEQ
ID NO:48,
respectively, and conservative amino acid substitutions thereof, and light
chain CDR1, CDR2 and
CDR3 domains having the sequences set forth in SEQ ID NO:49, SEQ ID NO:50, and
SEQ ID
NO :51, respectively, and conservative amino acid substitutions thereof..
[00774] In some embodiments, the 4-1BB agonist is a 4-1BB agonist biosimilar
monoclonal
antibody approved by drug regulatory authorities vvith reference to
utomilumab. In some
embodiments, the biosimilar monoclonal antibody comprises an 4-1BB antibody
comprising an amino
acid sequence which has at least 97% sequence identity, e.g., 97%, 98%, 99% or
100% sequence
identity, to the amino acid sequence of a reference medicinal product or
reference biological product
and which comprises one or more post-translational modifications as compared
to the reference
medicinal product or reference biological product, wherein the reference
medicinal product or
reference biological product is utomilumab. In some embodiments, the one or
more post-translational
modifications are selected from one or more of: glycosylation, oxidation,
deamidation, and truncation.
In some embodiments, the biosimilar is a 4-1BB agonist antibody authorized or
submitted for
authorization, wherein the 4-1BB agonist antibody is provided in a formulation
which differs from the
formulations of a reference medicinal product or reference biological product,
wherein the reference
medicinal product or reference biological product is utomilumab. The 4-1BB
agonist antibody may be
authorized by a drug regulatory authority such as the U.S. FDA and/or the
European Union's EMA. In
some embodiments, the biosimilar is provided as a composition which further
comprises one or more
excipients, wherein the one or more excipients are the same or different to
the excipients comprised in
a reference medicinal product or reference biological product, wherein the
reference medicinal
product or reference biological product is utomilumab. In some embodiments,
the biosimilar is
provided as a composition which further comprises one or more excipients,
wherein the one or more
excipients are the same or different to the excipients comprised in a
reference medicinal product or
reference biological product, wherein the reference medicinal product or
reference biological product
is utomilumab.
TABLE 6. Amino acid sequences for 4-1BB agonist antibodies related to
utomilumab.
Identifier Sequence (One-Letter Amino Acid
Symbols)
SEQ ID NO:42 EVQLVQSGAE VINKPGESLRI SCKGSGYSFS TYWISWVRQM
PGKGLEWMGK IYPGDSYTNY 60
heavy chain for
utomilumab SPSYQGQVTi SADASiSTAY LQWSSLKASO TAMYYCARGY
GIEDYWGQGT LVTVSSASTA 120
GPSVEPLAPC SRSTSESTAA LGCLVKDYFP EPVTVSWNSG ALTSGVHTFP AVIQSSGLYS
180
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LSSVVTVPSS NFGTQTYTCN VDHKPSNTKV DKTVERKCCV ECPPCPAPPV AGPSVFLFPP
240
KPKDTLMISR TPEVTCVVVD VSHEDPEVQF NWYVDGVEVH NAKTKPREEQ FNSTFRVVSV
300
LTVVHQDWLN GKEYKCKVSN KGLPAPIEKT ISKTKGQPRE PQVYTLPPSR EEMTKNQVSL
360
TCLVKGE,YPS D1AVEWESNG QPENNYKTTP E'MLDSDGSEtf LYSALTVIJKS RWQQGNVP'SC
420
SVMHEALHNH YTQSLSLSP G
441
SEQ ID NO:43 SYELTQPPSV SVSPGQTASI TCSGDNIGDQ YANWYQQKPG
QSPVLV=YQD KNRPSGIPER 60
light chain for
utomilumab FSGSNSGNTA TLTISGTQAM DEADYYCATY TGFGSLAVFG
GGTKLTVLGQ PKAAPSVTLF 120
PPSSEELQAN KATLVCLISD FYPGAVTVAW KADSSPVKAG VETTTPSKQS NNKYAASSYL
180
SLTPEQWKSH RSYSCQVTHE GSTVEKTVAP DECO
214
SEQ ID NO:44 EVQLVQSGAE VKKPGESLRI SCKGSGYSFS TYWISWVR0,4
PGKGLEWMG KIYPGDSYTN 60
heavy chain
variable Legion YSPSFQGQVT ISADKSISTA YLQWSSLKAS DTAMYYCARG YGIFDYWGQ
GTLVTVSS 118
for utomilumab
SEQ ID NO:45 SYELTQPPSV SVSPGQTASI TCSGDNIGDQ YAHWYQQKPG
QSPVLV=YQD KNRPSGIPER 60
light chain
variable region FSGSNSGNTA TLTISGTQAM DEADYYCATY TGEGSLAVFG GGTHLTVL
108
for utomilumab
SEQ ID NO:46 STYWIS
6
heavy chain CDR1
for uLomilumab
SEQ ID NO:47 KIYPGDSYTN YSPSFQG
17
heavy chain CDR2
for utomilumab
SEQ ID NO:48 RGYGIFDY
8
heavy chain CDR3
for utomilumab
SEQ ID NO:49 SGDNIGDQYA H
11
light chain CDR1
for utomilumab
SEQ ID NO:50 QDKNRPS
7
light chain CDR2
for utomilumab
SEQ ID NO:51 ATYTGFGSLA V
11
light chain CDR3
for utomilumab
[00775] In some embodiments, the 4-1BB agonist is the monoclonal antibody
urelumab, also known
as BMS-663513 and 20H4.9.h4a, or a fragment, derivative, variant, or
biosimilar thereof. Urelumab is
available from Bristol-Myers Squibb, Inc., and Creative Biolabs, Inc. Urelumab
is an immunoglobulin
G4-kappa, anti-Wool sapiens TNFRSF9 (tumor necrosis factor receptor
superfamily member 9, 4-
1BB, T cell antigen ILA, CD137)1, Homo sapiens (fully human) monoclonal
antibody. The amino
acid sequences of urelumab are set forth in Table 7. Urelumab comprises N-
glycosylation sites at
positions 298 (and 298"); heavy chain intrachain disulfide bridges at
positions 22-95 (VH-V-1.), 148-
204 (CH1-CL), 262-322(C2) and 368-426 (CH3) (and at positions 22"-95", 148"-
204", 262"-322",
and 368"-426"); light chain intrachain disulfide bridges at positions 23'-88'
(VH-VL) and 136'-196'
(CH1-CL) (and at positions 23"-88" and 136"-196¨); interchain heavy chain-
heavy chain
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disulfide bridges at positions 227-227" and 230-230 and interchain heavy chain-
light chain
disulfide bridges at 135-216' and 135"-216¨. The preparation and properties of
urelumab and its
variants and fragments are described in U.S. Patent Nos. 7,288,638 and
8,962,804, the disclosures of
which are incorporated by reference herein. The preclinical and clinical
characteristics of urelumab
are described in Segal, et al., Clin. Cancer Res. 2016, available at
http:/dx.doi.org/ 10.1158/1078-
0432.CCR-16-1272. Current clinical trials of urclumab in a variety of
hematological and solid tumor
indications include U.S. National Institutes of Health clinicaltrials.gov
identifiers NCT01775631,
NCT02110082, NCT02253992, and NCT01471210.
[00776] In some embodiments, a 4-1BB agonist comprises a heavy chain given by
SEQ ID NO:52
and a light chain given by SEQ ID NO:53. In some embodiments, a 4-1BB agonist
comprises heavy
and light chains having the sequences shown in SEQ ID NO:52 and SEQ ID NO:53,
respectively, or
antigen binding fragments, Fab fragments, single-chain variable fragments
(scFv), variants, or
conjugates thereof. In some embodiments, a 4-1BB agonist comprises heavy and
light chains that are
each at least 99% identical to the sequences shown in SEQ ID NO:52 and SEQ ID
NO:53,
respectively. In some embodiments, a 4-1BB agonist comprises heavy and light
chains that are each at
least 98% identical to the sequences shown in SEQ ID NO:52 and SEQ ID NO:53,
respectively. In
some embodiments, a 4-1BB agonist comprises heavy and light chains that are
each at least 97%
identical to the sequences shown in SEQ ID NO:52 and SEQ ID NO:53,
respectively. In some
embodiments, a 4-1BB agonist comprises heavy and light chains that are each at
least 96% identical
to the sequences shown in SEQ TD NO:52 and SEQ ID NO:53, respectively. In
sonic embodiments, a
4-1BB agonist comprises heavy and light chains that are each at least 95%
identical to the sequences
shown in SEQ ID NO:52 and SEQ ID NO:53, respectively.
[00777] In some embodiments, the 4-1BB agonist comprises the heavy and light
chain CDRs or
variable regions (VRs) of urelumab. In some embodiments, the 4-1BB agonist
heavy chain variable
region (VH) comprises the sequence shown in SEQ ID NO:54, and the 4-1BB
agonist light chain
variable region (VL) comprises the sequence shown in SEQ ID NO:55, and
conservative amino acid
substitutions thereof. In some embodiments, a 4-1BB agonist comprises Vii and
VL regions that arc
each at least 99% identical to the sequences shown in SEQ ID NO:54 and SEQ ID
NO:55,
respectively. In some embodiments, a 4-1BB agonist comprises VH and VL regions
that are each at
least 98% identical to the sequences shown in SEQ ID NO:54 and SEQ ID NO:55,
respectively. In
some embodiments, a 4-1BB agonist comprises VH and VL regions that are each at
least 97% identical
to the sequences shown in SEQ ID NO:54 and SEQ ID NO:55, respectively. In some
embodiments, a
4-1BB agonist comprises VH and VL regions that arc each at least 96% identical
to the sequences
shown in SEQ ID NO:54 and SEQ ID NO:55, respectively. In some embodiments, a 4-
1BB agonist
comprises VH and VL regions that are each at least 95% identical to the
sequences shown in SEQ ID
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NO:54 and SEQ ID NO:55, respectively. In some embodiments, a 4-1BB agonist
comprises an scFy
antibody comprising VH and VL regions that are each at least 99% identical to
the sequences shown in
SEQ ID NO:54 and SEQ ID NO:55.
[00778] In some embodiments, a 4-1BB agonist comprises heavy chain CDR1, CDR2
and CDR3
domains having the sequences set forth in SEQ ID NO:56, SEQ ID NO:57, and SEQ
ID NO:58,
respectively, and conservative amino acid substitutions thereof, and light
chain CDR1, CDR2 and
CDR3 domains having the sequences set forth in SEQ ID NO:59, SEQ ID NO:60, and
SEQ ID
NO:61, respectively, and conservative amino acid substitutions thereof.
[00779] In some embodiments, the 4-1BB agonist is a 4-1BB agonist biosimilar
monoclonal
antibody approved by drug regulatory authorities with reference to urelumab.
In some embodiments,
the biosimilar monoclonal antibody comprises an 4-1BB antibody comprising an
amino acid sequence
which has at least 97% sequence identity, e.g., 97%, 98%, 99% or 100% sequence
identity, to the
amino acid sequence of a reference medicinal product or reference biological
product and which
comprises one or more post-translational modifications as compared to the
reference medicinal
product or reference biological product, wherein the reference medicinal
product or reference
biological product is urelumab. In some embodiments, the one or more post-
translational
modifications are selected from one or more of: glycosylation, oxidation,
deamidation, and truncation.
In some embodiments, the biosimilar is a 4-1BB agonist antibody authorized or
submitted for
authorization, wherein the 4-1BB agonist antibody is provided in a formulation
which differs from the
formulations of a reference medicinal product or reference biological product,
wherein the reference
medicinal product or reference biological product is urelumab. The 4-1BB
agonist antibody may be
authorized by a drug regulatory authority such as the U.S. FDA and/or the
European Union's EMA. In
some embodiments, the biosimilar is provided as a composition which further
comprises one or more
excipients, wherein the one or more excipients are the same or different to
the excipients comprised in
a reference medicinal product or reference biological product, wherein the
reference medicinal
product or reference biological product is urelumab. In some embodiments, the
biosimilar is provided
as a composition which further comprises one or more excipients, wherein the
one or more excipients
are the same or different to the excipients comprised in a reference medicinal
product or reference
biological product, wherein the reference medicinal product or reference
biological product is
urelumab.
TABLE 7: Amino acid sequences for 4-1BB agonist antibodies related to
urelumab.
Identifier Sequence (One-Letter Amino Acid
Symbols)
SEQ ill N0:52 QVQLQQWGAG LLKPSETLSL TCAVYGGSN'S GYYWSWiRQS
PKGLEW_LC;E INHGGYVTYN 60
heavy chain for PSLESRVTIS VDTSKNQFSL KLSSVTAADT AVYYCARDYG
PGNYDWYFDL WGRGILVIVS 120
urelumab SASTKGPSVF PLAPCSRSTS ESTAALGCLV KDYFPEPVTV
SWNSGALTSG VHTFDAVLQS 180
SGLYSLSSVV TVRSSSIGTH TYIGNVDEKP SNTKVDKRVE SHYGPPCP2C PAPEFIGGPS
240
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VFLFPPKPKD TLMISRTPEV TCVVVDVSQE DPEVQFNWYV DGVEVHNAKT KPREEQFNST
300
YRVVSVLTVL HQDWLNGKEY KCKVSEKGLP SSIEKTISKA EGQPREPQVY TLPPSQEEMT
360
KNQVSLTCLV KGFYPSDIAV EWESNGOPEN NYKTTPPVLD SDGSFFLYSR LTVDHSRWQE
420
GNVFSCSVMH EALHNHYTQK SLSLSLGY.
448
SEQ ID NO:53 EIVLTQSPAT LSLSFGERAT LSCRASQSVS SYLAWYQQHP
GQAPRLLIYD ASNRATGIPA 60
light chain for R314SGSGf I L SSLEE EDFAVYYCQQ RSNWPPALTF CGGTKVE100
VAAOSVI 1 20
urelumab PPS2EQLKSG TASVVCLLNN .YPHEAKVQW KV2NALQSGN
SQESVTEQDS K2STYSLSST 180
LTLSKADYEK NKVYACEVM QGLSSPVTKS FNRGEC
216
SEQ ID NO:54 MKHLWFFLLL VAAPRWVLSQ VQLQQWGAGL LKPSETLSLT
CAVYGGSFSG YYWSWIRQSP 60
variable heavy EHGLEWIGEI NNGGYVTYNP SLESRVTISV DTSKNQFSLK
LSSVTAADTA VYYCARDYGP 120
chain for
urelumab
SEQ ID NO:55 MEAPAQLLFL LLLWLPD=G EIVLTQSPAT LSLSPGERAT LSCRASQSVS
SYLAWYQQKP 60
variable light GQAPRLLIYD ASNRATGIPA RFSGSGSGTD FTLTISSLEP
EDFAVYYCQQ 110
chain for
urelumab
SEQ ID NO:56 GYYWS
5
heavy chain CDR1
for urelumab
SEQ ID NO:57 EINHGGYVTY NPSLES
16
heavy chain CD02
for urelumab
SEQ ID NO:58 DYGPGNYDWY FDL
13
heavy chain CD03
for urelumab
SEQ ID NO:59 RASQSVSSYL A
11
light chain CDR1
for urelumab
SEQ ID 110:60 DASNRAT
7
light chain CD02
for urelumab
SEQ ID 00:61 QQRSDWPPAL T
11
uhain CDR3
for urelumab
[00780] In some embodiments, the 4-1BB agonist is selected from the group
consisting of 1D8,
3Elor, 4B4 (BioLegend 309809), H4-1BB-M127 (BD Pharmingen 552532), BBK2
(Thermo Fisher
MS621PABX), 145501 (Leinco Technologies B591), the antibody produced by cell
line deposited as
ATCC No. HB-11248 and disclosed in U.S. Patent No. 6,974,863, 5F4 (BioLegend
31 1503), C65-
485 (BD Pharmingen 559446), antibodies disclosed in U.S. Patent Application
Publication No. US
2005/0095244, antibodies disclosed in U.S. Patent No. 7,288,638 (such as
20H4.9-IgG1 (BMS-
663031)), antibodies disclosed in U.S. Patent No. 6,887,673 (such as 4E9 or
BMS-554271),
antibodies disclosed in U.S. Patent No. 7,214,493, antibodies disclosed in
U.S. Patent No. 6,303,121,
antibodies disclosed in U.S. Patent No. 6,569,997, antibodies disclosed in
U.S. Patent No. 6,905,685
(such as 4E9 or BMS-554271), antibodies disclosed in U.S. Patent No. 6,362,325
(such as 1D8 or
BMS-469492; 3H3 or BMS-469497; or 3E1), antibodies disclosed in U.S. Patent
No. 6,974,863 (such
as 53A2); antibodies disclosed in U.S. Patent No. 6,210,669 (such as 1D8, 3B8,
or 3E1), antibodies
described in U.S. Patent No. 5,928,893, antibodies disclosed in U.S. Patent
No. 6,303,121, antibodies
disclosed in U.S. Patent No. 6,569,997, antibodies disclosed in International
Patent Application
Publication Nos. WO 2012/177788, WO 2015/119923, and WO 2010/042433, and
fragments,
derivatives, conjugates, variants, or biosimilars thereof, wherein the
disclosure of each of the
foregoing patents or patent application publications is incorporated by
reference here.
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1007811 In some embodiments, the 4-1BB agonist is a 4-1BB agonistic fusion
protein described in
International Patent Application Publication Nos. WO 2008/025516 Al, WO
2009/007120 Al, WO
2010/003766 Al, WO 2010/010051 Al, and W02010/078966 Al; U.S. Patent
Application
Publication Nos. US 2011/0027218 Al, US 2015/0126709 Al, US 2011/0111494 Al,
US
2015/0110734 Al, and US 2015/0126710 Al; and U.S. Patent Nos. 9,359,420,
9,340,599, 8,921,519,
and 8,450,460, the disclosures of which are incorporated by reference herein.
[00782] In some embodiments, the 4-1BB agonist is a 4-1BB agonistic fusion
protein as depicted in
Structure I-A (C-terminal Fc-antibody fragment fusion protein) or Structure I-
B (N-terminal Fc-
antibody fragment fusion protein), or a fragment, derivative, conjugate,
variant, or biosimilar thereof
(see, Figure 18). In structures I-A and I-B, the cylinders refer to individual
polypeptide binding
domains. Structures 1-A and 1-B comprise three linearly-linked 'TNFRSF binding
domains derived
from e.g., 4-1BBL (4-1BB ligand, CD137 ligand (CD137L), or tumor necrosis
factor superfamily
member 9 (TNFSF9)) or an antibody that binds 4-1BB, which fold to form a
trivalent protein, which
is then linked to a second triavelent protein through IgGl-Fc (including CH3
and CH2 domains) is then
used to link two of the trivalent proteins together through disulfide bonds
(small elongated ovals),
stabilizing the structure and providing an agonists capable of bringing
together the intracellular
signaling domains of the six receptors and signaling proteins to form a
signaling complex. The
TNFRSF binding domains denoted as cylinders may be seFy domains comprising,
e.g., a VH and a VL
chain connected by a linker that may comprise hydrophilic residues and Gly and
Ser sequences for
flexibility, as well as Glu and Lys for solubility. Any scFy domain design may
be used, such as those
described in de Marco, Microbial Cell Factories, 2011, 10, 44; Ahmad, et al.,
Clin. & Dev. Immunol.
2012, 980250; Monnier, et al. ,Ant/bodies, 2013, 2, 193-208; or in references
incorporated elsewhere
herein. Fusion protein stmctures of this form are described in U.S. Patent
Nos. 9,359,420, 9,340,599,
8,921,519, and 8,450,460, the disclosures of which are incorporated by
reference herein.
[00783] Amino acid sequences for the other polypeptide domains of structure I-
A given in Figure 18
are found in Table 8. The Fc domain preferably comprises a complete constant
domain (amino acids
17-230 of SEQ ID NO:62) the complete hinge domain (amino acids 1-16 of SEQ ID
NO:62) or a
portion of the hinge domain (e.g., amino acids 4-16 of SEQ ID NO:62).
Preferred linkers for
connecting a C-terminal Fc-antibody may be selected from the embodiments given
in SEQ ID NO:63
to SEQ ID NO:72, including linkers suitable for fusion of additional
polypeptides.
TABLE 8: Amino acid sequences for TNFRSF agonist fusion proteins, including 4-
1BB agonist
fusion proteins, with C-terminal Fc-antibody fragment fusion protein design
(structure I-A).
Identifier Sequence (One-Letter Amino Acid
Symbols)
SEQ ID NO:62 KSCDKTHTCP PCPAPELLGG PSVFLFPPKP KDTLMISRTP
EVTCVVVDVS HEDPEVKFNW 60
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Fc domain YVDGVEVHNA KTKPREEQYN STYRVVSVLT VLHQDWLNGK
EYKCKVSNKA LPAPIEKTIS 120
KAKGQPREPQ VYTLPPSREE MTKNQVSLTC LVEGFYPSEI AVEWESNGQP ENNYKTTPPV
180
LDSDGSFFLY SHLTVDHSRW QQGNVFSCSV MNEALHNNYT QHSLSLSPGH
230
SEQ ID NO:63 GGPGSSKSCD KTHTCPPCPA PE
22
linker
SEQ ID NO:64 GGSGSSKSCD KTHTCPPCPA PE
22
linker
SEQ ID NO:65 GGPGSSSSSS SKSCDHTH= PPCPAPE
27
linker
SEQ ID NO:66 GGSGSSSSSS SKSCDHTHTC PPCPAPE
27
linker
SEQ ID NO:67 GGPGSSSSSS SSSHSCD= TCPPCPAPE
29
linker
SEQ ID NO:68 GGSGSSSSSS SSSHSCDEM TCPPCPAPE
29
linker
SEQ ID NO:69 GGPGSSGSGS SDETNTCPPC aAPE
24
linker
SEQ ID NO:70 GGPGSSGSGS DKTHTCPPCP APE
23
linker
SEQ ID NO:71 GGPSSSGSDK THTCPPCPAP E
21
linker
SEQ ID NO:72 GGSSSSSSSS GSEKTNTCPP CPAPE
25
linker
1007841 Amino acid sequences for the other polypeptide domains of structure I-
B given in Figure 18
are found in Table 9. If an Fc antibody fragment is fused to the N-terminus of
an TNRFSF fusion
protein as in structure I-B, the sequence of the Fc module is preferably that
shown in SEQ ID NO:73,
and the linker sequences are preferably selected from those embodiments set
forth in SED ID NO:74
to SEQ ID NO:76.
TABLE 9: Amino acid sequences for TNFRSF agonist fusion proteins, including 4-
1BB agonist
fusion proteins, with N-terminal Fe-antibody fragment fusion protein design
(structure I-B).
Identifier Sequence (One-Letter Amino Acid
Symbols)
SEQ ID NO:73 METDTLLLWV LLLWVPAGNG DHTHTCPPCP APELLGGPSV
FLEPPHPKDT LMISRTPEVT 60
Pc domain CVVVDVSNED PEVAFNWYVD GVEVNNAKTK PREEQYNSTY
RVVSVLI'VLH QDWLNGKEYA 120
CKVSNKALPA PIEKTISKAK GQPREPQVYT LPPSREEMTH NQVSLTCLVE GFYPSDIAME
180
WESNGQPENN YKTTPPVLDS DGSFYLYSKL TVDESRWQQG NVFSCSVMNE ALIINHYTQKS
240
LSLSPG
246
SEQ ID NO:74 SGSGSGSGSG S
11
linker
SEQ ID ND: 75 SSSSSSGSGS GS
12
linker
SEQ ID NO:76 SSSSSSGSGS GSGSGS
16
linker
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[00785] In some embodiments, a 4-1BB agonist fusion protein according to
structures I-A or I-B
comprises one or more 4-1BB binding domains selected from the group consisting
of a variable heavy
chain and variable light chain of utomilumab, a variable heavy chain and
variable light chain of
urelumab, a variable heavy chain and variable light chain of utomilumab, a
variable heavy chain and
variable light chain selected from the variable heavy chains and variable
light chains described in
Table 5, any combination of a variable heavy chain and variable light chain of
the foregoing, and
fragments, derivatives, conjugates, variants, and biosimilars thereof.
[00786] In some embodiments, a 4-1BB agonist fusion protein according to
structures I-A or I-B
comprises one or more 4-1BB binding domains comprising a 4-1BBL sequence. In
some
embodiments, a 4-1BB agonist fusion protein according to structures I-A or I-B
comprises one or
more 4-1BB binding domains comprising a sequence according to SEQ ID NO:77. In
some
embodiments, a 4-1BB agonist fusion protein according to structures I-A or I-B
comprises one or
more 4-1BB binding domains comprising a soluble 4-1BBL sequence. In some
embodiments, a 4-
1BB agonist fusion protein according to structures I-A or I-B comprises one or
more 4-1BB binding
domains comprising a sequence according to SEQ ID NO:78.
[00787] In some embodiments, a 4-1BB agonist fusion protein according to
structures I-A or I-B
comprises one or more 4-1BB binding domains that is a scFy domain comprising
VH and VL regions
that arc each at least 95% identical to the sequences shown in SEQ ID NO:43
and SEQ ID NO:44,
respectively, wherein the Vi4 and VL domains are connected by a linker. In
some embodiments, a 4-
1BB agonist fusion protein according to structures I-A or I-B comprises one or
more 4-1BB binding
domains that is a scFy domain comprising VII and VL regions that are each at
least 95% identical to
the sequences shown in SEQ ID NO:54 and SEQ ID NO:55, respectively, wherein
the VH and VL
domains are connected by a linker. In some embodiments, a 4-1BB agonist fusion
protein according
to structures I-A or I-B comprises one or more 4-1BB binding domains that is a
scFy domain
comprising VH and VL regions that are each at least 95% identical to the VH
and VL sequences given
in Table 10, wherein the VH and VL domains are connected by a linker.
TABLE 10: Additional polypeptide domains useful as 4-1BB binding domains in
fusion proteins or as
scFy 4-IBB agonist antibodies.
Identifier Sequence (One-Letter Amino Acid
Symbols)
SEQ ID NO:77 MEYASDASLD PEAPWPPAPR ARACRVLPWA LVAGLLLLLL
LAAACAVFLA CPWAVSGARA 60
4-13BL SPGSAASPRL REGPELSPDD RAGLLDLRQG MEAQLVAQNV
LLIDGPLSWY SDPGLAGVSL 120
TGGLSYKEDT KELVVAKAGV YYVFFQLELR RVVAGEGSGS VSLALHLQPL RSAAGAAALA
180
LTVDLPPASS EARNSAFGFQ GRLLHLSAGQ RLGVHLHTEA RARNAWQLTQ aATVLGLERV
240
TPEIPAGLPS PRSE
254
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SEQ ID NO:78 LRQGMFAQLV AQNVLLIDGP LSWYSDPGLA GVSLTGGLSY
KEDTKELVVA KAGVYYVFFQ 60
4-1BEL soluble LELRRVVAGE GSGSVSLALH LQPLRSAAGA AALALTVDLP
PASSEARNSA FCFQGRLLHL 120
domain
aAGQRLGVHL HTEARARHAW QLTQGATVLG LFRVTPEIPA GLPSPRSE
168
SEQ ID NO:79 QVQLQQPGAE LVKPGASVKL SCKASGYTFS SYWMHWVKQR
PGQVLEWIGE INPGNGHTNY 60
variable heavy
chain for 454-1- NEKFKSKATL TVD-ASSSTAY MQLSSLTSED SAVYYCARSF TTARGFAYWG
QGTLVTVS 118
^ version 1
SEQ ID 50:00 DIVMTQSPAT QSVTPGDRVS LSCRASQTIS DYLHWYQQKS
NESPRLLIKY ASQSISGIPS 60
variable light
chain for 154-1- RFSGSGSGSD FTLSINSVEP EDVGVYYCQD GHSFPPTFGG GTKLEIK
107
= version 1
SEQ ID 50:81 QVQLQQPGAE LVKPGASVKL SCKASGYTFS SYWMHWVKQR
PGQVLEWIGE INPGNGHTNY 60
variable heavy
chain for 4134-1- NEKFKSKATL TVDASSSTAY MQLSSLTSED SAVYYCARSF TTARGFAYWG
QGTLVTVSA 119
= version 2
SEQ ID NO:82 DIVMTQSPAT QSVTPGDRVS LSCRASQTIS DYLHWYQQKS
NESPRLLIKY ASQSISGIPS 60
variable light
chain for 454-1- RFSGSGSGSD FTLSINSVEP EDVGVYYCQD GHSFPPTFGG GTKLEIKR
108
^ version 2
SEQ ID 50:83 MDWTWRILFL VAAATGAHSE VQLVESGGGL VQPGGSLRLS
CAASGFTESD YWMSWVRQAP 60
variable heavy
chain fo, H39E3_ GKGLEWVADI KNDGSYTNYA PSLTNRFTIS RDNAKNSLYL QMNSLRAEDT
AVYYCARELT 120
2
SEQ ID NO:04 MEAPAQLLFL LLLWLPDTTG DIVMTQSPDS LAVSLGERAT
INCKSSQSLL SSGNQKNYL 60
variable light
chain for H39E3- WYQQKPGQPP ALLIYYASDR QSGV2DRESG SG5501i0fLT
ISSLQAELVA 110
2
[00788] In some embodiments, the 4- I BB agonist is a 4-1BB agonistic single-
chain fusion
polypeptide comprising (i) a first soluble 4-1BB binding domain, (ii) a first
peptide linker, (iii) a
second soluble 4-1BB binding domain, (iv) a second peptide linker, and (v) a
third soluble 4-1BB
binding domain, further comprising an additional domain at the N-terminal
and/or C-terminal end,
and wherein the additional domain is a Fab or Fc fragment domain. In some
embodiments, the 4-1BB
agonist is a 4-1BB agonistic single-chain fusion polypeptide comprising (i) a
first soluble 4-1BB
binding domain, (ii) a first peptide linker, (iii) a second soluble 4-1BB
binding domain, (iv) a second
peptide linker, and (v) a third soluble 4-1BB binding domain, further
comprising an additional domain
at the N-terminal and/or C-terminal end, wherein the additional domain is a
Fab or Fc fragment
domain, wherein each of the soluble 4-1BB domains lacks a stalk region (which
contributes to
trimerisation and provides a certain distance to the cell membrane, but is not
part of the 4-1BB
binding domain) and the first and the second peptide linkers independently
have a length of 3-8 amino
acids.
[00789] In some embodiments, the 4-1BB agonist is a 4-1BB agonistic single-
chain fusion
polypeptide comprising (i) a first soluble tumor necrosis factor (TNF)
superfamily cytokine domain,
(ii) a first peptide linker, (iii) a second soluble TNF superfamily cytokine
domain, (iv) a second
peptide linker, and (v) a third soluble TNT superfamily cytokine domain,
wherein each of the soluble
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TNF superfamily cytokine domains lacks a stalk region and the first and the
second peptide linkers
independently have a length of 3-8 amino acids, and wherein each TNF
superfamily cytokine domain
is a 4-1BB binding domain.
[00790] In some embodiments, the 4-1BB agonist is a 4-1BB agonistic scFy
antibody comprising
any of the foregoing VH domains linked to any of the foregoing VL domains.
[00791] In some embodiments, the 4-1BB agonist is BPS Bioscience 4-1BB agonist
antibody
catalog no. 79097-2, commercially available from BPS Bioscience, San Diego,
CA, USA. In some
embodiments, the 4-1BB agonist is Creative Biolabs 4-1BB agonist antibody
catalog no. MOM-
18179, commercially available from Creative Biolabs, Shirley, NY, USA.
3. 0X40 (CD134) Agonists
[00792] In some embodiments, the TNFRSF agonist is an 0X40 (CD134) agonist.
The 0X40
agonist may be any 0X40 binding molecule known in the art. The 0X40 binding
molecule may be a
monoclonal antibody or fusion protein capable of binding to human or mammalian
0X40. The 0X40
agonists or 0X40 binding molecules may comprise an immunoglobulin heavy chain
of any isotype
(e.g., IgG, IgE, IgM, IgD, IgA, and IgY), class (e.g., IgGl, IgG2, IgG3, IgG4,
IgAl and IgA2) or
subclass of immunoglobulin molecule. The 0X40 agonist or 0X40 binding molecule
may have both a
heavy and a light chain. As used herein, the term binding molecule also
includes antibodies (including
full length antibodies), monoclonal antibodies (including full length
monoclonal antibodies),
polyclonal antibodies, multispecific antibodies (e.g., bispecific antibodies),
human, humanized or
chimeric antibodies, and antibody fragments, e.g., Fab fragments, F(ab')
fragments, fragments
produced by a Fab expression library, epitope-binding fragments of any of the
above, and engineered
forms of antibodies, e.g., scFv molecules, that bind to 0X40. In some
embodiments, the 0X40
agonist is an antigen binding protein that is a fully human antibody. In some
embodiments, the 0X40
agonist is an antigen binding protein that is a humanized antibody. In some
embodiments, 0X40
agonists for use in the presently disclosed methods and compositions include
anti-0X40 antibodies,
human anti-0X40 antibodies, mouse anti-0X40 antibodies, mammalian anti-0X40
antibodies,
monoclonal anti-0X40 antibodies, polyclonal anti-0X40 antibodies, chimeric
anti-0X40 antibodies,
anti-0X40 adncctins, anti-0X40 domain antibodies, single chain anti-0X40
fragments, heavy chain
anti-0X40 fragments, light chain anti-0X40 fragments, anti-0X40 fusion
proteins, and fragments,
derivatives, conjugates, variants, or biosimilars thereof. In some
embodiments, the 0X40 agonist is an
agonistic, anti-0X40 humanized or fully human monoclonal antibody (i.e., an
antibody derived from
a single cell line).
[00793] In some embodiments, the 0X40 agonist or 0X40 binding molecule may
also be a fusion
protein. 0X40 fusion proteins comprising an Fe domain fused to OX4OL are
described, for example,
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in Sadun, et al., J. Immunother. 2009, 182, 1481-89. In some embodiments, a
multimeric 0X40
agonist, such as a trimeric or hexameric 0X40 agonist (with three or six
ligand binding domains),
may induce superior receptor (0X4OL) clustering and internal cellular
signaling complex formation
compared to an agonistic monoclonal antibody, which typically possesses two
ligand binding
domains. Trimeric (trivalent) or hexameric (or hexavalent) or greater fusion
proteins comprising three
'INFRSF binding domains and IgGI-Fc and optionally further linking two or more
of these fusion
proteins are described, e.g., in Gieffers, et al., Mol. Cancer Therapeutics
2013,12, 2735-47.
[00794] Agonistic 0X40 antibodies and fusion proteins are known to induce
strong immune
responses. Curti, et al., Cancer Res. 2013, 73, 7189-98. In some embodiments,
the 0X40 agonist is a
monoclonal antibody or fusion protein that binds specifically to 0X40 antigen
in a manner sufficient
to reduce toxicity. In some embodiments, the 0X40 agonist is an agonistic 0X40
monoclonal
antibody or fusion protein that abrogates antibody-dependent cellular toxicity
(ADCC), for example
NK cell cytotoxicity. hi some embodiments, the 0X40 agonist is an agonistic
0X40 monoclonal
antibody or fusion protein that abrogates antibody-dependent cell phagocytosis
(ADCP). In some
embodiments, the 0X40 agonist is an agonistic 0X40 monoclonal antibody or
fusion protein that
abrogates complement-dependent cytotoxicity (CDC). In some embodiments, the
0X40 agonist is an
agonistic 0X40 monoclonal antibody or fusion protein which abrogates Fe region
functionality.
[00795] In some embodiments, the 0X40 agonists are characterized by binding to
human 0X40
(SEQ ID NO:85) with high affinity and agonistic activity. In some embodiments,
the 0X40 agonist is
a binding molecule that binds to human 0X40 (SEQ ID NO:85). In some
embodiments, the 0X40
agonist is a binding molecule that binds to murinc 0X40 (SEQ ID NO:86). The
amino acid sequences
of 0X40 antigen to which an 0X40 agonist or binding molecule binds are
summarized in Table 11.
TABLE 11: Amino acid sequences of 0X40 antigens.
Identifier Sequence (One-Letter Amino Acid
Symbols)
SEQ ill 00:85 MCVGARRLGR GPCAALLLLG LGLSTVTGLH CVGDTYPSND
RCCHECRPGN GMVSRCSRSQ 60
human 0X40
NTVCRPCGPG FYNDVVSSKP CKPCTWCNLR SGSERKOLCT ATODTVCRCR AGTOPLDSYK
120
;Homo sapiens)
PGVDCAPCPP GHFSPGDNQA CKPWTNCTLA GKHTLQPASN SSDAICEDRD PPATQPOETO
100
GPPARPITVQ PTEAWPRTSQ GPSTRPVEVP GGRAVAAILG LGLVLGLLGP LAILLALYLL
240
RRDQRLPPDA HKPPGGGSFR TPIQEEQADA HSTLAKI
277
SEQ ID NO:86 MYVWVQQPTA LLLLGLTLGV TARRLNCVKII TYPSGHKCCR
EQOPGIIGMVS RCDITTRDTLC 60
ri ne 0X4
HPCETGFYNE AVNYDTCHQC TQCNHRSGSE LKQNCTPTCD TVCRCRPGTQ PRQDSGYKLG
120
;Mus musculus)
VDCVPCPPGH FSPGNNQACK PWTNCTLSGK QTRHPASDSL DAVCEDRSLL ATLLWETQRP
180
TFRPTTVQST TVWPRTSELP SPPTLVTPEG PAFAVLLGLG LGLLAPLTVL LALYLLRKAW
240
RLE'NTPKPCW GNSFRTPIQE EHTDAHFTLA NI
272
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[00796] In some embodiments, the compositions, processes and methods described
include a 0X40
agonist that binds human or murine 0X40 with a KD of about 100 pM or lower,
binds human or
murine 0X40 with a KD of about 90 pM or lower, binds human or murine 0X40 with
a KD of about
80 pM or lower, binds human or murine 0X40 with a KD of about 70 pM or lower,
binds human or
murine 0X40 with a KD of about 60 pM or lower, binds human or murine 0X40 with
a KD of about
50 pM or lower, binds human or murine 0X40 with a KD of about 40 pM or lower,
or binds human or
murine 0X40 with a KD of about 30 pM or lower.
[00797] In some embodiments, the compositions, processes and methods described
include a 0X40
agonist that binds to human or murine 0X40 with a kassoc of about 7.5 x 105
1/M- s or faster, binds to
human or murine 0X40 with a kassoc of about 7.5 x 105 1/M- s or faster, binds
to human or murine
0X40 with a kass.c of about 8 x 105 1/M. s or faster, binds to human or murine
0X40 with a kassoc of
about 8.5 x 105 1/Ms or faster, binds to human or murine 0X40 with a kassoc of
about 9 x 105 1/Ms
or faster, binds to human or murine 0X40 with a kassoc of about 9.5 x 105 1/M-
s or faster, or binds to
human or murine 0X40 with a kaoc of about 1 x 106 1/M- s or faster.
[00798] In some embodiments, the compositions, processes and methods described
include a 0X40
agonist that binds to human or murine 0X40 with a kdiõoe of about 2 x 10-5 1/s
or slower, binds to
human or murine 0X40 with a kdiõ., of about 2.1 x 10-5 1/s or slower, binds to
human or murine
0X40 with a kdissoc of about 2.2 x 10-5 1/s or slower, binds to human or
murine 0X40 with a kaissoc of
about 2.3 x 10-5 1/s or slower, binds to human or murine 0X40 with a kdissoc
of about 2.4 x 10 1/s or
slower, binds to human or murine 0X40 with a kdissoc of about 2.5 x 10-5 1/s
or slower, binds to human
or murine 0X40 with a kdissoc of about 2.6>< 10-5 1/s or slower or binds to
human or murine 0X40
with a kaissoc of about 2.7 x 10' 1/s or slower, binds to human or murine 0X40
with a kdissoc of about
2.8 x 10-5 1/s or slower, binds to human or murine 0X40 with a kdissoc of
about 2.9 x 10-5 1/s or
slower, or binds to human or murine 0X40 with a kdissoc of about 3 x 10' 1/s
or slower.
[00799] In some embodiments, the compositions, processes and methods described
include 0X40
agonist that binds to human or murine 0X40 with an IC50 of about 10 nM or
lower, binds to human or
murine 0X40 with an IC50 of about 9 nM or lower, binds to human or murine 0X40
with an IC50 of
about 8 nM or lower, binds to human or murine 0X40 with an IC50 of about 7 nM
or lower, binds to
human or murine 0X40 with an 1050 of about 6 nM or lower, binds to human or
murine 0X40 with an
1050 of about 5 nM or lower, binds to human or murine 0X40 with an 1050 of
about 4 nM or lower,
binds to human or murine 0X40 with an 1050 of about 3 nM or lower, binds to
human or murine
0X40 with an IC50 of about 2 nM or lower, or binds to human or murine 0X40
with an IC50 of about
1 nM or lower.
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[00800] In some embodiments, the 0X40 agonist is tavolixizumab, also known as
MEDI0562 or
MEDI-0562. Tavolixizumab is available from the MedImmune subsidiary of
AstraZeneca, Inc.
Tavolixizumab is immunoglobulin Gl-kappa, anti-[Homo sapiens TNFRSF4 (tumor
necrosis factor
receptor (TNFR) superfamily member 4, 0X40, CD134)1, humanized and chimeric
monoclonal
antibody. The amino acid sequences of tavolixizumab are set forth in Table 12.
Tavolixizumab
comprises N-glycosylation sites at positions 301 and 301", with fucosylated
complex bi-antennary
CHO-type glycans; heavy chain intrachain disulfide bridges at positions 22-95
(VH-VL), 148-204
(CH1-CL), 265-325 (CH2) and 371-429 (CH3) (and at positions 22"-95", 148"-
204", 265"-325", and
371"-429"); light chain intrachain disulfide bridges at positions 23'-88' (VH-
VI,) and 134'-194'
(CH1-CL) (and at positions 23"-88" and 134"-194"); interchain heavy chain-
heavy chain
disulfide bridges at positions 230-230" and 233-233"; and interchain heavy
chain-light chain
disulfide bridges at 224-214' and 224--214". Current clinical trials of
tavolixizumab in a variety of
solid tumor indications include U.S. National Institutes of Health
clinicaltrials.gov identifiers
NCT02318394 and NCT02705482.
[00801] In some embodiments, a 0X40 agonist comprises a heavy chain given by
SEQ ID NO:87
and a light chain given by SEQ ID NO:88. In some embodiments, a 0X40 agonist
comprises heavy
and light chains having the sequences shown in SEQ ID NO:87 and SEQ ID NO:88,
respectively, or
antigen binding fragments, Fab fragments, single-chain variable fragments
(scFy), variants, or
conjugates thereof In some embodiments, a 0X40 agonist comprises heavy and
light chains that are
each at least 99% identical to the sequences shown in SEQ ID NO:87 and SEQ ID
NO:88,
respectively. In some embodiments, a 0X40 agonist comprises heavy and light
chains that are each at
least 98% identical to the sequences shown in SEQ ID NO:87 and SEQ ID NO:88,
respectively. In
some embodiments, a 0X40 agonist comprises heavy and light chains that are
each at least 97%
identical to the sequences shown in SEQ ID NO:87 and SEQ ID NO:88,
respectively. In some
embodiments, a OX40 agonist comprises heavy and light chains that are each at
least 96% identical to
the sequences shown in SEQ ID NO:87 and SEQ ID NO:88, respectively. In some
embodiments, a
0X40 agonist comprises heavy and light chains that are each at least 95%
identical to the sequences
shown in SEQ ID NO:87 and SEQ ID NO:88, respectively.
[00802] In some embodiments, the 0X40 agonist comprises the heavy and light
chain CDRs or
variable regions (VRs) of tavolixizumab. In some embodiments, the 0X40 agonist
heavy chain
variable region (Vu) comprises the sequence shown in SEQ ID NO:89, and the
0X40 agonist light
chain variable region (VL) comprises the sequence shown in SEQ ID NO:90, and
conservative amino
acid substitutions thereof. In some embodiments, a 0X40 agonist comprises VE
and VL regions that
are each at least 99% identical to the sequences shown in SEQ ID NO: 89 and
SEQ ID NO:90,
respectively. In some embodiments, a 0X40 agonist comprises Vri and VL regions
that are each at
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least 98% identical to the sequences shown in SEQ ID NO:89 and SEQ ID NO:90,
respectively. In
some embodiments, a 0X40 agonist comprises VH and VL regions that are each at
least 97% identical
to the sequences shown in SEQ TD NO:89 and SEQ ID NO:90, respectively. In some
embodiments, a
0X40 agonist comprises VH and VL regions that are each at least 96% identical
to the sequences
shown in SEQ ID NO:89 and SEQ ID NO:90, respectively. In some embodiments, a
0X40 agonist
comprises VH and VI, regions that are each at least 95% identical to the
sequences shown in SEQ ID
NO:89 and SEQ ID NO:90, respectively. In some embodiments, an 0X40 agonist
comprises an scFy
antibody comprising VH and VL regions that are each at least 99% identical to
the sequences shown in
SEQ ID NO:89 and SEQ ID NO:90.
[00803] In some embodiments, a 0X40 agonist comprises heavy chain CDR1, CDR2
and CDR3
domains having the sequences set forth in SEQ ID NO:91, SEQ ID NO:92, and SEQ
ID NO:93,
respectively, and conservative amino acid substitutions thereof, and light
chain CDR1, CDR2 and
CDR3 domains having the sequences set forth in SEQ TD NO:94, SEQ TD NO:95, and
SEQ ID
NO:96, respectively, and conservative amino acid substitutions thereof.
[00804] In an embodiment, the 0X40 agonist is a 0X40 agonist biosimilar
monoclonal antibody
approved by drug regulatory authorities with reference to tavolixizumab. in an
embodimentin some
embodiments, the biosimilar monoclonal antibody comprises an 0X40 antibody
comprising an amino
acid sequence which has at least 97% sequence identity, e.g., 97%, 98%, 99% or
100% sequence
identity, to the amino acid sequence of a reference medicinal product or
reference biological product
and which comprises one or more post-translational modifications as compared
to the reference
medicinal product or reference biological product, wherein the reference
medicinal product or
reference biological product is tavolixizumab. In some embodiments, the one or
more post-
translational modifications are selected from one or more of: glycosylation,
oxidation, deamidation,
and truncation. In some embodiments, the biosimilar is a 0X40 agonist antibody
authorized or
submitted for authorization, wherein the 0X40 agonist antibody is provided in
a formulation which
differs from the formulations of a reference medicinal product or reference
biological product,
wherein the reference medicinal product or reference biological product is
tavolixizumab. The 0X40
agonist antibody may be authorized by a drug regulatory authority such as the
U.S. FDA and/or the
European Union's EMA. In some embodiments, the biosimilar is provided as a
composition which
further comprises one or more excipients, wherein the one or more excipients
are the same or different
to the excipients comprised in a reference medicinal product or reference
biological product, wherein
the reference medicinal product or reference biological product is
tavolixizumab. In some
embodiments, the biosimilar is provided as a composition which further
comprises one or more
excipients, wherein the one or more excipients are the same or different to
the excipients comprised in
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a reference medicinal product or reference biological product, wherein the
reference medicinal
product or reference biological product is tavolixizumab.
TABLE 12: Amino acid sequences for 0X40 agonist antibodies related to
tavolixizumab.
Identifier Sequence (One-Letter Amino Acid
Symbols)
SEQ ID NO:87 QVQLQESGPG LVHPSQTLSL TCAVYGGSFS SGYWNWIRKH
PGHGLEYIGY ISYNGITYHN 60
heavy chain for
tavolixizumab PSLKSRITIN RDTSENQYSL QLNSVTPEDT AVYYCARYKY
DYDGGHAMDY WGQGTLVTVS 120
SASTKGPSVE. PLAPSSESYS GGTAALGCLV KDYEPEPVTV SWNSGALTSG VliTAVLQS
180
SGLYSLSSVV TVPSSSLGTQ TYICNVNEXP SNTKVDKRVE PKSCDKTHTC PPCPAPELLG
240
GPSVFLFPFK PKDTLMISRT PEVTCVVVDV SHEDREVKFN WYVDGVEVHN AKTKPREEQY
300
NSTYRVVSVL TVLHQDWLNG KEYKCKVSNK ALPARIEKTI SKAKGQPREP QVYTLEPSRE
360
EMTKNQVSLT CLVHGFYPSD LAVEWESNGQ PENNYKTTPP VIDSDGSFEL YSKLTVDKSR
420
WQQGNVFSCS VMHEALHNHY TQHSLSLSPG K
451
SEQ ID NO:88 DIQMTQSPSS LSASVGDRVT ITCRASQDIS NYLNWYQQKP
GHAPHLLIYY TSKLHSGVPS 60
light chain for
tavolixizumab RFSGSGSGTD YTLTISSLQP EDFATYYCQQ GSALPWTFGQ
GTKVEIKRTV AAPSVFIFPP 120
SDEQLHSGTA SVVCLLNNFY PREAKVQWKV DNALQSGNSQ ESVTEQDSKD STYSLSSTLT
180
LSKADYEKHK VYACEVTHQG LSSPVTKSYN RGEC
214
SEQ ID NO:89 QVQLQESGPG LVKPSQTLSL TCAVYGGSFS SGYWNWIRKH
PGKGLEYIGY ISYNGITYHN 60
heavy chain
variable region PSLKSRITIN RDTSKNQYSL QLNSVTPEDT AVYYCARYKY
DYDGGHAMDY WGQGTLVT 118
for
tavolixizumah
SEQ ID NO: 00 D1QM1QSPSS LSASVGDRVT ITCHASQDIS NYLNWYQQIKP
G:tenLYKLL_LYY TSKLHSGVPS 60
light chain
variable region RFSGSGSGTD YTLTISSLQP EDFATYYCQQ GSALPWTFGQ GTKVEIKR
108
for
tavolixizumab
SEQ ID NO:91 GSESSGYWN
9
heavy chain CDR1
for
tavolixizumab
SEQ ID NO:92 YIGYISYNGI TYH
13
heavy chain CDR2
for
tavolixizumab
SEQ ID NO:93 RYKYDYDGGH AMDY
14
heavy chain CDR3
for
Lavolixizumab
SEQ ID NO:94 QDISNYLN
8
light chain CDRi
for
tavolixizumab
SEQ ID NO:95 LLIYYTSKLH S
11
light chain CDR2
for
tavolixizumab
SEQ ID NO:96 QQGSALPW
8
light chain CDR3
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for
tavolixizumab
[00805] In some embodiments, the 0X40 agonist is 11D4, which is a fully human
antibody
available from Pfizer, Inc. The preparation and properties of 11D4 are
described in U.S. Patent Nos.
7,960,515; 8,236,930; and 9,028,824, the disclosures of which are incorporated
by reference herein.
The amino acid sequences of 11D4 are set forth in Table 13.
[00806] In some embodiments, a 0X40 agonist comprises a heavy chain given by
SEQ ID NO:97
and a light chain given by SEQ ID NO:98. In some embodiments, a 0X40 agonist
comprises heavy
and light chains having the sequences shown in SEQ ID NO:97 and SEQ ID NO:98,
respectively, or
antigen binding fragments, Fab fragments, single-chain variable fragments
(scFv), variants, or
conjugates thereof. In some embodiments, a 0X40 agonist comprises heavy and
light chains that are
each at least 99% identical to the sequences shown in SEQ ID NO:97 and SEQ ID
NO:98,
respectively. In some embodiments, a 0X40 agonist comprises heavy and light
chains that arc each at
least 98% identical to the sequences shown in SEQ ID NO:97 and SEQ ID NO:98,
respectively. In
some embodiments, a 0X40 agonist comprises heavy and light chains that are
each at least 97%
identical to the sequences shown in SEQ ID NO:97 and SEQ ID NO:98,
respectively. In some
embodiments, a 0X40 agonist comprises heavy and light chains that are each at
least 96% identical to
the sequences shown in SEQ ID NO:97 and SEQ ID NO:98, respectively. In some
embodiments, a
0X40 agonist comprises heavy and light chains that are each at least 95%
identical to the sequences
shown in SEQ ID NO:97 and SEQ ID NO:98, respectively.
[00807] In some embodiments, the 0X40 agonist comprises the heavy and light
chain CDRs or
variable regions (VRs) of 11D4. In some embodiments, the 0X40 agonist heavy
chain variable region
(VII) comprises the sequence shown in SEQ ID NO:99, and the 0X40 agonist light
chain variable
region (VL) comprises the sequence shown in SEQ ID NO:100, and conservative
amino acid
substitutions thereof In some embodiments, a 0X40 agonist comprises VH and VL
regions that are
each at least 99% identical to the sequences shown in SEQ ID NO:99 and SEQ ID
NO:100,
respectively. In some embodiments, a 0X40 agonist comprises VH and VL regions
that are each at
least 98% identical to the sequences shown in SEQ ID NO:99 and SEQ ID NO:100,
respectively. In
some embodiments, a 0X40 agonist comprises VII and VL regions that are each at
least 97% identical
to the sequences shown in SEQ ID NO:99 and SEQ ID NO:100, respectively. In
some embodiments,
a 0X40 agonist comprises VH and VL regions that are each at least 96%
identical to the sequences
shown in SEQ ID NO:99 and SEQ ID NO: 100, respectively. In some embodiments, a
0X40 agonist
comprises VH and VL regions that are each at least 95% identical to the
sequences shown in SEQ ID
NO:99 and SEQ ID NO: 100, respectively.
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[00808] In some embodiments, a 0X40 agonist comprises heavy chain CDR1, CDR2
and CDR3
domains having the sequences set forth in SEQ ID NO:101, SEQ ID NO:102, and
SEQ ID NO:103,
respectively, and conservative amino acid substitutions thereof, and light
chain CDR1, CDR2 and
CDR3 domains having the sequences set forth in SEQ ID NO:104, SEQ ID NO:105,
and SEQ ID
NO:106, respectively, and conservative amino acid substitutions thereof
[00809] In some embodiments, the 0X40 agonist is a 0X40 agonist biosimilar
monoclonal antibody
approved by drug regulatory authorities with reference to 11D4. In some
embodiments, the biosimilar
monoclonal antibody comprises an 0X40 antibody comprising an amino acid
sequence which has at
least 97% sequence identity, e.g., 97%, 98%, 99% or 100% sequence identity, to
the amino acid
sequence of a reference medicinal product or reference biological product and
which comprises one or
more post-translational modifications as compared to the reference medicinal
product or reference
biological product, wherein the reference medicinal product or reference
biological product is 11D4.
In some embodiments, the one or more post-translational modifications are
selected from one or more
of glycosylation, oxidation, deamidation, and truncation. In some embodiments,
the biosimilar is a
0X40 agonist antibody authorized or submitted for authorization, wherein the
0X40 agonist antibody
is provided in a formulation which differs from the formulations of a
reference medicinal product or
reference biological product, wherein the reference medicinal product or
reference biological product
is 11D4. The 0X40 agonist antibody may be authorized by a drug regulatory
authority such as the
U.S. FDA and/or the European Union's EMA. In some embodiments, the biosimilar
is provided as a
composition which further comprises one or more excipients, wherein the one or
more excipients are
the same or different to the excipients comprised in a reference medicinal
product or reference
biological product, wherein the reference medicinal product or reference
biological product is 11D4.
In some embodiments, the biosimilar is provided as a composition which further
comprises one or
more excipients, wherein the one or more excipients are the same or different
to the excipients
comprised in a reference medicinal product or reference biological product,
wherein the reference
medicinal product or reference biological product is 11D4.
TABLE 13: Amino acid sequences for 0X40 agonist antibodies related to 11D4.
Identifier Sequence (One-Letter Amino Acid
Symbols)
SEQ ID ND:97 EVQLVESGGG LVQPGGSLRL SCAASGFTFS SYSMNWVRQA
PGKGLEWVSY ISSSSSTIDY 60
heavy chain for ADSVKGRFTI SRDNAKNSLY LQMNSLRDED TAVYYCARES
GWYLFDYWGQ GTLVTVSSAS 120
:104
TKGPSVFPLA PCSRSTSEST AALGCLVEDY FPEPVTVSWN SGALTSGVHT FPAYLQSSGL 180
YSLSSVVTVP SSNEGTQTYT CNVDHKPSNT KVDKTVERKC CVECPPCPAP PVAGPSVFLF
240
PPKPKDTLMI SRTPEVTCVV VDVSHEDPEV QFNWYVDGVE VHNAKTKPRE EQFNSTFRVV
300
SVLTVVHQDW LNGXEYKCKV SNKGLPAPIE KTISKTKGQP REPQVY=PP SREEMTKNQV
360
SLTCLVKGFY PSDIAV-EWES NGQPENNYHT TPPMLDSDGS FFLYSKLTVD KSRWQQGNVF
420
SCSVMHEALH NHYTQKSLSL SPGH
444
SEQ ID NO:98 DIQMTQSFSS LSASVGDRVT I2CRASQGS5 SWLAWYQQKP
EKAPKSLIYA ASSLQSGVPS 60
light chain for R.SGSGSGTJJ 2.C.L15.51,Q2 EDFATYYCQc YNSY22TFGG
G:KVEIKKTV A.A.25V22 120
:104
SDEQLKSGTA SVVCLLNNFY PREAKVQWKV DNALQSGNSQ ESVTEQDSKD STYSLSSTL? 150
LSKAllYEKHK VYACEVTHQG LSSPVTKSPV RGEC
214
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SEQ ID NO:99 EVQLVESGGG LVQPGGSLRL SCAASGFTES SYSMNWVRQA
PGKGLEWVSY ISSSSSTIDY 60
heavy chain ADSVXGRFTI SRDNAHNSLY LQMNSLRDED TAVYYaARES
GWYLFDYWGQ GTLVTVSS 118
variable region
for 11D4
SEQ ID N0:100 DIQMTQSPSS LSASVGDRVT ITCRASQGIS SWLAWYQQHP
EHAPHSLIYA ASSIQSGVPS 60
light chain KTSGSGSGTD _"T_L11SSLQP Eat'ATYYCQQ YNSYPPTGG GTKVE1K
107
variable region
for 11D4
SEQ ID NO:101 SYSMN
5
heavy chain CDR1
for 11D4
SEQ ID NO:102 YISSSSSTID YADSVIKG
17
heavy chain CDR2
for 11D4
SEQ ID NO:103 ESGWYLFDY
9
heavy chain CU-3
for 11D4
SEQ ID NO:104 RASQGISSWL A
11
light chain CDR1
for 11D4
SEQ it 50:105 AASSLQS
7
light chain CD02
for 11D4
SEQ ID NO:106 QQYNSYPPT
9
light chain C2,23
for 1104
[00810] In some embodiments, the 0X40 agonist is 18D8, which is a fully human
antibody
available from Pfizer, Inc. The preparation and properties of 18D8 are
described in U.S. Patent Nos.
7,960,515; 8,236,930; and 9,028,824, the disclosures of which are incorporated
by reference herein.
The amino acid sequences of 18D8 are set forth in Table 14.
[00811] In some embodiments, a 0X40 agonist comprises a heavy chain given by
SEQ ID NO:107
and a light chain given by SEQ ID NO: 108. In some embodiments, a 0X40 agonist
comprises heavy
and light chains having the sequences shown in SEQ ID NO:107 and SEQ ID
NO:108, respectively,
or antigen binding fragments, Fab fragments, single-chain variable fragments
(scFv), variants, or
conjugates thereof. In some embodiments, a OX40 agonist comprises heavy and
light chains that are
each at least 99% identical to the sequences shown in SEQ ID NO: 107 and SEQ
TD NO:108,
respectively. In some embodiments, a 0X40 agonist comprises heavy and light
chains that are each at
least 98% identical to the sequences shown in SEQ ID NO: 107 and SEQ ID
NO:108, respectively. In
some embodiments, a 0X40 agonist comprises heavy and light chains that are
each at least 97%
identical to the sequences shown in SEQ ID NO:107 and SEQ ID NO: 108,
respectively. In some
embodiments, a OX40 agonist comprises heavy and light chains that are each at
least 96% identical to
the sequences shown in SEQ ID NO: 107 and SEQ ID NO:108, respectively. In some
embodiments, a
0X40 agonist comprises heavy and light chains that are each at least 95%
identical to the sequences
shown in SEQ ID NO: 107 and SEQ ID NO:108, respectively.
[00812] In some embodiments, the 0X40 agonist comprises the heavy and light
chain CDRs or
variable regions (VRs) of 18D8. In some embodiments, the 0X40 agonist heavy
chain variable region
(VII) comprises the sequence shown in SEQ ID NO: 109, and the OX40 agonist
light chain variable
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region (VL) comprises the sequence shown in SEQ ID NO:110, and conservative
amino acid
substitutions thereof. In some embodiments, a 0X40 agonist comprises VH and VL
regions that are
each at least 99% identical to the sequences shown in SEQ ID NO: 109 and SEQ
TD NO:110,
respectively. In some embodiments, a 0X40 agonist comprises VH and VL regions
that are each at
least 98% identical to the sequences shown in SEQ ID NO: 109 and SEQ ID
NO:110, respectively. In
some embodiments, a 0X40 agonist comprises VH and VI, regions that are each at
least 97% identical
to the sequences shown in SEQ ID NO:109 and SEQ ID NO: 110, respectively. In
some embodiments,
a 0X40 agonist comprises VH and VL regions that are each at least 96%
identical to the sequences
shown in SEQ ID NO: 109 and SEQ ID NO:110, respectively. In some embodiments,
a OX40 agonist
comprises VH and VL regions that are each at least 95% identical to the
sequences shown in SEQ ID
NO:109 and SEQ ID NO:110, respectively.
1008131 In some embodiments, a 0X40 agonist comprises heavy chain CDR1, CDR2
and CDR3
domains having the sequences set forth in SEQ ID NO:111, SEQ ID NO:112, and
SEQ ID NO:113,
respectively, and conservative amino acid substitutions thereof, and light
chain CDR1, CDR2 and
CDR3 domains having the sequences set forth in SEQ ID NO:114, SEQ ID NO: 115,
and SEQ ID
NO:116, respectively, and conservative amino acid substitutions thereof.
1008141 In some embodiments, the 0X40 agonist is a OX40 agonist biosimilar
monoclonal antibody
approved by drug regulatory authorities with reference to 18D8. In sonic
embodiments, the biosimilar
monoclonal antibody comprises an 0X40 antibody comprising an amino acid
sequence which has at
least 97% sequence identity, e.g., 97%, 98%, 99% or 100% sequence identity, to
the amino acid
sequence of a reference medicinal product or reference biological product and
which comprises one or
more post-translational modifications as compared to the reference medicinal
product or reference
biological product, wherein the reference medicinal product or reference
biological product is 18D8.
In some embodiments, the one or more post-translational modifications are
selected from one or more
of: glyeosylation, oxidation, deamidation, and truncation. In some
embodiments, the biosimilar is a
0X40 agonist antibody authorized or submitted for authorization, wherein the
OX40 agonist antibody
is provided in a formulation which differs from the formulations of a
reference medicinal product or
reference biological product, wherein the reference medicinal product or
reference biological product
is 18D8. The OX40 agonist antibody may be authorized by a drug regulatory
authority such as the
U.S. FDA and/or the European Union's EMA. in some embodiments, the biosimilar
is provided as a
composition which further comprises one or more excipients, wherein the one or
more excipients are
the same or different to the excipients comprised in a reference medicinal
product or reference
biological product, wherein the reference medicinal product or reference
biological product is 18D8.
In some embodiments, the biosimilar is provided as a composition which further
comprises one or
more excipients, wherein the one or more excipients are the same or different
to the excipients
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comprised in a reference medicinal product or reference biological product,
wherein the reference
medicinal product or reference biological product is 18D8.
TABLE 14: Amino acid sequences for 0X40 agonist antibodies related to 18D8.
Identifier Sequence (One-Letter Amino Acid
Symbols)
SEQ ID NO:107 EVQLVESGGG LVQPGRSLRL SCAASGFTFD DYAMHWVRQA
PGIKGLEWVSG ISWNSGSIGY 60
heavy chain for
18D8 ADSVKGRFTI SRDNAKNSLY LQMNSLRAED TALYYCAKEQ
STADYYFYYG MDVWGQGTTV 120
1VSSAS1KG2 SVLA2CSR STSESTAALG CLVKOYEP VTVSWNSGAL TSGVHTFPAV
180
LQSSGLYSLS SVVTVPSSNF GTQTYTCNVD HKPSNTKVDK TVERKCCVEC PPCPAPPVAG
240
PSVELFPPHP KDTLMISRTP EVTCVVVDVS NEDPEVQFNW YVDGVEVIINA KTKPREEQFN
300
STERVVSVLT VVHQDWLNGH EYKCKVSNKG LPAPIEKTIS KTKGQPREPQ VYTLPESREE
360
MTKNQVSLTC LVKGFYPSDI AVEWESNGQP ENNYKTTPPM LDSDGSFFLY SKLTVDIKSRW
420
QQGNVFSCSV MHEALHNHYT QKSLSLSPGK
450
SEQ ID NO:108 EIVVTQSPAT LSLSPGERAT LSCRASQSVS SYLAWYQQKP
GQAPRLLIYD ASNRATGIPA 60
light chain for
18:0 RFSGSGSGTD FTLTISSLEP EDFAVYYCQQ RSNWPTFGQG
TKVEIKRTVA A2SVFIFPPS 120
DEQLKSGTAS VVCLLNNFYP REAKVQWKVD NALQSGNSQE SVTEQDSKDS TYSLSSTLTL
180
SHADYEKHKV YACEVTHQGL SSPVTKSFNR GEC
213
SEQ ID NO:109 EVQLVESGGG LVQPGRSLRL SCAASGFTED DYAMHWVRQA
PGKGLEWVSG ISWNSGSIGY 60
heavy chain
variable region ADSVKGRFTI SRDNAHNSLY LQMNSLRAED TALYYCAKDQ
STADYYFYYG MDVWGQGTTV 120
for 18D8
TVSS
124
SEQ ID NO:110 EIVVTQSPAT LSLSPGERAT LSCRASQSVS SYLAWYQQKP
GQAPRLLIYD ASMRATGIPA 60
light chain
variable region RFSGSGSGTD FTLTISSLEP EDFAVYYCQQ RSNWPTFGQG TKVEIK
106
for 18D8
SEQ ID NO:111 DYAMH
5
heavy chain Cprd
for 18D8
SEQ ID NO:112 GISWNSGSIG YADSVKG
17
heavy chain CDR2
for 18D8
SEQ ID NO:113 DQSTADYYFY YGMDV
15
heavy chain CDR3
for 18DO
SEQ ID NO:114 RASQSVSSYL A
11
light chain CDR1
for 18D8
SEQ ID NO:115 DASNRAT
7
light chain CDR2
for 18D8
SEQ ID NO:116 QQRSNWPT
8
light_ chain CDRS
for 18178
[00815] hi some embodiments, the 0X40 agonist is Hu119-122, which is a
humanized antibody
available from GlaxoSmithKline plc. The preparation and properties of Hu119-
122 are described in
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U.S. Patent Nos. 9,006,399 and 9,163,085, and in International Patent
Publication No. WO
2012/027328, the disclosures of which are incorporated by reference herein.
The amino acid
sequences of Hu119-122 are set forth in Table 15.
[00816] In some embodiments, the 0X40 agonist comprises the heavy and light
chain CDRs or
variable regions (VRs) of Hu119-122. In some embodiments, the 0X40 agonist
heavy chain variable
region (VII) comprises the sequence shown in SEQ ID NO:117, and the 0X40
agonist light chain
variable region (VL) comprises the sequence shown in SEQ ID NO:118, and
conservative amino acid
substitutions thereof. In some embodiments, a 0X40 agonist comprises VH and VL
regions that are
each at least 99% identical to the sequences shown in SEQ ID NO: 117 and SEQ
ID NO:118.
respectively. In some embodiments, a 0X40 agonist comprises VII and VL regions
that are each at
least 98% identical to the sequences shown in SEQ ID NO: 117 and SEQ ID
NO:118, respectively. In
some embodiments, a 0X40 agonist comprises VH and VL regions that are each at
least 97% identical
to the sequences shown in SEQ TD NO:117 and SEQ ID NO:118, respectively. In
some embodiments,
a 0X40 agonist comprises VH and VL regions that are each at least 96%
identical to the sequences
shown in SEQ ID NO: 117 and SEQ ID NO:118, respectively. In some embodiments,
a 0X40 agonist
comprises VH and VL regions that are each at least 95% identical to the
sequences shown in SEQ ID
NO:117 and SEQ ID NO:118, respectively.
[00817] In some embodiments, a 0X40 agonist comprises heavy chain CDR1, CDR2
and CDR3
domains having the sequences set forth in SEQ ID NO:119, SEQ ID NO: 120, and
SEQ ID NO:121,
respectively, and conservative amino acid substitutions thereof, and light
chain CDR1, CDR2 and
CDR3 domains having the sequences set forth in SEQ ID NO:122, SEQ ID NO:123,
and SEQ ID
NO:124, respectively, and conservative amino acid substitutions thereof.
[00818] In some embodiments, the 0X40 agonist is a OX40 agonist biosimilar
monoclonal antibody
approved by drug regulatory authorities with reference to Hu119-122. In some
embodiments, the
biosimilar monoclonal antibody comprises an 0X40 antibody comprising an amino
acid sequence
which has at least 97% sequence identity, e.g., 97%, 98%, 99% or 100% sequence
identity, to the
amino acid sequence of a reference medicinal product or reference biological
product and which
comprises one or more post-translational modifications as compared to the
reference medicinal
product or reference biological product, wherein the reference medicinal
product or reference
biological product is Hu119-122. In some embodiments, the one or more post-
translational
modifications are selected from one or more of: glycosylation, oxidation,
dcamidation, and truncation.
In some embodiments, the biosimilar is a 0X40 agonist antibody authorized or
submitted for
authorization, wherein the 0X40 agonist antibody is provided in a formulation
which differs from the
formulations of a reference medicinal product or reference biological product,
wherein the reference
medicinal product or reference biological product is Hu119-122. The 0X40
agonist antibody may be
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authorized by a drug regulatory authority such as the U.S. FDA and/or the
European Union's EMA. In
some embodiments, the biosimilar is provided as a composition which further
comprises one or more
excipients, wherein the one or more excipients are the same or different to
the excipients comprised in
a reference medicinal product or reference biological product, wherein the
reference medicinal
product or reference biological product is Hu119-122. In some embodiments, the
biosimilar is
provided as a composition which further comprises one or more excipients,
wherein the one or more
excipients are the same or different to the excipients comprised in a
reference medicinal product or
reference biological product, wherein the reference medicinal product or
reference biological product
is Hu119-122.
TABLE 15: Amino acid sequences for 0X40 agonist antibodies related to Hul 19-
122.
Identifier Sequence (One-Letter Amino Acid
Symbols)
SEQ Ill 60:117 EVQLVESGGG LVQ200SLRL SCAASEYE.HP SH2MSWVRQA
2GKGLELVAA INS2GGSTYY .. 60
heavy chain PDTMERRFTI SRDNAKNSLY LQMNSLRAED TAVYYCARHY
DDYYAWFAYW GQGTMVTVSS 120
variable region
for Hu119-122
SEQ ill 60:118 E1VLTQSPAK LSESFGERAT LSCRASKSVS TSGYSIMHWY
GOKYGOAFRL I1YLASNLES 60
light chain GVPARFSGSG SGTDFTLTIS SLEPEDFAVY YCQHSRELPL
TFGGGTKVEI K 111
variable region
for Hu119-122
SEQ ID NO:119 SHDMS
5
heavy chain CDR1
for Hu119-122
SEQ ID NO:120 AINSDGGSTY YPDTMER
17
heavy chain CDR2
for Hul19 122
SEQ 10 NO: 121 HYDOYYAWFA Y
11
heavy chain C003
for Hu119-122
SEQ ID NO:122 RASKSVSTSG YSYMH
15
light chain CDR1
for Hu119-122
SEQ ID NO:123 LASNLES
7
light chain CDR2
for Hu119-122
SEQ ID 60:124 QHSRELPIT
9
light chain ClFrL3
for Hu119-122
1008191 In some embodiments, the 0X40 agonist is Hu106-222, which is a
humanized antibody
available from GlaxoSmithKline plc. The preparation and properties of Hu106-
222 are described in
U.S. Patent Nos. 9,006,399 and 9,163,085, and in International Patent
Publication No. WO
2012/027328, the disclosures of which are incorporated by reference herein.
The amino acid
sequences of Hu106-222 are set forth in Table 16.
1008201 In some embodiments, the 0X40 agonist comprises the heavy and light
chain CDRs or
variable regions (VRs) of Hu106-222. In some embodiments, the 0X40 agonist
heavy chain variable
region (VH) comprises the sequence shown in SEQ ID NO:125, and the 0X40
agonist light chain
variable region (VL) comprises the sequence shown in SEQ ID NO:126, and
conservative amino acid
substitutions thereof In some embodiments, a 0X40 agonist comprises Vu and VI,
regions that are
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each at least 99% identical to the sequences shown in SEQ ID NO: 125 and SEQ
ID NO:126,
respectively. In some embodiments, a 0X40 agonist comprises VH and VL regions
that are each at
least 98% identical to the sequences shown in SEQ ID NO: 125 and SEQ ID
NO:126, respectively. In
some embodiments, a 0X40 agonist comprises VH and VL regions that are each at
least 97% identical
to the sequences shown in SEQ ID NO:125 and SEQ ID NO: 126, respectively. In
some embodiments,
a 0X40 agonist comprises VH and VL regions that are each at least 96%
identical to the sequences
shown in SEQ ID NO: 125 and SEQ ID NO:126, respectively. In some embodiments,
a 0X40 agonist
comprises VH and VL regions that are each at least 95% identical to the
sequences shown in SEQ ID
NO:125 and SEQ ID NO:126, respectively.
[00821] In some embodiments, a 0X40 agonist comprises heavy chain CDR1, CDR2
and CDR3
domains having the sequences set forth in SEQ ID NO:127, SEQ ID NO:128, and
SEQ ID NO:129,
respectively, and conservative amino acid substitutions thereof, and light
chain CDR1, CDR2 and
CDR3 domains having the sequences set forth in SEQ TD NO:130, SEQ ID NO:131,
and SEQ ID
NO:132, respectively, and conservative amino acid substitutions thereof.
[00822] In some embodiments, the OX40 agonist is a 0X40 agonist biosimilar
monoclonal antibody
approved by drug regulatory authorities with reference to Hu106-222. In some
embodiments, the
biosimilar monoclonal antibody comprises an 0X40 antibody comprising an amino
acid sequence
which has at least 97% sequence identity, e.g., 97%, 98%, 99% or 100% sequence
identity, to the
amino acid sequence of a reference medicinal product or reference biological
product and which
comprises one or more post-translational modifications as compared to the
reference medicinal
product or reference biological product, wherein the reference medicinal
product or reference
biological product is Hu106-222. In some embodiments, the one or more post-
translational
modifications are selected from one or more of: glycosylation, oxidation,
deamidation, and truncation.
In some embodiments, the biosimilar is a 0X40 agonist antibody authorized or
submitted for
authorization, wherein the 0X40 agonist antibody is provided in a formulation
which differs from the
formulations of a reference medicinal product or reference biological product,
wherein the reference
medicinal product or reference biological product is Hu106-222. The OX40
agonist antibody may be
authorized by a drug regulatory authority such as the U.S. FDA and/or the
European Union's EMA. In
some embodiments, the biosimilar is provided as a composition which further
comprises one or more
excipients, wherein the one or more excipients are the same or different to
the excipients comprised in
a reference medicinal product or reference biological product, wherein the
reference medicinal
product or reference biological product is Hu106-222. In some embodiments, the
biosimilar is
provided as a composition which further comprises one or more excipients,
wherein the one or more
excipients are the same or different to the excipients comprised in a
reference medicinal product or
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reference biological product, wherein the reference medicinal product or
reference biological product
is Hu106-222.
TABLE 16: Amino acid sequences for 0X40 agonist antibodies related to Hu106-
222.
Identifier Sequence (One-Letter Amino Acid
Symbols)
SEQ ID NO:125 QVQLVQSGSE LKKPGASVKV SCKASGYTFT DYSMHWVRQA
PGQGLKWMSW INTETGEPTY 60
heavy chain
variable recp.on ADDFKGRFVE SLDTSVSTAY LQISSLKAED TAVYYCANPY
YDYVSYYAMD YWGQGTTVTV 120
for Hu106 222
SS
122
SEQ ID NO:126 DIQMTQSPSS LSASVGDRVT ITCKASQDVS TAVAWYQQKP
GKAPHLLIYS ASYLYTGVPS 60
light chain
variable region RFSGSGSGTD FTFTISSLQP EDIATYYCQQ HYSTPRTFGQ GTKLEIK
107
for Hu106 222
SEQ ID NO:127 DYSMH
5
heavy chain CDR1
for Hu106-222
SEQ Ill NO: 128 WINTETGEP1 YADJ_HKG
17
heavy chain CDR2
for Hu106-222
SEQ ID NO:129 PYYDYVSYYA MDY
13
heavy chain CDR3
for Hu106-222
SEQ ID NO:130 KASQDVSTAV A
11
light chain CDR1
for Hu106-222
SEQ ID NO:131 SASYLYT
7
light chain CDR2
for Hu106-222
SEQ ID NO:132 QQHYSTPRT
9
light chain CDR3
for Hu106-222
[00823] In some embodiments, the 0X40 agonist antibody is MEDI6469 (also
referred to as 9B12).
MEDI6469 is a murine monoclonal antibody. Weinberg, et al., I Immunother.
2006, 29, 575-585. In
some embodiments the 0X40 agonist is an antibody produced by the 9B12
hybridoma, deposited with
Biovest Inc. (Malvern, MA, USA), as described in Weinberg, et al.,.I.
Immunother. 2006, 29, 575-
585, the disclosure of which is hereby incorporated by reference in its
entirety. In some embodiments,
the antibody comprises the CDR sequences of MEDI6469. In some embodiments, the
antibody
comprises a heavy chain variable region sequence and/or a light chain variable
region sequence of
MEDI6469.
[00824] In some embodiments, the 0X40 agonist is L106 BD (Pharmingen Product
#340420). In
some embodiments, the 0X40 agonist comprises the CDRs of antibody L106 (BD
Pharmingen
Product #340420). In some embodiments, the 0X40 agonist comprises a heavy
chain variable region
sequence and/or a light chain variable region sequence of antibody L106 (BD
Pharmingen Product
#340420). In some embodiments, the 0X40 agonist is ACT35 (Santa Cruz
Biotechnology, Catalog
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#20073). In some embodiments, the 0X40 agonist comprises the CDRs of antibody
ACT35 (Santa
Cruz Biotechnology, Catalog #20073). In some embodiments, the 0X40 agonist
comprises a heavy
chain variable region sequence and/or a light chain variable region sequence
of antibody ACT35
(Santa Cruz Biotechnology, Catalog #20073). In some embodiments, the 0X40
agonist is the murine
monoclonal antibody anti-mCD134/m0X40 (clone 0X86), commercially available
from InVivoMAb,
BioXcell Inc, West Lebanon, NH.
[00825] In some embodiments, the 0X40 agonist is selected from the 0X40
agonists described in
International Patent Application Publication Nos. WO 95/12673, WO 95/21925, WO
2006/121810,
WO 2012/027328, WO 2013/028231, WO 2013/038191, and WO 2014/148895; European
Patent
Application EP 0672141; U.S. Patent Application Publication Nos. US
2010/136030, US
2014/377284, US 2015/190506, and US 2015/132288 (including clones 20E5 and
12H3); and U.S.
Patent Nos. 7,504,101, 7,550,140, 7,622,444, 7,696,175, 7,960,515, 7,961,515,
8,133,983, 9,006,399,
and 9,163,085, the disclosure of each of which is incorporated herein by
reference in its entirety.
[00826] In some embodiments, the 0X40 agonist is an 0X40 agonistic fusion
protein as depicted in
Structure I-A (C-terminal Fe-antibody fragment fusion protein) or Structure I-
B (N-terminal Fe-
antibody fragment fusion protein), or a fragment, derivative, conjugate,
variant, or biosimilar thereof.
The properties of structures I-A and I-B are described above and in U.S.
Patent Nos. 9,359,420,
9,340,599, 8,921,519, and 8,450,460, the disclosures of which are incorporated
by reference herein.
Amino acid sequences for the polypeptide domains of structure I-A given in
Figure 18 are found in
Table 9. The Fe domain preferably comprises a complete constant domain (amino
acids 17-230 of
SEQ ID NO:62) the complete hinge domain (amino acids 1-16 of SEQ ID NO:62) or
a portion of the
hinge domain (e.g., amino acids 4-16 of SEQ ID NO:62). Preferred linkers for
connecting a C-
terrninal Fe-antibody may be selected from the embodiments given in SEQ ID
NO:63 to SEQ ID
NO:72, including linkers suitable for fusion of additional polypeptides.
Likewise, amino acid
sequences for the polypeptide domains of structure I-B given in Figure 18 are
found in Table 9. If an
Fe antibody fragment is fused to the N-terminus of an TNRFSF fusion protein as
in structure I-B, the
sequence of the Fe module is preferably that shown in SEQ ID NO:73, and the
linker sequences arc
preferably selected from those embodiments set forth in SED ID NO:74 to SEQ ID
NO:76.
[00827] In some embodiments, an 0X40 agonist fusion protein according to
structures I-A or I-B
comprises one or more 0X40 binding domains selected from the group consisting
of a variable heavy
chain and variable light chain of tavolixizumab, a variable heavy chain and
variable light chain of
11D4, a variable heavy chain and variable light chain of 18D8, a variable
heavy chain and variable
light chain of Hu119-122, a variable heavy chain and variable light chain of
Hu106-222, a variable
heavy chain and variable light chain selected from the variable heavy chains
and variable light chains
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described in Table 17, any combination of a variable heavy chain and variable
light chain of the
foregoing, and fragments, derivatives, conjugates, variants, and biosimilars
thereof.
[00828] In some embodiments, an 0X40 agonist fusion protein according to
structures I-A or I-B
comprises one or more 0X40 binding domains comprising an OX4OL sequence. In
some
embodiments, an 0X40 agonist fusion protein according to structures I-A or I-B
comprises one or
more 0X40 binding domains comprising a sequence according to SEQ ID NO:133. In
some
embodiments, an 0X40 agonist fusion protein according to structures I-A or I-B
comprises one or
more 0X40 binding domains comprising a soluble 0X401, sequence. In some
embodiments, a 0X40
agonist fusion protein according to structures I-A or I-B comprises one or
more 0X40 binding
domains comprising a sequence according to SEQ ID NO:134. In some embodiments,
a OX40 agonist
fusion protein according to structures I-A or I-B comprises one or more 0X40
binding domains
comprising a sequence according to SEQ ID NO:135.
[00829] In some embodiments, an 0X40 agonist fusion protein according to
structures I-A or I-B
comprises one or more 0X40 binding domains that is a scFv domain comprising VH
and VL regions
that are each at least 95% identical to the sequences shown in SEQ ID NO:89
and SEQ ID NO:90,
respectively, wherein the VH and VL domains are connected by a linker In some
embodiments, an
0X40 agonist fusion protein according to structures I-A or I-B comprises one
or more OX40 binding
domains that is a scFv domain comprising VI; and VL regions that are each at
least 95% identical to
the sequences shown in SEQ ID NO:99 and SEQ ID NO:100, respectively, wherein
the VH and
domains are connected by a linker. In some embodiments, an 0X40 agonist fusion
protein according
to structures 1-A or 1-B comprises one or more OX40 binding domains that is a
scFv domain
comprising VH and VL regions that are each at least 95% identical to the
sequences shown in SEQ ID
NO:109 and SEQ ID NO:110, respectively, wherein the VE and VL domains are
connected by a linker.
In some embodiments, an 0X40 agonist fusion protein according to structures I-
A or I-B comprises
one or more 0X40 binding domains that is a scFv domain comprising VH and VL
regions that are each
at least 95% identical to the sequences shown in SEQ ID NO:127 and SEQ ID
NO:128, respectively,
wherein the VII and VL domains are connected by a linker. In some embodiments,
an 0X40 agonist
fusion protein according to structures I-A or I-B comprises one or more 0X40
binding domains that is
a say domain comprising VH and VL regions that are each at least 95% identical
to the sequences
shown in SEQ ID NO: 125 and SEQ ID NO:126, respectively, wherein the Vi and VL
domains are
connected by a linker. In some embodiments, an OX40 agonist fusion protein
according to structures
I-A or I-B comprises one or more OX40 binding domains that is a scFv domain
comprising Vu and
VL regions that arc each at least 95% identical to the VH and VL sequences
given in Table 17, wherein
the VH and VL domains are connected by a linker.
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TABLE 17: Additional polypeptide domains useful as 0X40 binding domains in
fusion proteins (e.g.,
structures I-A and I-B) or as scFy 0X40 agonist antibodies.
Identifier Sequence (One-Letter Amino Acid
Symbols)
SEQ ID NO:133 MERVQPLEEN VGNAARPRFE RNKLLLVASV IQGLGLLLCF
TYICLHFSAL QVSHRYPRIQ 60
0X40L SIKVQFTEYK KEKGEILTSO KEDEIMKVQN NSVIINCDGF
YLISLKGYFS QEVNISLHYQ 120
KDEEPLFQLK KVRSVNSLMV ASLTYKDKVY LNVTTDNTSL DDFHVNGGEL ILIHQNPGEF
180
CVL
183
SEQ ID NO:134 SHRYFRIQSI KVQFTEYKKE KGFILTSQKE DEIMKVQNNS
VIINCDGFYL ISLKGYFSQE 60
OX4OL soluble VNISLHYQKD EEPLEQLKKV RSVNSLMVAS LTYKDKVYLN
VTTDNTSLDD FHVNGGELIL 120
domain IHQNPGEFCV L
131
SEQ ID NO:135 YPRIQSIKVQ FTEYKKEKGF ILTSQKEDEI MKVQNNSVII
NCDGFYLISL KGYFSQEVNI 60
OX4OL soluble SLHYQKDEEP LFQLEKVRSV NSLMVASLTY KDKVYLNVTT
DNTSLDDFHV NGGELILIHQ 120
domain NPGEFCVL
128
;alternative)
SEQ ID NO:136 EVQLVESGGG LVQPGGSLRL SCAASGFTES NYTMNWVRQA
PGKGLEWVSA ISGSGGSTYY 60
variable heavy ADSVKGRFTI SRDNSKNTLY LQMNSLRAED TAVYYCAKER
YSQVHYALDK WGQGTLVTVS 120
chain for 008
SEQ ID 50:137 2IVMTQS2DS DPVT2GEPAS ISCKSSQLDL HSNGYNYLEW
YLQKAGQ52Q LLIYLGSNKA 60
variable light SGVPDRFSGS GSGTDFTLKI SRVEAEDVGV YYCQQYYNHP TTFGQGTK
108
chain for 008
SEQ ID 50:138 EVQLVESGGG VVQPGRSLRL SCAASGFTES DYTMNWVRQA
PGKGLEWVSS ISGGSTYYAD 60
variable heavy SRKGRFTISR DNSHNTLYLQ MNNLRAEDTA VYYCARDRYF
RQQNAFDYWG QGTLVTVSSA 120
chain for 011
SEQ ID 50:139 DIVMTQSPDS LPVTPGEPAS ISCRSSQSLL HSNGYNYLEW
YLQKAGQSPQ LLIYLGSNRA 60
variable light SGVPDRFSGS GSGTDFTLKI SRVEAEDVGV YYCQQYYNHP TTFGQGTH
108
chain for 011
SEQ Ill 50:140 EVQLVESGGG DVQPRGSDRL 0CAASGJ2'2FS SYAMNWVKQA
2GKGLEWVAV ISY2GSNKYY 60
variable heavy ADSVKGRFTI SRDNSKNTLY LQMNSLRAED TAVYYCAKER
YITLPNALDY WGQGTLVTVS 120
chain for 021
SEQ ID NO:141 DIQMTQSPVS LPVTPGEPAS ISCRSSQSLL HSNGYNYLEW
YLQKPGQSPQ LLIYLGSNRA 60
variable light SGVPDRFSGS GSGTDFTLKI SRVEAEDVGV YYCQQYKSNP PTEGQGTK
108
chain for 021
SEQ ID NO:142 EVQLVESGGG LVHPGGSLRL SCAGSGFTFS SYAMHWVRQA
PGKGLEWVSA IGTGGGTYYA 60
variable heavy DSVMGRFTIS RDNSKNTLYL QMNSLRAEDT AVYYCARYEN
VMGLYWFDYW GQGTLVTVSS 120
chain for 023
SEQ ID NO:143 EIVLTQSPAT LSLSPGERAT LSCRASQSVS SYLAWYQKP GQAPRLLIYD
ASNRATGIPA 60
variable light RFSGSGSGTD FTLTISSLEP EDFAVYYCQQ RSNWPPAFGG GTHVEIKR
108
chain for 023
SEQ ID NO:144 EVQLQQSGPE LVKPGASVKM SCKASGYTFT SYVMHWVKQK
PGQGLEWIGY INPYNDGTKY 60
heavy chain NEKFKGKATL TSDHSSSTAY MELSSLTSED SAVYYCANYY
GSSLSMDYWG QGTSVTVSS 119
variable region
SEQ TO 50:145 DIQMTQTTSS LSASIGURVT ISCRASQUIS NYLNWIQDKP
DGTVKILTYY TSKIHSGVPS 60
light chain RFSGSGSGTD YSLTISNLEQ EDIATYFCQQ GNTLPWTFGG GTKLEIKR
108
variable region
SEQ ID NO:146 EVQLQQSGPE LVKPGASVKI SCKTSGYTFK DYTMHWVKQS
HGKSLEWIGG IYPNNGGSTY 60
heavy chain NQNFKDKATL TVDXSSSTAY MEFRSLTSED SAVYYCARMG
YHGPHLDFDV WGAGTTVTVS 120
variable region p
121
SEQ ID NO:147 DIVMTQSHKE MSTSLGDRVS ITCHASQDVG AAVAWYQQHF
GQSPHLLIYW ASTRHTGVPD 60
lighL chain RFTGGGSGTD FTLTISNVQS EDLTDYFCQQ YINYPLTFGG GTKLEIKR
108
variable region
SEQ ID 50:148 QIQLVQSGFE LKHFGETVKI SCKASGYTFT DYSMHWVKQA
FGHGLKWMGW INTETGEPTY 60
heavy chain ADDFKGRFAF SLETSASTAY LQINNLKNED TATYFCANFY
YDYVSYYAMD YWGHGTSVTV 120
variable region SS
122
of humanized
antibody
SEQ ID NO:149 QVQLVQSGSE LKKPGASVKV SCKASGYTFT DYSMHWVRQA
PGQGLKWMGW INTETGEPTY 60
heavy chain AD0FKGRFVF SLDTSVSTAY LQ1SSLKAED TAVYYCANPY
YJYVSYYAMD YWGQGTTVTV 120
variable region SS
122
of humanized
antibody
SEQ ID NO:150 DIVMTQSHKE MSTSVRDRVS ITCHASQDVS TAVAWYQQKP
GQSPHLLIYS ASYLYTGVPD 60
light chain RFTGSGSGTD FTFTISSVQA EDLAVYYCQQ HYSTPRTFGG GTKLEIK
107
variable region
of humanized
antibody
SEQ ID NO:151 DIVMTQSHKE MSTSVRDRVS ITCHASQDVS TAVAWYQQKP
GQSPHLLIYS ASYLYTGVPD 60
light chain RFTGSGSGTD FTFTISSVQA EDLAVYYCQQ HYSTPRTFGG GTKLEIK
107
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variable region
of humanized
antibody
SEQ ID NO:152 EVQLVESGGG LVOPGESLKL SCESNEYEFP SHDMSWVRKT
PEKRLELVAA INSDGGSTYY 60
heavy chain PDTMERRFII SRDNTKIKTLY LOMSSLRSED TALYYaARHY
DDYYAWFAYW GOGTLVTVSA 120
variable region
of humanized
antibody
SEQ ID NO:153 EVQLVESGGG LVQPGGSLRL SCAASEYEFP SHDMSWVRQA
PGKGLELVAA INSDGGSTYY 60
heavy chain PDTMERRFTI SRDNAKNSLY LQMNSLRAED TAVYYaARHY
DDYYAWFAYW GQGTMVTVSS 120
variable region
of humanized
antibody
SEQ ID NO:154 DIVLTQSPAS LAVSLGQRAT ISCRASKSVS TSGYSYMHWY
QQKPGQPPKL LIYLASNLES 60
light chain GVPARFSGSG SGTDFTLN=H PVEEEDAATY YCQHSRELPL
TFGAGTKLEL K 111
variable region
of humanized
antibody
SEQ ID NO:155 EIVLTQSPAT LSLSFGERAT LSCRASKSVS TSGYSYMHWY
QQKPGQAPRL LIYLASNLES 60
light chain GVPARFSGSG SGTDFTLT=S SLEPEDFAVY YCQHSRELPL
TFGGGTKVEI K 111
variable region
of humanized
antibody
SEQ ID 50:156 MYLGLNYVN'i Vn,LNGVQSE VKLEZSGGGL VQPGGSMKLS CAASGID
AWMDVIVRQSP 60
heavy chain EKGLEWVAEI RSKANNHATY YAESVNGRFT ISRDDSKSSV
YLQMNSLRAE DTGIYYCTWG 120
variable region EVEYFDYWGQ GTTLTVSS
138
SEQ ID NO:157 MRPSIQFLGL LLFWLHGAQC DIQMTQSPSS LSASLGGKVT
ITCKSSQDIN KYIAWYQHKP 60
light chain GHGPRLLIHY TSTLQPGIPS RFSGSGSGRD YSFSISNLEP
EDIATYYCLQ YDNLLTFGAG 120
variable region
THLELK
126
[00830] In some embodiments, the 0X40 agonist is a 0X40 agonistic single-chain
fusion
polypeptide comprising (i) a first soluble 0X40 binding domain, (ii) a first
peptide linker, (iii) a
second soluble 0X40 binding domain, (iv) a second peptide linker, and (v) a
third soluble 0X40
binding domain, further comprising an additional domain at the N-tenninal
and/or C-tenninal end,
and wherein the additional domain is a Fab or Fc fragment domain. In some
embodiments, the 0X40
agonist is a 0X40 agonistic single-chain fusion polypeptide comprising (i) a
first soluble 0X40
binding domain, (ii) a first peptide linker, (iii) a second soluble 0X40
binding domain, (iv) a second
peptide linker, and (v) a third soluble 0X40 binding domain, further
comprising an additional domain
at the N-terminal and/or C-terminal end, wherein the additional domain is a
Fab or Fe fragment
domain wherein each of the soluble 0X40 binding domains lacks a stalk region
(which contributes to
trimerisation and provides a certain distance to the cell membrane, but is not
part of the 0X40 binding
domain) and the first and the second peptide linkers independently have a
length of 3-8 amino acids.
[00831] In some embodiments, the 0X40 agonist is an 0X40 agonistic single-
chain fusion
polypeptide comprising (i) a first soluble tumor necrosis factor (TNF)
superfamily cytokine domain,
(ii) a first peptide linker, (iii) a second soluble TNF superfamily cytokine
domain, (iv) a second
peptide linker, and (v) a third soluble TNF superfamily cytokine domain,
wherein each of the soluble
TNF superfamily cytokine domains lacks a stalk region and the first and the
second peptide linkers
independently have a length of 3-8 amino acids, and wherein the TNF
superfamily cytokine domain is
an 0X40 binding domain.
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[00832] In some embodiments, the 0X40 agonist is MEDI6383. MEDI6383 is an 0X40
agonistic
fusion protein and can be prepared as described in U.S. Patent No. 6,312,700,
the disclosure of which
is incorporated by reference herein.
[00833] In some embodiments, the 0X40 agonist is an 0X40 agonistic scFy
antibody comprising
any of the foregoing VH domains linked to any of the foregoing VL domains.
[00834] In some embodiments, the 0X40 agonist is Creative Biolabs 0X40 agonist
monoclonal
antibody MOM-18455, commercially available from Creative Biolabs, Inc.,
Shirley, NY, USA.
[00835] In some embodiments, the 0X40 agonist is 0X40 agonistic antibody clone
Ber-ACT35
commercially available from BioLegend, Inc., San Diego, CA, USA.
C. Optional Cell Viability Analyses
[00836] Optionally, a cell viability assay can be performed after the priming
first expansion
(sometimes referred to as the initial bulk expansion), using standard assays
known in the art. Thus, in
certain embodiments, the method comprises performing a cell viability assay
subsequent to the
priming first expansion. For example, a trypan blue exclusion assay can be
done on a sample of the
bulk TILs, which selectively labels dead cells and allows a viability
assessment. Other assays for use
in testing viability can include but are not limited to the Alamar blue assay;
and the MTT assay.
1. Cell Counts, Viability, Flow Cytometry
[00837] In some embodiments, cell counts and/or viability are measured. The
expression of markers
such as but not limited CD3, CD4, CD8, and CD56, as well as any other
disclosed or described
herein, can be measured by flow cytometry with antibodies, for example but not
limited to those
commercially available from BD Bio-sciences (BD Biosciences, San Jose, CA)
using a FACSCantoTM
flow cytometer (BD Biosciences). The cells can be counted manually using a
disposable c-chip
hemocytometer (VWR, Batavia, IL) and viability can be assessed using any
method known in the art,
including but not limited to trypan blue staining. The cell viability can also
be assayed based on U.S.
Patent Application Publication No. 2018/0282694, incorporated by reference
herein in its entirety.
Cell viability can also be assayed based on U.S. Patent Application
Publication No. 2018/0280436 or
International Patent Application Publication No. WO/2018/081473, both of which
are incorporate
herein in their entireties for all purposes.
[00838] In some cases, the bulk TIL population can be cryopreserved
immediately, using the
protocols discussed below. Alternatively, the bulk T1L population can be
subjected to REP and then
cryopre served as discussed below. Similarly, in the case where genetically
modified TILs will be used
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in therapy, the bulk or REP TIL populations can be subjected to genetic
modifications for suitable
treatments.
2. Cell Cultures
[00839] In some embodiments, a method for expanding TILs, including those
discussed above as
well as exemplified in Figures 1 and 8, in particular, e.g.. Figure 8A and/or
Figure 8B and/or Figure
8C and/or Figure 8D, may include using about 5,000 mL to about 25,000 mL of
cell medium, about
5,000 mL to about 10,000 mL of cell medium, or about 5,800 mL to about 8,700
mL of cell medium.
In some embodiments, the media is a serum free medium. In some embodiments,
the media in the
priming first expansion is serum free. In some embodiments, the media in the
second expansion is
serum free. In some embodiments, the media in the priming first expansion and
the second expansion
(also referred to as rapid second expansion) are both serum free. In some
embodiments, expanding the
number of TILs uses no more than one type of cell culture medium. Any suitable
cell culture medium
may be used, e.g., AIM-V cell medium (L-glutaminc, 50 tM streptomycin sulfate,
and 10 uM
gentamicin sulfate) cell culture medium (Invitrogen, Carlsbad CA). In this
regard, the inventive
methods advantageously reduce the amount of medium and the number of types of
medium required
to expand the number of TIL. In some embodiments, expanding the number of TIL
may comprise
feeding the cells no more frequently than every third or fourth day. Expanding
the number of cells in a
gas permeable container simplifies the procedures necessary to expand the
number of cells by
reducing the feeding frequency necessary to expand the cells.
[00840] In some embodiments, the cell culture medium in the first and/or
second gas perrneable
container is unfiltered. The use of unfiltered cell medium may simplify the
procedures necessary to
expand the number of cells. In some embodiments, the cell medium in the first
and/or second gas
permeable container lacks beta-mercaptoethanol (BME).
[00841] In some embodiments, the duration of the method comprising obtaining a
tumor tissue
sample from the mammal; culturing the tumor tissue sample in a first gas
permeable container
containing cell medium including IL-2, IX antigen-presenting feeder cells, and
OKT-3 for a duration
of about 1 to 8 days, e.g., about 7 days as a priming first expansion, or
about 8 days as a priming first
expansion; transferring the TILs to a second gas permeable container and
expanding the number of
TILs in the second gas permeable container containing cell medium including IL-
2, 2X antigen-
presenting feeder cells, and OKT-3 for a duration of about 7 to 9 days, e.g.,
about 7 days, about 8
days, or about 9 days.
[00842] In some embodiments, the duration of the method comprising obtaining a
tumor tissue
sample from the mammal; culturing the tumor tissue sample in a first gas
permeable container
containing cell medium including IL-2, 1X antigen-presenting feeder cells, and
OKT-3 for a duration
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of about 1 to 7 days, e.g., about 7 days as a priming first expansion;
transferring the TILs to a second
gas permeable container and expanding the number of TILs in the second gas
permeable container
containing cell medium including 1L-2, 2X antigen-presenting feeder cells, and
OKT-3 for a duration
of about 7 to 14 days, or about 7 to 9 days, e.g., about 7 days, about 8 days,
or about 9 days, about 10
days, or about 11 days.
[00843] In some embodiments, the duration of the method comprising obtaining a
tumor tissue
sample from the mammal; culturing the tumor tissue sample in a first gas
permeable container
containing cell medium including H,-2, 1X antigen-presenting feeder cells, and
OKT-3 for a duration
of about 1 to 7 days, e.g., about 7 days, as a priming first expansion;
transferring the TILs to a second
gas permeable container and expanding the number of TILs in the second gas
permeable container
containing cell medium including IL-2, 2X antigen-presenting feeder cells, and
OKT-3 for a duration
of about 7 to 11 days, e.g., about 7 days, about 8 days, about 9 days, about
10, or about 11 days.
[00844] In some embodiments, TILs are expanded in gas-permeable containers.
Gas-permeable
containers have been used to expand TILs using PBMCs using methods,
compositions, and devices
known in the art, including those described in U.S. Patent Application
Publication No. 2005/0106717
Al, the disclosures of which are incorporated herein by reference. In some
embodiments, TILs are
expanded in gas-permeable bags. In some embodiments, TILs are expanded using a
cell expansion
system that expands TILs in gas permeable bags, such as the Xuri Cell
Expansion System W25 (GE
Healthcare). In some embodiments, TILs are expanded using a cell expansion
system that expands
TILs in gas permeable bags, such as the WAVE Bioreactor System, also known as
the Xuri Cell
Expansion System W5 (GE Healthcare). In some embodiments, the cell expansion
system includes a
gas permeable cell bag with a volume selected from the group consisting of
about 100 mL, about 200
mL, about 300 mL, about 400 mL, about 500 mL, about 600 mL, about 700 mL,
about 800 mL, about
900 mL, about 1 L, about 2 L, about 3 L, about 4 L, about 5 L, about 6 L,
about 7 L, about 8 L, about
9 L, and about 10 L.
[00845] In some embodiments, TILs can be expanded in G-REX flasks
(commercially available
from Wilson Wolf Manufacturing). Such embodiments allow for cell populations
to expand from
about 5 x 10 cells/cm' to between 10 x 10' and 30x 106 cells/cm'. In some
embodiments this is
without feeding. In some embodiments, this is without feeding so long as
medium resides at a height
of about 10 cm in the G-REX flask. In some embodiments this is without feeding
but with the
addition of one or more cytokines. In some embodiments, the cytokine can be
added as a bolus
without any need to mix the cytokine with the medium. Such containers,
devices, and methods are
known in the art and have been used to expand TILs, and include those
described in U.S. Patent
Application Publication No. US 2014/0377739A1, International Publication No.
WO 2014/210036
Al, U.S. Patent Application Publication No. us 2013/0115617 Al, International
Publication No. WO
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2013/188427 Al, U.S. Patent Application Publication No. US 2011/0136228 Al,
U.S. Patent No. US
8,809,050 B2, International publication No. WO 2011/072088 A2, U.S. Patent
Application
Publication No. US 2016/0208216 Al, U.S. Patent Application Publication No. US
2012/0244133
Al, International Publication No. WO 2012/129201 Al, U.S. Patent Application
Publication No. US
2013/0102075 Al, U.S. Patent No. US 8,956,860 B2, International Publication
No. WO 2013/173835
Al, U.S. Patent Application Publication No. US 2015/0175966 Al, the
disclosures of which are
incorporated herein by reference. Such processes are also described in Jin
etal., J. Immunotherapy,
2012, 35:283-292.
D. Optional Knockdown or Knockout of Genes in TILs
[00846] In some embodiments, the expanded TiLs of the present invention are
further manipulated
before, during, or after an expansion step, including during closed, sterile
manufacturing processes,
each as provided herein, in order to alter protein expression in a transient
manner. In some
embodiments, the transiently altered protein expression is due to transient
gene editing. In some
embodiments, the expanded TILs of the present invention are treated with
transcription factors (TFs)
and/or other molecules capable of transiently altering protein expression in
the TILs. In some
embodiments, the TFs and/or other molecules that are capable of transiently
altering protein
expression provide for altered expression of tumor antigens and/or an
alteration in the number of
tumor antigen-specific T cells in a population of TILs.
[00847] In certain embodiments, the method comprises genetically editing a
population of TILs. In
certain embodiments, the method comprises genetically editing the first
population of TILs, the
second population of TILs and/or the third population of TILs.
[00848] In some embodiments, the present invention includes genetic editing
through nucleotide
insertion, such as through ribonucleic acid (RNA) insertion, including
insertion of messenger RNA
(mRNA) or small (or short) interfering RNA (siRNA), into a population of TILs
for promotion of the
expression of one or more proteins or inhibition of the expression of one or
more proteins, as well as
simultaneous combinations of both promotion of one set of proteins with
inhibition of another set of
proteins.
[00849] In some embodiments, the expanded TiLs of the present invention
undergo transient
alteration of protein expression. In some embodiments, the transient
alteration of protein expression
occurs in the bulk TIL population prior to first expansion, including, for
example in the TIL
population obtained from for example, Step A as indicated in Figure 8
(particularly Figure 8A and/or
Figure 8B and/or Figure 8C and/or Figure 8D). In some embodiments, the
transient alteration of
protein expression occurs during the first expansion, including, for example
in the TIL population
expanded in for example, Step B as indicated in Figure 8 (for example Figure
8A and/or Figure 8B
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and/or Figure 8C and/or Figure 8D). In some embodiments, the transient
alteration of protein
expression occurs after the first expansion, including, for example in the TIL
population in transition
between the first and second expansion (e.g., the second population of TILs as
described herein), the
TIL population obtained from for example, Step B and included in Step C as
indicated in Figure 8. In
some embodiments, the transient alteration of protein expression occurs in the
bulk TIL population
prior to second expansion, including, for example in the IlL population
obtained from for example,
Step C and prior to its expansion in Step D as indicated in Figure 8. In some
embodiments, the
transient alteration of protein expression occurs during the second expansion,
including, for example
in the TIL population expanded in for example, Step D as indicated in Figure 8
(e.g., the third
population of TILs). In some embodiments, the transient alteration of protein
expression occurs after
the second expansion, including, for example in the TIL population obtained
from the expansion in
for example, Step D as indicated in Figure 8.
[00850] In some embodiments, a method of transiently altering protein
expression in a population of
TILs includes the step of electroporation. Electroporation methods are known
in the art and are
described, e.g, in Tsong, Biophys. 1 1991, 60, 297-306, and U.S. Patent
Application Publication No.
2014/0227237 Al, the disclosures of each of which are incorporated by
reference herein. In some
embodiments, a method of transiently altering protein expression in population
of TILs includes the
step of calcium phosphate transfection. Calcium phosphate transfection methods
(calcium phosphate
DNA precipitation, cell surface coating, and endocytosis) are known in the art
and are described in
Graham and van der Eb, Virology 1973, 52, 456-467, Wigler, et al., Proc. Natl.
Acad. Sci. 1979, 76,
1373-1376; and Chen and Okayarea,Mol. Cell. Biol. 1987, 7, 2745-2752; and in
U.S. Patent No.
5,593,875, the disclosures of each of which are incorporated by reference
herein. In some
embodiments, a method of transiently altering protein expression in a
population of TILs includes the
step of liposomal transfection. Liposomal transfection methods, such as
methods that employ a 1:1
(w/w) liposome formulation of the cationic lipid N41-(2,3-dioleyloxy)propyll-
n,n,n-
trimethylammonium chloride (DOTMA) and dioleoyl phophotidylethanolamine (DOPE)
in filtered
water, are known in the art and are described in Rose, et al., Biotechniques
1991, /0, 520-525 and
Feigner, et al., Proc. Natl. Acad. Sci. USA, 1987, 84, 7413-7417 and in U.S.
Patent Nos. 5,279,833;
5,908,635; 6,056,938; 6,110,490; 6,534,484; and 7,687,070, the disclosures of
each of which are
incorporated by reference herein. In some embodiments, a method of transiently
altering protein
expression in a population of TILs includes the step of transfection using
methods described in U.S.
Patent Nos. 5,766,902; 6,025,337; 6,410,517; 6,475,994; and 7,189,705; the
disclosures of each of
which are incorporated by reference herein.
[00851] In some embodiments, transient alteration of protein expression
results in an increase in
Stem Memory T cells (TSCMs). TSCMs are early progenitors of antigen-
experienced central memory
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T cells. TSCMs generally display the long-term survival, self-renewal, and
multipotency abilities that
define stem cells, and are generally desirable for the generation of effective
TIL products. TSCM have
shown enhanced anti-tumor activity compared with other T cell subsets in mouse
models of adoptive
cell transfer. In some embodiments, transient alteration of protein expression
results in a TIL
population with a composition comprising a high proportion of TSCM. In some
embodiments,
transient alteration of protein expression results in an at least 5%, at least
10%, at least 10%, at least
20%, at least 25%, at least 30%, at least 35%, at least 40%, at least 45%, at
least 50%, at least 55%, at
least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least
85%, at least 90%, or at least
95% increase in TSCM percentage. In some embodiments, transient alteration of
protein expression
results in an at least a 1-fold, 2-fold, 3-fold, 4-fold, 5-fold, or 10-fold
increase in TSCMs in the TIL
population. In some embodiments, transient alteration of protein expression
results in a TIL
population with at least at least 5%, at least 10%, at least 10%, at least
20%, at least 25%, at least
30%, at least 35%, at least 40%, at least 45%, at least 50%, at least 55%, at
least 60%, at least 65%, at
least 70%, at least 75%, at least 80%, at least 85%, at least 90%, or at least
95% TSCMs. In some
embodiments, transient alteration of protein expression results in a
therapeutic TIL population with at
least at least 5%, at least 10%, at least 10%, at least 20%, at least 25%, at
least 30%, at least 35%, at
least 40%, at least 45%, at least 50%, at least 55%, at least 60%, at least
65%, at least 70%, at least
75%, at least 80%, at least 85%, at least 90%, or at least 95% TSCMs.
1008521 In some embodiments, transient alteration of protein expression
results in rejuvenation of
antigen-experienced T-cells. In some embodiments, rejuvenation includes, for
example, increased
proliferation, increased T-cell activation, and/or increased antigen
recognition.
1008531 In some embodiments, transient alteration of protein expression alters
the expression in a
large fraction of the T-cells in order to preserve the tumor-derived TCR
repertoire. In some
embodiments, transient alteration of protein expression does not alter the
tumor-derived TCR
repertoire. In some embodiments, transient alteration of protein expression
maintains the tumor-
derived TCR repertoire.
1008541 In some embodiments, transient alteration of protein results in
altered expression of a
particular gene. In some embodiments, the transient alteration of protein
expression targets a gene
including but not limited to PD-1 (also referred to as PDCD1 or CC279),
TGFBR2, CCR4/5, CBLB
(CBL-B), CISH, CCRs (chimeric co-stimulatory receptors), IL-2, IL-12, IL-15,
IL-21, NOTCH 1/2
1CD, CTLA-4, T1M3, LA(i3, TIGIT, TET2, TGFP, CCR2, CCR4, CCR5, CXCR1, CXCR2,
CSCR3,
CCL2 (MCP-1), CCL3 (MIP-1a), CCL4 (MIP1-f3), CCL5 (RANTES), CXCL1/CXCL8,
CCL22,
CCL17, CXCL1/CXCL8, VHL, CD44, PIK3CD, SOCS1, thymocyte selection associated
high
mobility group (HMG) box (TOX), ankyrin repeat domain 11 (ANKRD11), BCL6 co-
repressor
(BCOR), and/or cAMP protein kinase A (PKA). In some embodiments, the transient
alteration of
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protein expression targets a gene selected from the group consisting of PD-1,
TGFBR2, CCR4/5,
CTLA-4, CBLB (CBL-B), CISH, CCRs (chimeric co-stimulatory receptors), IL-2, IL-
12, IL-15, IL-
21, NOTCH 1/2 ICD, TIM3, LAG3, TIGIT, TET2, TGF(3, CCR2, CCR4, CCR5, CXCR1,
CXCR2,
CSCR3, CCL2 (MCP-1), CCL3 (MIP-1a), CCL4 (MIP1-13), CCL5 (RANTES).
CXCL1/CXCL8,
CCL22, CCL17, CXCL1/CXCL8, VHL, CD44, PIK3CD, SOCS1, thymocyte selection
associated
high mobility group (HMG) box (TOX), ankyrm repeat domain 11 (ANKR1)11), BCL6
co-repressor
(BCOR), and/or cAMP protein kinase A (PKA). In some embodiments, the transient
alteration of
protein expression targets PD-1. In some embodiments, the transient alteration
of protein expression
targets TGFBR2. In some embodiments, the transient alteration of protein
expression targets CCR4/5.
In some embodiments, the transient alteration of protein expression targets
CTLA-4. In some
embodiments, the transient alteration of protein expression targets CBLB. In
some embodiments, the
transient alteration of protein expression targets CISH. In some embodiments,
the transient alteration
of protein expression targets CCRs (chimeric co-stimulatory receptors). In
some embodiments, the
transient alteration of protein expression targets IL-2. In some embodiments,
the transient alteration of
protein expression targets TL-12. In some embodiments, the transient
alteration of protein expression
targets IL-15. In some embodiments, the transient alteration of protein
expression targets IL-21. In
some embodiments, the transient alteration of protein expression targets NOTCH
1/2 ICD. In some
embodiments, the transient alteration of protein expression targets TIM3. In
some embodiments, the
transient alteration of protein expression targets LAG3. In some embodiments,
the transient alteration
of protein expression targets TIGIT. In some embodiments, the transient
alteration of protein
expression targets TET2. In some embodiments, the transient alteration of
protein expression targets
TGF13. In some embodiments, the transient alteration of protein expression
targets CCR1. In some
embodiments, the transient alteration of protein expression targets CCR2. In
some embodiments, the
transient alteration of protein expression targets CCR4. In some embodiments,
the transient alteration
of protein expression targets CCR5. In some embodiments, the transient
alteration of protein
expression targets CXCR1. In some embodiments, the transient alteration of
protein expression
targets CXCR2. In some embodiments, the transient alteration of protein
expression targets CSCR3.
In some embodiments, the transient alteration of protein expression targets
CCL2 (MCP-1). In some
embodiments, the transient alteration of protein expression targets CCL3 (MIP-
1a). In some
embodiments, the transient alteration of protein expression targets CCL4 (MIP1-
13). In some
embodiments, the transient alteration of protein expression targets CCL5
(RANTES). In some
embodiments, the transient alteration of protein expression targets CXCL1. In
some embodiments, the
transient alteration of protein expression targets CXCL8. In some embodiments,
the transient
alteration of protein expression targets CCL22. In some embodiments, the
transient alteration of
protein expression targets CCL17. In some embodiments, the transient
alteration of protein expression
targets VHL. In some embodiments, the transient alteration of protein
expression targets CD44. In
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some embodiments, the transient alteration of protein expression targets P11(3
CD. In some
embodiments, the transient alteration of protein expression targets SOCS1. In
some embodiments, the
transient alteration of protein expression targets thymocyte selection
associated high mobility group
(HMG) box (TOX). In some embodiments, the transient alteration of protein
expression targets
ankyrin repeat domain 11 (ANKRD11). In some embodiments, the transient
alteration of protein
expression targets BCL6 co-repressor (BCOR). In some embodiments, the
transient alteration of
protein expression targets cAMP protein kinase A (PKA).
[00855] In some embodiments, the transient alteration of protein expression
results in increased
and/or overexpression of a chemokine receptor. In some embodiments, the
chemokine receptor that is
oyerexpressed by transient protein expression includes a receptor with a
ligand that includes but is not
limited to CCL2 (MCP-1), CCL3 (MIP-1a), CCL4 (MIP1-13), CCL5 (RANTES), CXCL1,
CXCL8,
CCL22, and/or CCL17.
[00856] In some embodiments, the transient alteration of protein expression
results in a decrease
and/or reduced expression of PD-1, CTLA-4, CBLB, CISH, TIM-3, LAG-3, TIGIT,
TET2, TGF13R2,
and/or TGFI3 (including resulting in, for example, TGFI3 pathway blockade). In
some embodiments,
the transient alteration of protein expression results in a decrease and/or
reduced expression of PD-1.
In some embodiments, the transient alteration of protein expression results in
a decrease and/or
reduced expression of CBLB (CBL-B). In some embodiments, the transient
alteration of protein
expression results in a decrease and/or reduced expression of CISH. In some
embodiments, the
transient alteration of protein expression results in a decrease and/or
reduced expression of TIM-3. In
some embodiments, the transient alteration of protein expression results in a
decrease and/or reduced
expression of LAG-3. In some embodiments, the transient alteration of protein
expression results in a
decrease and/or reduced expression of TIGIT. In some embodiments, the
transient alteration of
protein expression results in a decrease and/or reduced expression of TET2. In
some embodiments,
the transient alteration of protein expression results in a decrease and/or
reduced expression of
TGFDR2. In some embodiments, the transient alteration of protein expression
results in a decrease
and/or reduced expression of TGFI3.
[00857] In some embodiments, the transient alteration of protein expression
results in increased
and/or overexpression of chemokine receptors in order to, for example, improve
TIL trafficking or
movement to the tumor site. In some embodiments, the transient alteration of
protein expression
results in increased and/or overexpression of a CCR (chimeric co-stimulatory
receptor). In some
embodiments, the transient alteration of protein expression results in
increased and/or overexpression
of a chemokine receptor selected from the group consisting of CCR1, CCR2,
CCR4, CCR5, CXCR1,
CXCR2, and/or CSCR3.
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[00858] In some embodiments, the transient alteration of protein expression
results in increased
and/or overexpression of an interleukin. In some embodiments, the transient
alteration of protein
expression results in increased and/or overexpression of an interleukin
selected from the group
consisting of IL-2, IL-12, IL-15, and/or IL-21.
[00859] In some embodiments, the transient alteration of protein expression
results in increased
and/or overexpression of NOTCH 1/2 ICD. In some embodiments, the transient
alteration of protein
expression results in increased and/or overexpression of VHL. In some
embodiments, the transient
alteration of protein expression results in increased and/or overexpression of
CD44. In some
embodiments, the transient alteration of protein expression results in
increased and/or overexpression
of PIK3CD. In some embodiments, the transient alteration of protein expression
results in increased
and/or overexpression of SOCS1,
[00860] In some embodiments, the transient alteration of protein expression
results in decreased
and/or reduced expression of cAMP protein kinase A (PKA).
[00861] In some embodiments, the transient alteration of protein expression
results in decreased
and/or reduced expression of a molecule selected from the group consisting of
PD-1, LAG3, TIM3,
CTLA-4, TIGIT, TET2, CISH, TGFI3R2, PKA, CBLB, BAFF (BR3), and combinations
thereof. In
some embodiments, the transient alteration of protein expression results in
decreased and/or reduced
expression of two molecules selected from the group consisting of PD-1, LAG3,
TIM3, CTLA-4,
TIGIT, TET2, CISH, TGFPR2, PKA, CBLB, BAFF (BR3), and combinations thereof. In
some
embodiments, the transient alteration of protein expression results in
decreased and/or reduced
expression of PD-1 and one molecule selected from the group consisting of
LAG3, TIM3, CTLA-4,
TIGIT, TET2, CISH, TGFI3R2, PKA, CBLB, BAFF (BR3), and combinations thereof.
In some
embodiments, the transient alteration of protein expression results in
decreased and/or reduced
expression of PD-1, CTLA-4, LAG-3, CISH, CBLB, TIM3, TIGIT, TET2 and
combinations thereof.
In some embodiments, the transient alteration of protein expression results in
decreased and/or
reduced expression of PD-1 and one of CTLA-4, LAG3, CISH, CBLB, TIM3, TIGIT,
TET2 and
combinations thereof. In some embodiments, the transient alteration of protein
expression results in
decreased and/or reduced expression of PD-1 and CTLA-4. In some embodiments,
the transient
alteration of protein expression results in decreased and/or reduced
expression of PD-1 and LAG3. In
some embodiments, the transient alteration of protein expression results in
decreased and/or reduced
expression of PD-1 and CISH. In some embodiments, the transient alteration of
protein expression
results in decreased and/or reduced expression of PD-1 and CBLB. In some
embodiments, the
transient alteration of protein expression results in decreased and/or reduced
expression of PD-1 and
TIM3. In some embodiments, the transient alteration of protein expression
results in decreased and/or
reduced expression of PD-1 and TIGIT. In some embodiments, the transient
alteration of protein
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expression results in decreased and/or reduced expression of PD-1 and TET2.
IIn some embodiments,
the transient alteration of protein expression results in decreased and/or
reduced expression of CTLA-
4 and LAG3. In some embodiments, the transient alteration of protein
expression results in decreased
and/or reduced expression of CTLA-4 and CISH. In some embodiments, the
transient alteration of
protein expression results in decreased and/or reduced expression of CTLA-4
and CBLB. In some
embodiments, the transient alteration of protein expression results in
decreased and/or reduced
expression of CTLA-4 and TIM3. In some embodiments, the transient alteration
of protein expression
results in decreased and/or reduced expression of CTLA-4 and TIGIT. In some
embodiments, the
transient alteration of protein expression results in decreased and/or reduced
expression of CTLA-4
and TET2. In some embodiments, the transient alteration of protein expression
results in decreased
and/or reduced expression of LAG3 and CISH. In some embodiments, the transient
alteration of
protein expression results in decreased and/or reduced expression of LAG3 and
CBLB. In some
embodiments, the transient alteration of protein expression results in
decreased and/or reduced
expression of LAG3 and TIM3. In some embodiments, the transient alteration of
protein expression
results in decreased and/or reduced expression of LAG3 and TIGIT. In some
embodiments, the
transient alteration of protein expression results in decreased and/or reduced
expression of LAG3 and
TET2. In some embodiments, the transient alteration of protein expression
results in decreased and/or
reduced expression of CISH and CBLB. In some embodiments, the transient
alteration of protein
expression results in decreased and/or reduced expression of CISH and TIM3. In
some embodiments,
the transient alteration of protein expression results in decreased and/or
reduced expression of CISH
and TIGIT. In some embodiments, the transient alteration of protein expression
results in decreased
and/or reduced expression of CISH and TET2. In some embodiments, the transient
alteration of
protein expression results in decreased and/or reduced expression of CBLB and
TIM3. In some
embodiments, the transient alteration of protein expression results in
decreased and/or reduced
expression of CBLB and TIGIT. In some embodiments, the transient alteration of
protein expression
results in decreased and/or reduced expression of CBLB and TET2. In some
embodiments, the
transient alteration of protein expression results in decreased and/or reduced
expression of TIM3 and
PD-1. In some embodiments, the transient alteration of protein expression
results in decreased and/or
reduced expression of TIM3 and LAG3. In some embodiments, the transient
alteration of protein
expression results in decreased and/or reduced expression of TIM3 and CISH. In
some embodiments,
the transient alteration of protein expression results in decreased and/or
reduced expression of TIM3
and CBLB. In some embodiments, the transient alteration of protein expression
results in decreased
and/or reduced expression of TIM3 and TIGIT. In some embodiments, the
transient alteration of
protein expression results in decreased and/or reduced expression of TIM3 and
TET2.
[00862] In some embodiments, an adhesion molecule selected from the group
consisting of CCR2,
CCR4, CCR5, CXCR2, CXCR3, CX3CR1, and combinations thereof, is inserted by a
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gammaretroviral or lentiviral method into the first population of TILs, second
population of TILs, or
harvested population of TILs (e.g., the expression of the adhesion molecule is
increased).
1008631 In some embodiments, the transient alteration of protein expression
results in decreased
and/or reduced expression of a molecule selected from the group consisting of
PD-1, LAG3, TIM3,
CTLA-4, TIGIT, TET2, CISH, TGF13R2, PKA, CBLB, BAFF (BR3), and combinations
thereof, and
increased and/or enhanced expression of CCR2, CCR4, CCR5, CXCR2, CXCR3,
CX3CR1, and
combinations thereof In some embodiments, the transient alteration of protein
expression results in
decreased and/or reduced expression of a molecule selected from the group
consisting of PD-1,
CTLA-4, LAG3, TIM3, CISH, CBLB, TIGIT, TET2 and combinations thereof, and
increased and/or
enhanced expression of CCR2, CCR4, CCR5, CXCR2, CXCR3, CX3CR1, and
combinations thereof.
[00864] In some embodiments, there is a reduction in expression of about 5%,
about 10%, about
10%, about 20%, about 25%, about 30%, about 35%, about 40%, about 45%, about
50%, about 55%,
about 60%, about 65%, about 70%, about 75%, about 80%, about 85%, about 90%,
or about 95%. In
some embodiments, there is a reduction in expression of at least about 65%,
about 70%, about 75%,
about 80%, about 85%, about 90%, or about 95%. In some embodiments, there is a
reduction in
expression of at least about 75%, about 80%, about 85%, about 90%, or about
95%. In some
embodiments, there is a reduction in expression of at least about 80%, about
85%, about 90%, or
about 95%. In some embodiments, there is a reduction in expression of at least
about 85%, about
90%, or about 95%. In some embodiments, there is a reduction in expression of
at least about 80%. In
some embodiments, there is a reduction in expression of at least about 85%, In
some embodiments,
there is a reduction in expression of at least about 90%. In some embodiments,
there is a reduction in
expression of at least about 95%. In some embodiments, there is a reduction in
expression of at least
about 99%.
[00865] In some embodiments, there is an increase in expression of about 5%,
about 10%, about
10%, about 20%, about 25%, about 30%, about 35%, about 40%, about 45%, about
50%, about 55%,
about 60%, about 65%, about 70%, about 75%, about 80%, about 85%, about 90%,
or about 95%. In
some embodiments, there is an increase in expression of at least about 65%,
about 70%, about 75%,
about 80%, about 85%, about 90%, or about 95%. in some embodiments, there is
an increase in
expression of at least about 75%, about 80%, about 85%, about 90%, or about
95%. In some
embodiments, there is an increase in expression of at least about 80%, about
85%, about 90%, or
about 95%. In some embodiments, there is an increase in expression of at least
about 85%, about
90%, or about 95%. In some embodiments, there is an increase in expression of
at least about 80%. In
some embodiments, there is an increase in expression of at least about 85%, In
some embodiments,
there is an increase in expression of at least about 90%. In some embodiments,
there is an increase in
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expression of at least about 95%. In some embodiments, there is an increase in
expression of at least
about 99%.
[00866] In some embodiments, transient alteration of protein expression is
induced by treatment of
the TILs with transcription factors (TFs) and/or other molecules capable of
transiently altering protein
expression in the TILs. In some embodiments, the SQZ vector-free microfluidic
platform is employed
for intracellular delivery of the transcription factors (TFs) and/or other
molecules capable of
transiently altering protein expression. Such methods demonstrating the
ability to deliver proteins,
including transcription factors; to a variety of primary human cells,
including T cells (which have
been described in U.S. Patent Application Publication Nos. US 2019/0093073 Al,
US 2018/0201889
Al, and US 2019/0017072 Al, the disclosures of each of which are incorporated
herein by reference).
Such method can be employed with the present invention in order to expose a
population of TILs to
transcription factors (TFs) and/or other molecules capable of inducing
transient protein expression,
wherein said TFs and/or other molecules capable of inducing transient protein
expression provide for
increased expression of tumor antigens and/or an increase in the number of
tumor antigen-specific T
cells in the population of TILs, thus resulting in reprogramming of the TIL
population and an increase
in therapeutic efficacy of the reprogrammed TIL population as compared to a
non-reprogrammed TIL
population. In some embodiments, the reprogramming results in an increased
subpopulation of
effector T cells and/or central memory T cells relative to the starting or
prior population (i.e., prior to
reprogramming) population of TILs, as described herein.
[00867] In sonic embodiments, the transcription factor (TF) includes but is
not limited to TCF-1,
NOTCH 1/2 1CD, and/or MYB. In some embodiments, the transcription factor (TF)
is TCF-1. In
some embodiments, the transcription factor (TF) is NOTCH 1/2 ICD. In some
embodiments, the
transcription factor (TF) is MYB. In some embodiments, the transcription
factor (TF) is administered
with induced pluripotent stem cell culture (iPSC), such as the commercially
available KNOCKOUT
Serum Replacement (Gibco/ThermoFisher), to induce additional TIL
reprogramming. In some
embodiments, the transcription factor (TF) is administered with an iPSC
cocktail to induce additional
TIL reprogramming. In some embodiments, the transcription factor (TF) is
administered without an
iPSC cocktail. In some embodiments, reprogramming results in an increase in
the percentage of
TSCMs. In some embodiments, reprogramming results in an increase in the
percentage of TSCMs by
about 5%, about 10%, about 10%, about 20%, about 25%, about 30%, about 35%,
about 40%, about
45%, about 50%, about 55%, about 60%, about 65%, about 70%, about 75%, about
80%, about 85%,
about 90%, or about 95% TSCMs.
[00868] In some embodiments, a method of transient altering protein
expression, as described
above, may be combined with a method of genetically modifying a population of
TILs includes the
step of stable incorporation of genes for production of one or more proteins.
In certain embodiments,
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the method comprises a step of genetically modifying a population of TILs. In
certain embodiments,
the method comprises genetically modifying the first population of TILs, the
second population of
TILs and/or the third population of TILs. In some embodiments, a method of
genetically modifying a
population of TILs includes the step of retroviral transduction. In some
embodiments, a method of
genetically modifying a population of TILs includes the step of lentiviral
transduction. Lentiviral
transduction systems are known in the art and arc described, e.g., in Levine,
et at., Proc. Nat'l Acad.
Set. 2006, 103, 17372-77; Zufferey, et at., Nat. Biotechnol. 1997, 15, 871-75;
Dull, et at., I Virology
1998, 72, 8463-71, and U.S. Patent No. 6,627,442, the disclosures of each of
which are incorporated
by reference herein. In some embodiments, a method of genetically modifying a
population of TILs
includes the step of gamma-retroviral transduction. Gamma-retroyiral
transduction systems are known
in the art and are described, e.g., Cepko and Pear. Cur. Prot. Mol. Biol.
1996, 9.9.1-9.9.16, the
disclosure of which is incorporated by reference herein. In some embodiments,
a method of
genetically modifying a population of TILs includes the step of transposon-
mcdiatcd gene transfer.
Transposon-mediated gene transfer systems are known in the art and include
systems wherein the
transposase is provided as DNA expression vector or as an expressible RNA or a
protein such that
long-term expression of the transposase does not occur in the transgenic
cells, for example, a
transposase provided as an mRNA (e.g., an mRNA comprising a cap and poly-A
tail). Suitable
transposon-mediated gene transfer systems, including the salmonid-type Tel-
like transposase (SB or
Sleeping Beauty transposase), such as SB10, SB11, and SB100x, and engineered
enzymes with
increased enzymatic activity, are described in, e.g., Hackett, et al., Mol.
Therapy 2010, 18, 674-83 and
U.S. Patent No. 6,489,458, the disclosures of each of which are incorporated
by reference herein.
[00869]
[00870] In some embodiments, transient alteration of protein expression in
TILs is induced by small
interfering RNA (siRNA), sometimes known as short interfering RNA or silencing
RNA, which is a
double stranded RNA molecule, generally 19-25 base pairs in length. siRNA is
used in RNA
interference (RNAi), where it interferes with expression of specific genes
with complementary
nucleotide sequences. In some embodiments, transient alteration of protein
expression is a reduction
in expression. In some embodiments, transient alteration of protein expression
in TILs is induced by
self-delivering RNA interference (sdRNA), which is a chemically-synthesized
asymmetric siRNA
duplex with a high percentage of 2' -OH substitutions (typically fluorine or -
OCH3) which comprises a
20-nucleotide antisense (guide) strand and a 13 to 15 base sense (passenger)
strand conjugated to
cholesterol at its 3' end using a tetraethylenglycol (TEG) linker. Small
interfering RNA (siRNA),
sometimes known as short interfering RNA or silencing RNA, is a double
stranded RNA molecule,
generally 19-25 base pairs in length. siRNA is used in RNA interference
(RNAi), where it interferes
with expression of specific genes with complementary nucleotide sequences.
sdRNA arc covalently
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and hydrophobically modified RNAi compounds that do not require a delivery
vehicle to enter cells.
sdRNAs are generally asymmetric chemically modified nucleic acid molecules
with minimal double
stranded regions. sdRNA molecules typically contain single stranded regions
and double stranded
regionsand can contain a variety of chemical modifications within both the
single stranded and double
stranded regions of the molecule. Additionally, the sdRNA molecules can be
attached to a
hydrophobic conjugate such as a conventional and advanced sterol-type
molecule, as described herein.
sdRNAs and associated methods for making such sdRNAs have also been described
extensively in,
for example,U.S. Patent Application Publication Nos. US 2016/0304873 Al, US
2019/0211337 Al,
US 2009/0131360 Al, and US 2019/0048341 Al, and U.S. Patent Nos. 10,633,654
and
10,913,948B2, the disclosures of each of which are incorporated by reference
herein.To optimize
sdRNA structure, chemistry, targeting position, sequence preferences, and the
like, analgorithm has
been developed and utilized for sdRNA potency prediction. Based on these
analyses, functional
sdRNA sequences have been generally defined as having over 70% reduction in
expression at 1 iaM
concentration, with a probability over 40%.
[00871] Double stranded DNA (dsRNA) can be generally used to define any
molecule comprising a
pair of complementary strands of RNA, generally a sense (passenger) and
antisense (guide) strands,
and may include single-stranded overhang regions. The term dsRNA, contrasted
with siRNA,
generally refers to a precursor molecule that includes the sequence of an
siRNA molecule which is
released from the larger dsRNA molecule by the action of cleavage enzyme
systems, including Dicer.
[00872] In some embodiments, the method comprises transient alteration of
protein expression in a
population of TILs, including TILs modified to express a CCR, comprising the
use of self-delivering
RNA interference (sdRNA), which is for example, a chemically-synthesized
asymmetric siRNA
duplex with a high percentage of 2'-OH substitutions (typically fluorine or -
OCH3) which comprises a
20-nucleotide antisense (guide) strand and a 13 to 15 base sense (passenger)
strand conjugated to
cholesterol at its 3' end using a tetraethylenglycol (TEG) linker. Methods of
using siRNA and sdRNA
have been described in Khvorova and Watts, Nat. Biotechnol. 2017, 35, 238-248;
Byrne, etal., I
Ocul. Pharmcwol. Ther. 2013, 29, 855-864; and Ligtenberg, et al., Mol.
Therapy, 2018, 26, 1482-93,
the disclosures of which are incorporated by reference herein. In some
embodiments, delivery of
siRNA is accomplished using electroporation or cell membrane disruption (such
as the squeeze or
SQZ method). In some embodiments, delivery of sdRNA to a TIL population is
accomplished without
use of electroporation, SQZ, or other methods, instead using a 1 to 3 day
period in which a TIL
population is exposed to sdRNA at a concentration of 1 01/10,000 TILs in
medium. In certain
embodiments, the method comprises delivery or siRNA or sdRNA to a TILs
population comprising
exposing the TILs population to sdRNA at a concentration of 1 1.tM/10,000 TILs
in medium for a
period of between 1 to 3 days. In some embodiments, delivery of sdRNA to a TIL
population is
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accomplished using a 1 to 3 day period in which a TIL population is exposed to
sdRNA at a
concentration of 10 piM/10,000 TILs in medium. In some embodiments, delivery
of sdRNA to a TIL
population is accomplished using a 1 to 3 day period in which a Tit population
is exposed to sdRNA
at a concentration of 50 ptM/10,000 TILs in medium. In some embodiments,
delivery of sdRNA to a
TIL population is accomplished using a 1 to 3 day period in which a TIL
population is exposed to
sdRNA at a concentration of between 0.1 M/10,000 Ls and 50 1jM/10,000Ls in
medium. In
some embodiments, delivery of sdRNA to a TIL population is accomplished using
a 1 to 3 day period
in which a TIL population is exposed to sdRNA at a concentration of between
0.1 ptM/10,000 TILs
and 50 iuM/10,000 TILs in medium, wherein the exposure to sdRNA is performed
two, three, four, or
five times by addition of fresh sdRNA to the media. Other suitable processes
are described, for
example, in U.S. Patent Application Publication No. US 2011/0039914 Al, US
2013/0131141 Al,
and US 2013/0131142 Al, and U.S. Patent No. 9,080,171, the disclosures of
which are incorporated
by reference herein.
1008731 In some embodiments, siRNA or sdRNA is inserted into a population of
TILs during
manufacturing. In some embodiments, the sdRNA encodes RNA that interferes with
NOTCH 1/2
1CD, PD-1, CTLA-4 TIM-3, LAG-3, TIG1T, TGF13, TGFBR2, cAMP protein kinase A
(PKA), BAFF
BR3, CISH, and/or CBLB. In some embodiments, the reduction in expression is
determined based on
a percentage of gene silencing, for example, as assessed by flow cytometry
and/or qPCR. In some
embodiments, there is a reduction in expression of about 5%, about 10%, about
10%, about 20%,
about 25%, about 30%, about 35%, about 40%, about 45%, about 50%, about 55%,
about 60%, about
65%, about 70%, about 75%, about 80%, about 85%, about 90%, or about 95%. In
some
embodiments, there is a reduction in expression of at least about 65%, about
70%, about 75%, about
80%, about 85%, about 90%, or about 95%. In some embodiments, there is a
reduction in expression
of at least about 75%, about 80%, about 85%, about 90%, or about 95%. In some
embodiments, there
is a reduction in expression of at least about 80%, about 85%, about 90%, or
about 95%. In some
embodiments, there is a reduction in expression of at least about 85%, about
90%, or about 95%. In
some embodiments, there is a reduction in expression of at least about 80%. In
some embodiments,
there is a reduction in expression of at least about 85%, In some embodiments,
there is a reduction in
expression of at least about 90%. In some embodiments, there is a reduction in
expression of at least
about 95%. In some embodiments, there is a reduction in expression of at least
about 99%.
1008741 The self-deliverable RNAi technology based on the chemical
modification of siRNAs can
be employed with the methods of the present invention to successfully deliver
the sdRNAs to the TILs
as described herein. The combination of backbone modifications with asymmetric
siRNA structure
and a hydrophobic ligand (see, for example, Ligtenberg, et al.,Mol. Therapy,
2018, 26, 1482-93 and
U.S. Patent Application Publication No. 2016/0304873 Al, the disclosures of
which are incorporated
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by reference herein) allow sdRNAs to penetrate cultured mammalian cells
without additional
formulations and methods by simple addition to the culture media, capitalizing
on the nuclease
stability of sdRNAs. This stability allows the support of constant levels of
RNAi-mediated reduction
of target gene activity simply by maintaining the active concentration of
sdRNA in the media. While
not being bound by theory, the backbone stabilization of sdRNA provides for
extended reduction in
gene expression effects which can last for months in non-dividing cells.
[00875] In some embodiments, over 95% transfection efficiency of TILs and a
reduction in
expression of the target by various specific siRNAs or sdRNAs occurs. Tn some
embodiments,
siRNAs or sdRNAs containing several unmodified ribose residues were replaced
with fully modified
sequences to increase potency and/or the longevity of RNAi effect. In some
embodiments, a reduction
in expression effect is maintained for 12 hours, 24 hours, 36 hours, 48 hours,
5 days, 6 days, 7 days,
or 8 days or more. In some embodiments, the reduction in expression effect
decreases at 10 days or
more post siRNAs or sdRNA treatment of the TILs. In some embodiments, more
than 70% reduction
in expression of the target expression is maintained. In some embodiments,
more than 70% reduction
in expression of the target expression is maintained TILs. In some
embodiments, a reduction in
expression in the PD-1/PD-L1 pathway allows for the TILs to exhibit a more
potent in vivo effect,
which is in some embodiments, due to the avoidance of the suppressive effects
of the PD-1/PD-L1
pathway. In some embodiments, a reduction in expression of PD-1 by siRNAs or
sdRNA results in an
increase TIL proliferation.
[00876]
[00877] In some embodiments, the sdRNA sequences used in the invention exhibit
a 70% reduction
in expression of the target gene. In some embodiments, the sdRNA sequences
used in the invention
exhibit a 75% reduction in expression of the target gene.
In some embodiments, the sdRNA sequences used in the invention exhibit an 80%
reduction in
expression of the target gene. In some embodiments, the sdRNA sequences used
in the invention
exhibit an 85% reduction in expression of the target gene. In some
embodiments, the sdRNA
sequences used in the invention exhibit a 90% reduction in expression of the
target gene. In some
embodiments, the sdRNA sequences used in the invention exhibit a 95% reduction
in expression of
the target gene. In some embodiments, the sdRNA sequences used in the
invention exhibit a 99%
reduction in expression of the target gene. In some embodiments, the sdRNA
sequences used in the
invention exhibit a reduction in expression of the target gene when delivered
at a concentration of
about 0.25 M to about 4 M. In some embodiments, the sdRNA sequences used in
the invention
exhibit a reduction in expression of the target gene when delivered at a
concentration of about 0.25
M. In some embodiments, the sdRNA sequences used in the invention exhibit a
reduction in
expression of the target gene when delivered at a concentration of about 0.5
M. In some
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embodiments, the sdRNA sequences used in the invention exhibit a reduction in
expression of the
target gene when delivered at a concentration of about 0.75 p.M. In some
embodiments, the sdRNA
sequences used in the invention exhibit a reduction in expression of the
target gene when delivered at
a concentration of about 1.0 M. In some embodiments, the sdRNA sequences used
in the invention
exhibit a reduction in expression of the target gene when delivered at a
concentration of about 1.25
M. In some embodiments, the sdRNA sequences used in the invention exhibit a
reduction in
expression of the target gene when delivered at a concentration of about 1.5
M. In some
embodiments, the sdRNA sequences used in the invention exhibit a reduction in
expression of the
target gene when delivered at a concentration of about 1.75 MM. In some
embodiments, the sdRNA
sequences used in the invention exhibit a reduction in expression of the
target gene when delivered at
a concentration of about 2.0 M. In some embodiments, the sdRNA sequences used
in the invention
exhibit a reduction in expression of the target gene when delivered at a
concentration of about 2.25
M. In some embodiments, the sdRNA sequences used in the invention exhibit a
reduction in
expression of the target gene when delivered at a concentration of about 2.5
M. In some
embodiments, the sdRNA sequences used in the invention exhibit a reduction in
expression of the
target gene when delivered at a concentration of about 2.75 M. In some
embodiments, the sdRNA
sequences used in the invention exhibit a reduction in expression of the
target gene when delivered at
a concentration of about 3.0 M. In some embodiments, the sdRNA sequences used
in the invention
exhibit a reduction in expression of the target gene when delivered at a
concentration of about 3.25
M. In some embodiments, the sdRNA sequences used in the invention exhibit a
reduction in
expression of the target gene when delivered at a concentration of about 3.5
M. In some
embodiments, the sdRNA sequences used in the invention exhibit a reduction in
expression of the
target gene when delivered at a concentration of about 3.75 MM. In some
embodiments, the sdRNA
sequences used in the invention exhibit a reduction in expression of the
target gene when delivered at
a concentration of about 4.0 M.
1008781 In some emodiments, the siRNA or sdRNA oligonucleotide agents comprise
one or more
modification to increase stability and/or effectiveness of the therapeutic
agent, and to effect efficient
delivery of the oligonucleotide to the cells or tissue to be treated. Such
modifications can include a 2'-
0-methyl modification, a 2'-0-fluro modification, a diphosphorothioate
modification, 2' F modified
nucleotide, a2'-0-methyl modified and/or a 2'deoxy nucleotide. In some
embodiments, the
oligonucleotide is modified to include one or more hydrophobic modifications
including, for example,
sterol, cholesterol, vitamin D, naphtyl, isobutyl, benzyl, indol, tryptophane,
and/or phenyl. In asome
embodiments, chemically modified nucleotides are combination of
phosphorothioates, 2'-0-methyl,
2'deoxy, hydrophobic modifications and phosphorothioates. In some embodiments,
the sugars can be
modified and modified sugars can include but are not limited to D-ribosc, 2'-0-
alkyl (including 2'-0-
methyl and 2'-0-ethyl), i.e., 2'-alkoxy, 2'-amino, 2'-S-alkyl, 2'-halo
(including 2'-fluoro), T-
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methoxyethoxy, 2'-allyloxy (-0CH2CH=CH2), 2'-propargyl, 2'-propyl, ethynyl,
ethenyl, propenyl, and
cyano and the like. In some embodiments, the sugar moiety can be a hexose and
incorporated into an
oligonucleotide as described in Augustyns, et al., Nucl. Acids. Res. 1992, 18,
4711, the disclosure of
which is incorporated by reference herein.
[00879] In some embodiments, the double-stranded siRNA or sdRNA
oligonucleotide of the
invention is double-stranded over its entire length, i.e., with no overhanging
single-stranded sequence
at either end of the molecule, i.e., is blunt-ended. In some embodiments, the
individual nucleic acid
molecules can be of different lengths. In other words, a double-stranded siRNA
or sdRNA
oligonucleotide of the invention is not double-stranded over its entire
length. For instance, when two
separate nucleic acid molecules are used, one of the molecules, e.g., the
first molecule comprising an
antisense sequence, can be longer than the second molecule hybridizing thereto
(leaving a portion of
the molecule single-stranded). In some embodiments, when a single nucleic acid
molecule is used a
portion of the molecule at either end can remain single-stranded.
[00880] In some embodiments, a double-stranded siRNA or sdRNA oligonucleotide
of the invention
contains mismatches and/or loops or bulges, but is double-stranded over at
least about 70% of the
length of the oligonucleotide. In some embodiments, a double-stranded
oligonucleotide of the
invention is double-stranded over at least about 80% of the length of the
oligonucleotide. In other
embodiments, a double-stranded siRNA or sdRNA oligonucleotide of the invention
is double-
stranded over at least about 90%-95% of the length of the oligonucleotide. In
some embodiments, a
double-stranded oligonucleotide of the invention is double-stranded over at
least about 96%-98% of
the length of the oligonucicotidc. In some embodiments, the double-stranded
siRNA or sdRNA
oligonucleotide of the invention contains at least or up to 1, 2, 3, 4, 5, 6,
7, 8, 9, 10, 11, 12, 13, 14, or
15 mismatches.
[00881] In some embodiments, the siRNA or sdRNA oligonucleotide can be
substantially protected
from nucleases e.g., by modifying the 3' or 5' linkages, as described in U.S.
Patent. No. 5,849,902, the
disclosure of which is incorporated by reference herein). For example,
oligonucleotides can be made
resistant by the inclusion of a "blocking group." The term "blocking group" as
used herein refers to
substituents (e.g., other than OH groups) that can be attached to
oligonucleotides or nucleomonomers,
either as protecting groups or coupling groups for synthesis (e.g., FITC,
propyl (CH2-CH2-CH3),
glycol (-0-CH2-CH2-0-) phosphate (P032"), hydrogen phosphonate, or
phosphoramidite). "Blocking
groups" can also include "end blocking groups" or "exonuclease blocking
groups" which protect the
5' and 3' termini of the oligonucleotide, including modified nucleotides and
non-nucleotide
exonuclease resistant structures.
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[00882] In some embodiments, at least a portion of the contiguous
polynucleotides within the
siRNA or sdRNA are linked by a substitute linkage, e.g., a phosphorothioate
linkage.
[00883] In some embodiments, chemical modification can lead to at least a 1.5,
2, 3, 4, 5, 6, 7, 8, 9,
10, 15, 20, 25, 30, 35, 40, 45, 50, 55, 60, 65, 70, 75, 80, 85, 90, 95, 100,
105, 110, 115, 120, 125, 130,
135, 140, 145, 150, 155, 160, 165, 170, 175, 180, 185, 190, 195, 200, 225,
250, 275, 300, 325, 350,
375, 400, 425, 450, 475, or 500 percent in cellular uptake of an siRNA or
sdRNA. In some
embodiments, at least one of the C or U residues includes a hydrophobic
modification. In some
embodiments, a plurality of Cs and Us contain a hydrophobic modification. In
some embodiments, at
least 10%, 15%, 20%, 30%, 40%, 50%, 55%, 60% 65%, 70%, 75%, 80%, 85%, 90% or
at least 95%
of the Cs and Us can contain a hydrophobic modification. In some embodiments,
all of the Cs and Us
contain a hydrophobic modification.
[00884] In some embodiments, the siRNA or sdRNA molecules exhibit enhanced
endosomal release
through the incorporation of protonatable amines. In some embodiments,
protonatable amines are
incorporated in the sense strand (in the part of the molecule which is
discarded after RISC loading). In
some embodiments, the siRNA or sdRNA compounds of the invention comprise an
asymmetric
compound comprising a duplex region (required for efficient RISC entry of 10-
15 bases long) and
single stranded region of 4-12 nucleotides long; with a 13 nucleotide duplex.
In some embodiments, a
6 nucleotide single stranded region is employed. In some embodiments, the
single stranded region of
the siRNA or sdRNA comprises 2-12 phosphorothioate internucleotide linkages
(referred to as
phosphorothioate modifications). In some embodiments, 6-8 phosphorothioate
internucleotide
linkages are employed. In some embodiments, the siRNA or sdRNA compounds of
the invention also
include a unique chemical modification pattern, which provides stability and
is compatible with RISC
entry.
[00885] The guide strand, for example, may also be modified by any chemical
modification which
confirms stability without interfering with RISC entry. In some embodiments,
the chemical
modification pattern in the guide strand includes the majority of C and U
nucleotides being 2' F
modified and the 5 'end being phosphorylated.
[00886] In some embodiments, at least 30% of the nucleotides in the siRNA or
sdRNA ae modified.
In some embodiments, at least 30%, 31%, 32%, 33%, 34%, 35%, 36%, 37%, 38%,
39%, 40%, 41%,
42%, 43%, 44%, 45%, 46%, 47%, 48%, 49%, 50%, 51%, 52%, 53%, 54%, 55%, 56%,
57%, 58%,
59%, 60%, 61%, 62%, 63%, 64%, 65%, 66%, 67%, 68%, 69%, 70%, 71%, 72%, 73%,
74%, 75%,
76%, 77%, 78%, 79%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%,
91%, 92%,
93%, 94%, 95%, 96%, 97%, 98% or 99% of the nucleotides in the siRNA or sdRNA
are modified. In
some embodiments, 100% of the nucleotides in the siRNA or sdRNA are modified.
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[00887] In some embodiments, the sdRNA molecules have minimal double stranded
regions. In
some embodiments the region of the molecule that is double stranded ranges
from 8-15 nucleotides
long. In some embodiments, the region of the molecule that is double stranded
is 8, 9, 10, 11, 12, 13,
14 or 15 nucleotides long. In some embodiments the double stranded region is
13 nucleotides long.
There can be 100% complementarity between the guide and passenger strands, or
there may be one or
more mismatches between the guide and passenger strands. In some embodiments,
on one end of the
double stranded molecule, the molecule is either blunt-ended or has a one-
nucleotide overhang. The
single stranded region of the molecule is in some embodiments between 4-12
nucleotides long. In
some embodiments, the single stranded region can be 4, 5, 6, 7, 8, 9, 10, 11
or 12 nucleotides long. In
some embodiments, the single stranded region can also be less than 4 or
greater than 12 nucleotides
long. In certain embodiments, the single stranded region is 6 or 7 nucleotides
long.
[00888] In some embodiments, the siRNA or sdRNA molecules have increased
stability. In some
instances, a chemically modified siRNA or sdRNA molecule has a half-life in
media that is longer
than 1,2, 3,4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20,
21,22, 23, 24 or more than 24
hours, including any intermediate values. In some embodiments, the siRNA or
sdRNA has a half-life
in media that is longer than 12 hours.
[00889] In some embodiments, the siRNA or sdRNA is optimized for increased
potency and/or
reduced toxicity. In some embodiments, nucleotide length of the guide and/or
passenger strand, and/or
the number of phosphorothioate modifications in the guide and/or passenger
strand, can in some
aspects influence potency of the RNA molecule, while replacing 2'-fluoro (2'F)
modifications with 2'-
0-methyl (2'0Me) modifications can in some aspects influence toxicity of the
molecule. In some
embodiments, reduction in 2'F content of a molecule is predicted to reduce
toxicity of the molecule. In
some embodiments, the number of phosphorothioate modifications in an RNA
molecule can influence
the uptake of the molecule into a cell, for example the efficiency of passive
uptake of the molecule
into a cell. In some embodiments, the siRNA or sdRNA has no 2'F modification
and yet are
characterized by equal efficacy in cellular uptake and tissue penetration.
[00890] In some embodiments, a guide strand is approximately 18-19 nucleotides
in length and has
approximately 2-14 phosphate modifications. For example, a guide strand can
contain 2, 3, 4, 5, 6, 7,
8, 9, 10, 11, 12, 13, 14 or more than 14 nucleotides that are phosphate-
modified. The guide strand
may contain one or more modifications that confer increased stability without
interfering with RISC
entry. The phosphate modified nucleotides, such as phosphorothioate modified
nucleotides, can be at
the 3' end, 5' end or spread throughout the guide strand. In some embodiments,
the 3' terminal 10
nucleotides of the guide strand contain 1, 2, 3, 4, 5, 6, 7, 8, 9 or 10
phosphorothioate modified
nucleotides. The guide strand can also contain 2'F and/or 2'0Me modifications,
which can be located
throughout the molecule. In some embodiments, the nucleotide in position one
of the guide strand (the
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nucleotide in the most 5' position of the guide strand) is 2'0Me modified
and/or phosphorylated. C
and U nucleotides within the guide strand can be 2'F modified. For example, C
and U nucleotides in
positions 2-10 of a 19 nt guide strand (or corresponding positions in a guide
strand of a different
length) can be 2'F modified. C and U nucleotides within the guide strand can
also be 2'0Me modified.
For example, C and U nucleotides in positions 11-18 of a 19 nt guide strand
(or corresponding
positions in a guide strand of a different length) can be 2'0Me modified. In
some embodiments, the
nucleotide at the most 3' end of the guide strand is unmodified. In certain
embodiments, the majority
of Cs and Us within the guide strand are 2'F modified and the 5' end of the
guide strand is
phosphorylated. In other embodiments, position 1 and the Cs or Us in positions
11-18 are 2'0Me
modified and the 5' end of the guide strand is phosphorylated. In other
embodiments, position 1 and
the Cs or Us in positions 11-18 are 2'0Me modified, the 5' end of the guide
strand is phosphorylated,
and the Cs or Us in position 2-10 are 2'F modified.
[00891] The self-deliverable RNAi technology provides a method of directly
transfecting cells with
the RNAi agent (whether siRNA, sdRNA, or other RNAi agents), without the need
for additional
formulations or techniques. The ability to transfect hard-to-transfect cell
lines, high in vivo activity,
and simplicity of use, are characteristics of the compositions and methods
that present significant
functional advantages over traditional siRNA-based techniques, and as such,
the sdRNA methods are
employed in several embodiments related to the methods of reduction in
expression of the target gene
in the TILs of the present invention. The sdRNA method allows direct delivery
of chemically
synthesized compounds to a wide range of primary cells and tissues, both es-
viva and in vivo. The
sdRNAs described in some embodiments of the invention herein are commercially
available from
Advirna LLC, Worcester, MA, USA.
[00892] The siRNA or sdRNA may be formed as hydrophobically-modified siRNA-
antisense
oligonucleotide hybrid structures, and are disclosed, for example in Byrne ,
et al., J. Ocular
Phartnacol. Therape lit. 2013, 29, 855-864, the disclosure of which is
incorporated by reference
herein.
[00893] In some embodiments, the siRNA or sdRNA oligonucleotides can be
delivered to the TILs
described herein using sterile electroporation. In certain embodiments, the
method comprises sterile
electroporation of a population of TILs to deliver siRNA or sdRNA
oligonucleotides.
[00894] In some embodiments, the oligonucleotides can be delivered to the
cells in combination
with a transmembrane delivery system. In some embodiments, this transmembrane
delivery system
comprises lipids, viral vectors, and the like. In some embodiments, the
oligonucleotide agent is a self-
delivery RNAi agent, that does not require any delivery agents. In certain
embodiments, the method
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comprises use of a transmembrane delivery system to deliver siRNA or sdRNA
oligonucleotides to a
population of TILs.
[00895] Oligonucleotides and oligonucleotide compositions are contacted with
(e.g., brought into
contact with, also referred to herein as administered or delivered to) and
taken up by TILs described
herein, including through passive uptake by TILs. The sdRNA can be added to
the TILs as described
herein during the first expansion, for example Step B, after the first
expansion, for example, during
Step C, before or during the second expansion, for example before or during
Step D, after Step D and
before harvest in Step F, during or after harvest in Step F, before or during
final formulation and/or
transfer to infusion Bag in Step F, as well as before any optional
cryopreservation step in Step F.
Moreeover, sdRNA can be added after thawing from any cryopreservation step in
Step F. In some
embodiments, one or more sdRNAs targeting genes as described herein, including
PD-1, LAG-3,
TIM-3, CISH, CTLA-4, TIGIT, TET2 and CBLB, may be added to cell culture media
comprising
TILs and other agents at concentrations selected from the group consisting of
100 nM to 20 mM, 200
nM to 10 mM, 500 nm to 1 mM, I !AM to 100 JAM, and 1 p..M to 100 JAM. In some
embodiments, one
or more sdRNAs targeting genes as described herein, including PD-1, LAG-3, TIM-
3, CISH, CTLA-
4, TIG1T, TET2 and CBLB, may be added to cell culture media comprising TILs
and other agents at
amounts selected from the group consisting of 0.1 04 sdRNA/10,000 TILs/100
i.t.L media, 0.5 uM
sdRNA/10,000 TILs /1004 media, 0.75 vt,M sdRNA/10,000 TILs /1001AL media, 1 gM
sdRNA/10,000 TILs /100 L media, 1.25 vt,M sdRNA/10,000 TILs /1004 media, 1.5
1.11µ4
sdRNA/10,000 TILs /100 fiL media, 2 inM sdRNA/10,000 TiLs /100 [IL media, 5
viM sdRNA/10,000
TILs /100 1AL media, or 10 i.tM sdRNA/10,000 TILs /100 )1L media. In some
embodiments, one or
more sdRNAs targeting genes as described herein, including PD-1, LAG-3, TIM-3,
CISH, CTLA-4,
TIG1T, TET2 and CBLB, may be added to T1L cultures during the pre-REP or REP
stages twice a
day, once a day, every two days, every three days, every four days, every five
days, every six days, or
every seven days.
[00896] Oligonucleotide compositions of the invention, including sdRNA, can be
contacted with
TILs as described herein during the expansion process, for example by
dissolving sdRNA at high
concentrations in cell culture media and allowing sufficient time for passive
uptake to occur. In
certain embodiments, the method of the present invention comprises contacting
a population of TILs
with an oligonucleotide composition as described herein. In certain
embodiments, the method
comprises dissolving an oligonucleotide e.g. sdRNA in a cell culture media and
contacting the cell
culture media with a population of TILs. The TILs may be a first population, a
second population
and/or a third population as described herein.
[00897] In some embodiments, delivery of oligonucleotides into cells can be
enhanced by suitable
art recognized methods including calcium phosphate, DMSO, glycerol or dextran,
electroporation, or
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by transfection, e.g., using cationic, anionic, or neutral lipid compositions
or liposomes using methods
known in the art, such as those methods described in U.S. Patent Nos.
4,897,355; 5,459,127;
5,631,237; 5,955,365; 5,976,567; 10,087,464; and 10,155,945; and Bergan, et
al., Nucl. Acids Res.
1993, 21, 3567, the disclosures of each of which are incorporated by reference
herein.
1008981 In some embodiments, more than one siRNA or sdRNA is used to reduce
expression of a
target gene. In some embodiments, one or more of PD-1, TIM-3, CBLB, LAG3, CTLA-
4, TIGIT,
TET2 and/or CISH targeting siRNA or sdRNAs are used together. In some
embodiments, a PD-1
siRNA or sdRNA is used with one or more of TIM-3, CBLB, LAG3, TIGIT,
TET2 and/or
CISH in order to reduce expression of more than one gene target. In some
embodiments, a LAG3
siRNA or sdRNA is used in combination with a CISH targeting siRNA or sdRNA to
reduce gene
expression of both targets. In some embodiments, the siRNA or sdRNAs targeting
one or more of PD-
1, TIM-3, CBLB, LAG3, CTLA-4, TIGIT, TET2 and/or CISH herein are commercially
available
from Advirna LLC, Worcester, MA, USA.
1008991 In some embodiments, the siRNA or sdRNA targets a gene selected from
the group
consisting of PD-1, LAG3, TIM3, CTLA-4, TIGIT, TET2, CISH, TGF13R2, PKA, CBLB,
BAFF
(BR3), and combinations thereof. In some embodiments, the siRNA or sdRNA
targets a gene selected
from the group consisting of PD-1, LAG3, TIM3, CTLA-4, TIGIT, TET2, CISH,
TGFf3R2, PKA,
CBLB, BAFF (BR3), and combinations thereof, in some embodiments, one siRNA or
sdRNA targets
PD-1 and another siRNA or sdRNA targets a gene selected from the group
consisting of LAG3,
TIM3, CTLA-4, TIGIT, TET2, CISH, TGFf3R2, PKA, CBLB, BAFF (BR3), and
combinations
thereof In some embodiments, the siRNA or sdRNA targets a gene selected from
PD-1, LAG-3,
CISH, CBLB, TIM3, CTLA-4, TIGIT, TET2 and combinations thereof. In some
embodiments, the
siRNA or sdRNA targets a gene selected from PD-1 and one of LAG3, CISH, CBLB,
TIM3. CTLA-
4, TIGIT, TET2 and combinations thereof. In some embodiments, one siRNA or
sdRNA targets PD-1
and one siRNA or sdRNA targets LAG3. In some embodiments, one siRNA or sdRNA
targets PD-1
and one siRNA or sdRNA targets CISH. In some embodiments, one siRNA or sdRNA
targets PD-1
and one siRNA or sdRNA targets CBLB. In some embodiments, one siRNA or sdRNA
targets PD-1
and one siRNA or sdRNA targets TIM3. In some embodiments, one siRNA or sdRNA
targets PD-1
and one siRNA or sdRNA targets CTLA-4. In some embodiments, one siRNA or sdRNA
targets PD-1
and one siRNA or sdRNA targets TiGIT. In some embodiments, one siRNA or sdRNA
targets PD-1
and one siRNA or sdRNA targets TET2. In some embodiments, one siRNA or sdRNA
targets LAG3
and one siRNA or sdRNA targets CISH. In some embodiments, one siRNA or sdRNA
targets LAG3
and one siRNA or sdRNA targets CBLB. In some embodiments, one siRNA or sdRNA
targets LAG3
and one siRNA or sdRNA targets TIM3. In some embodiments, one siRNA or sdRNA
targets LAG3
and one siRNA or sdRNA targets CTLA-4. In some embodiments, one siRNA or sdRNA
targets
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LAG3 and one siRNA or sdRNA targets TIGIT. In some embodiments, one siRNA or
sdRNA targets
LAG3 and one siRNA or sdRNA targets TET2. In some embodiments, one siRNA or
sdRNA targets
CISH and one siRNA or sdRNA targets CBLB. in some embodiments, one siRNA or
sdRNA targets
CISH and one siRNA or sdRNA targets TIM3. In some embodiments, one siRNA or
sdRNA targets
CISH and one siRNA or sdRNA targets CTLA-4. In some embodiments, one siRNA or
sdRNA
targets CISH and one siRNA or sdRNA targets TIGIT. In some embodiments, one
siRNA or sdRNA
targets CISH and one siRNA or sdRNA targets TET2. In some embodiments, one
siRNA or sdRNA
targets CBLB and one siRNA or sdRNA targets TIM3. In some embodiments, one
siRNA or sdRNA
targets CBLB and one siRNA or sdRNA targets CTLA-4. In some embodiments, one
siRNA or
sdRNA targets CBLB and one siRNA or sdRNA targets TIGIT. In some embodiments,
one siRNA or
sdRNA targets CBLB and one siRNA or sdRNA targets TET2. In some embodiments,
one siRNA or
sdRNA targets TIM3 and one siRNA or sdRNA targets PD-1. In some embodiments,
one siRNA or
sdRNA targets TIM3 and one siRNA or sdRNA targets LAG3. In some embodiments,
one siRNA or
sdRNA targets TIM3 and one siRNA or sdRNA targets CISH. In some embodiments,
one siRNA or
sdRNA targets TIM3 and one siRNA or sdRNA targets CBLB. In some embodiments,
one siRNA or
sdRNA targets TIM3 and one siRNA or sdRNA targets CTLA-4. In some embodiments,
one siRNA
or sdRNA targets TIM3 and one siRNA or sdRNA targets TIGIT. In some
embodiments, one siRNA
or sdRNA targets TIM3 and one siRNA or sdRNA targets TET2. In some
embodiments, one siRNA
or sdRNA targets CTLA-4 and one siRNA or sdRNA targets TIGIT. In some
embodiments, one
siRNA or sdRNA targets CTLA-4 and one siRNA or sdRNA targets TET2. In some
embodiments,
one siRNA or sdRNA targets TIGIT and one siRNA or sdRNA targets TET2.
[00900] As discussed herein, embodiments of the present invention provide
tumor infiltrating
lymphocytes (TILs) that have been genetically modified via gene-editing to
enhance their therapeutic
effect. Embodiments of the present invention embrace genetic editing through
nucleotide insertion
(RNA or DNA) into a population of TILs for both promotion of the expression of
one or more
proteins and inhibition of the expression of one or more proteins, as well as
combinations thereof.
Embodiments of the present invention also provide methods for expanding TILs
into a therapeutic
population, wherein the methods comprise gene-editing the TILs. There are
several gene-editing
technologies that may be used to genetically modify a population of TILs,
which are suitable for use
in accordance with the present invention.
[00901] In some embodiments, the method comprises a method of genetically
modifying a
population of TILs in a first population, a second population and/or a third
population as described
herein. In some embodiments, a method of genetically modifying a population of
TILs includes the
step of stable incorporation of genes for production or inhibition (e.g.,
silencing) of one ore more
proteins. In some embodiments, a method of genetically modifying a population
of TILs includes the
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step of electroporation. Electroporation methods are known in the art and are
described, e.g., in
Tsong, Biophys. J. 1991, 60, 297-306, and U.S. Patent Application Publication
No. 2014/0227237 Al,
the disclosures of each of which are incorporated by reference herein. Other
electroporation methods
known in the art, such as those described in U.S. Patent Nos. 5,019,034;
5,128,257; 5,137,817;
5,173,158; 5,232,856; 5,273;525; 5,304,120; 5,318,514; 6,010,613 and
6,078;490, the disclosures of
which arc incorporated by reference herein, may be used. In some embodiments,
the electroporation
method is a sterile electroporation method. In some embodiments, the
electroporation method is a
pulsed electroporation method. In some embodiments, the electroporation method
is a pulsed
electroporation method comprising the steps of treating TILs with pulsed
electrical fields to alter,
manipulate, or cause defined and controlled, permanent or temporary changes in
the TILs, comprising
the step of applying a sequence of at least three single, operator-controlled,
independently
programmed, DC electrical pulses, having field strengths equal to or greater
than 100 V/cm, to the
TILs, wherein the sequence of at least three DC electrical pulses has one,
two, or three of the
following characteristics: (1) at least two of the at least three pulses
differ from each other in pulse
amplitude; (2) at least two of the at least three pulses differ from each
other in pulse width; and (3) a
first pulse interval for a first set of two of the at least three pulses is
different from a second pulse
interval for a second set of two of the at least three pulses. In some
embodiments, the electroporation
method is a pulsed electroporation method comprising the steps of treating
TILs with pulsed electrical
fields to alter, manipulate, or cause defined and controlled, permanent or
temporary changes in the
TILs, comprising the step of applying a sequence of at least three single,
operator-controlled,
independently programmed, DC electrical pulses, having field strengths equal
to or greater than 100
V/cm, to the TILs, wherein at least two of the at least three pulses differ
from each other in pulse
amplitude. In some embodiments, the electroporation method is a pulsed
electroporation method
comprising the steps of treating TILs with pulsed electrical fields to alter,
manipulate, or cause
defined and controlled, permanent or temporary changes in the TILs, comprising
the step of applying
a sequence of at least three single, operator-controlled, independently
programmed; DC electrical
pulses, having field strengths equal to or greater than 100 V/cm, to the TILs,
wherein at least two of
the at least three pulses differ from each other in pulse width. In some
embodiments, the
electroporation method is a pulsed electroporation method comprising the steps
of treating TILs with
pulsed electrical fields to alter, manipulate, or cause defined and
controlled, permanent or temporary
changes in the TILs, comprising the step of applying a sequence of at least
three single, operator-
controlled, independently programmed, DC electrical pulses, having field
strengths equal to or greater
than 100 V/cm, to the TILs, wherein a first pulse interval for a first set of
two of the at least three
pulses is different from a second pulse interval for a second set of two of
the at least three pulses. In
some embodiments, the electroporation method is a pulsed electroporation
method comprising the
steps of treating TILs with pulsed electrical fields to induce pore formation
in the TILs, comprising
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the step of applying a sequence of at least three DC electrical pulses, having
field strengths equal to or
greater than 100 V/cm, to TILs, wherein the sequence of at least three DC
electrical pulses has one,
two, or three of the following characteristics: (1) at least two of the at
least three pulses differ from
each other in pulse amplitude; (2) at least two of the at least three pulses
differ from each other in
pulse width; and (3) a first pulse interval for a first set of two of the at
least three pulses is different
from a second pulse interval for a second set of two of the at least three
pulses, such that induced
pores are sustained for a relatively long period of time, and such that
viability of the TILs is
maintained. In some embodiments, a method of genetically modifying a
population of TILs includes
the step of calcium phosphate transfection. Calcium phosphate transfection
methods (calcium
phosphate DNA precipitation, cell surface coating, and endocytosis) are known
in the art and are
described in Graham and van der Eb, Virology 1973, 52, 456-467; Wigler, et al.
, Proc. Natl. Acad.
Sci. 1979, 76, 1373-1376; and Chen and Okayarea,Mol. Cell. Biol. 1987, 7, 2745-
2752; and in U.S.
Patent No. 5,593,875, the disclosures of each of which arc incorporated by
reference herein. In some
embodiments, a method of genetically modifying a population of TILs includes
the step of liposomal
transfection. Liposomal transfection methods, such as methods that employ a
1:1 (w/w) liposome
formulation of the cationic lipid Nt 1-(2,3-dioleyloxy)propy1]-71,71,71-
trimethylammonium chloride
(DOTMA) and dioleoyl phophotidylethanolamine (DOPE) in filtered water, are
known in the art and
are described in Rose, etal., Biotechniques 1991, /0, 520-525 and Felgner,
etal., Proc. Natl. Acad.
Sci. USA, 1987, 84, 7413-7417 and in U.S. Patent Nos. 5,279,833; 5,908,635;
6,056,938; 6,110,490;
6,534,484; and 7,687,070, the disclosures of each of which are incorporated by
reference herein. In
some embodiments, a method of genetically modifying a population of TILs
includes the step of
transfection using methods described in U.S. Patent Nos. 5,766,902; 6,025,337;
6,410,517; 6,475,994;
and 7,189,705; the disclosures of each of which are incorporated by reference
herein. The TILs may
be a first population, a second population and/or a third population of TILs
as described herein.
1009021 According to an embodiment, the gene-editing process may comprise the
use of a
programmable nuclease that mediates the generation of a double-strand or
single-strand break at one
or more immune checkpoint genes. Such programmable nucleases enable precise
genome editing by
introducing breaks at specific genomic loci, i.e., they rely on the
recognition of a specific DNA
sequence within the genome to target a nuclease domain to this location and
mediate the generation of
a double-strand break at the target sequence. A double-strand break in the DNA
subsequently recruits
endogenous repair machinery to the break site to mediate genome editing by
either non-homologous
end-joining (NHEJ) or homology-directed repair (HDR). Thus, the repair of the
break can result in the
introduction of insertion/deletion mutations that disrupt (e.g., silence,
repress, or enhance) the target
gene product.
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[00903] Major classes of nucleases that have been developed to enable site-
specific genomic editing
include zinc finger nucleases (ZFNs), transcription activator-like nucleases
(TALENs), and CRISPR-
associated nucleases (e.g., CRISPR/Cas9). These nuclease systems can be
broadly classified into two
categories based on their mode of DNA recognition: ZFNs and TALENs achieve
specific DNA
binding via protein-DNA interactions, whereas CRISPR systems, such as Cas9,
are targeted to
specific DNA sequences by a short RNA guide molecule that base-pairs directly
with the target DNA
and by protein-DNA interactions. See, e.g., Cox et al., Nature Medicine, 2015,
Vol. 21, No. 2.
[00904] Non-limiting examples of gene-editing methods that may be used in
accordance with TIL
expansion methods of the present invention include CRISPR methods, TALE
methods, and ZFN
methods, which are described in more detail below. According to an embodiment,
a method for
expanding TILs into a therapeutic population may be carried out in accordance
with any embodiment
of the methods described herein (e.g., Gen 3) or as described in U.S. Patent
Application Publication
Nos. US 2020/0299644 Al and US 2020/0121719 Al and U.S. Patent No. 10,925,900,
the disclosures
of which are incorporated by reference herein, wherein the method further
comprises gene-editing at
least a portion of the TILs by one or more of a CRISPR method, a TALE method
or a ZFN method, in
order to generate TILs that can provide an enhanced therapeutic effect.
According to an embodiment,
gene-edited TILs can be evaluated for an improved therapeutic effect by
comparing them to non-
modified TILs in vitro, e.g., by evaluating in vitro effector function,
cytokine profiles, etc. compared
to unmodified TILs. In certain embodiments, the method comprises gene editing
a population of TILs
using CRISPR, TALE and/ or ZFN methods.
[00905] In some embodiments of the present invention, electroporation is used
for delivery of a
gene editing system, such as CRISPR, TALEN, and ZEN systems. In some
embodiments of the
present invention, the electroporation system is a flow electroporation
system. An example of a
suitable flow electroporation system suitable for use with some embodiments of
the present invention
is the commercially-available MaxCyte STX system. There are several
alternative commercially-
available electroporation instruments which may be suitable for use with the
present invention, such
as the AgilePul se system or ECM 830 available from BTX-Harvard Apparatus,
Cellaxess Elektra
(Cellectricon), Nucleofector (Lonza/Amaxa), GenePulser MXcell (BIORAD),
iPorator-96 (Primax) or
siPORTer96 (Ambion). In some embodiments of the present invention, the
electroporation system
forms a closed, sterile system with the remainder of the TIL expansion method.
In some embodiments
of the present invention, the electroporation system is a pulsed
electroporation system as described
herein, and forms a closed, sterile system with the remainder of the TIL
expansion method.
[00906] A method for expanding TILs into a therapeutic population may be
carried out in
accordance with any embodiment of the methods described herein (e.g., Gen 3)
or as described in
U.S. Patent Application Publication Nos. US 2020/0299644 Al and US
2020/0121719 Al and U.S.
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Patent No. 10,925,900, the disclosures of which are incorporated by reference
herein, wherein the
method further comprises gene-editing at least a portion of the TILs by a
CRISPR method (e.g.,
CRISPR/Cas9 or CRISPR/Cpfl). According to particular embodiments, the use of a
CRISPR method
during the TIL expansion process causes expression of one or more immune
checkpoint genes to be
silenced or reduced in at least a portion of the therapeutic population of
TILs. Alternatively, the use of
a CRISPR method during the T1L expansion process causes expression of one or
more immune
checkpoint genes to be enhanced in at least a portion of the therapeutic
population of TILs.
[00907] CRISPR stands for -clustered regularly interspaced short palindromic
repeats." A method
of using a CRISPR system for gene editing is also referred to herein as a
CRISPR method. There are
three types of CRISPR systems which incorporate RNAs and Cas proteins, and
which may be used in
accordance with the present invention: Types I, II, and III. The Type II
CRISPR (exemplified by
Cas9) is one of the most well-characterized systems.
[00908] CRISPR technology was adapted from the natural defense mechanisms of
bacteria and
archaea (the domain of single-celled microorganisms). These organisms use
CRISPR-derived RNA
and various Cas proteins, including Cas9, to foil attacks by viruses and other
foreign bodies by
chopping up and destroying the DNA of a foreign invader. A CRISPR is a
specialized region of DNA
with two distinct characteristics: the presence of nucleotide repeats and
spacers. Repeated sequences
of nucleotides are distributed throughout a CRISPR region with short segments
of foreign DNA
(spacers) interspersed among the repeated sequences. In the type II CRISPR/Cas
system, spacers are
integrated within the CRISPR genomic loci and transcribed and processed into
short CRISPR RNA
(crRNA). These crRNAs anneal to trans-activating crRNAs (tracrRNAs) and direct
sequence-specific
cleavage and silencing of pathogenic DNA by Cas proteins. Target recognition
by the Cas9 protein
requires a "seed- sequence within the crRNA and a conserved dinucleotide-
containing protospacer
adjacent motif (PAM) sequence upstream of the crRNA-binding region. The
CRISPR/Cas system can
thereby be retargeted to cleave virtually any DNA sequence by redesigning the
crRNA. The crRNA
and tracrRNA in the native system can be simplified into a single guide RNA
(sgRNA) of
approximately 100 nucleotides for usc in genetic engineering. The CRISPR/Cas
system is directly
portable to human cells by co-delivery of plasmids expressing the Cas9 endo-
nuclease and the
necessary crRNA components. Different variants of Cas proteins may be used to
reduce targeting
limitations (e.g., orthologs of Cas9, such as Cpfl).
[00909] Non-limiting examples of genes that may be silenced or inhibited by
permanently gene-
editing TILs via a CRISPR method include PD-1, CTLA-4, LAG-3, HAVCR2 (TIM-3),
Cish, TGFI3,
PKA, CBL-B, PPP2CA, PPP2CB, PTPN6, PTPN22, PDCD1, BTLA, CD160, TIGIT, TET2,
CD96,
CRTAM, LAIR1, SIGLEC7, SIGLEC9, CD244, TNFRSF10B, TNFRSF10A, CASP8, CASP10,
CASP3, CASP6, CASP7, FADD, FAS, SMAD2, SMAD3, SMAD4, SMAD10, SKI, SKIL, TGIF1,
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ILlORA, ILlORB, HMOX2, IL6R, IL6ST, EIF2AK4, CSK, PAG1, SIT1, FOXP3, PRDM1,
BATF,
GUCY1A2, GUCY1A3, GUCY1B2, GUCY1B3, TOX, SOCS1, ANKRD11, and BCOR.
[00910] Non-limiting examples of genes that may be enhanced by penuanently
gene-editing TILs
via a CRISPR method include CCR2, CCR4, CCR5, CXCR2, CXCR3, CX3CR1, 1L-2,
1L12, IL-15,
and IL-21.
[00911] Examples of systems, methods, and compositions for altering the
expression of a target
gene sequence by a CRISPR method, and which may be used in accordance with
embodiments of the
present invention, are described in U.S. Patent Nos, 8,697,359; 8,993,233;
8,795,965; 8,771,945;
8,889,356; 8,865,406; 8,999,641; 8,945,839; 8,932,814; 8,871,445; 8,906,616;
and 8,895,308, the
disclosures of each of which are incorporated by reference herein. Resources
for carrying out CRISPR
methods, such as plasmids for expressing CR1SPR/Cas9 and CRISPR/Cpfl, are
commercially
available from companies such as GenScript.
[00912] In some embodiments, genetic modifications of populations of TILs, as
described herein,
may be performed using the CRISPR/Cpfl system as described in U.S. Patent No.
US 9790490, the
disclosure of which is incorporated by reference herein.
[00913] A method for expanding TILs into a therapeutic population may be
carried out in
accordance with any embodiment of the methods described herein (e.g., Gen 2)
or as described in
U.S. Patent Application Publication Nos. US 2020/0299644 Al and US
2020/0121719 Al and U.S.
Patent No. 10,925,900, the disclosures of which arc incorporated by reference
herein, wherein the
method further comprises gene-editing at least a portion of the TILs by a TALE
method. According to
particular embodiments, the use of a TALE method during the TIL expansion
process causes
expression of one or more immune checkpoint genes to be silenced or reduced in
at least a portion of
the therapeutic population of TILs. Alternatively, the use of a TALE method
during the TIL
expansion process causes expression of one or more immune checkpoint genes to
be enhanced in at
least a portion of the therapeutic population of TILs.
[00914] TALE stands for transcription activator-like effector `"' proteins,
which include
transcription activator-like effector nucleases (TALENs'). A method of using a
TALE system for
gene editing may also be referred to herein as a TALE method. TALEs are
naturally occurring
proteins from the plant pathogenic bacteria genus Xanthomonas, and contain DNA-
binding domains
composed of a series of 33-35-amino-acid repeat domains that each recognizes a
single base pair.
TALE specificity is determined by two hypervariable amino acids that are known
as the repeat-
variable di-residues (RVDs). Modular TALE repeats are linked together to
recognize contiguous
DNA sequences. A specific RVD in the DNA-binding domain recognizes a base in
the target locus,
providing a structural feature to assemble predictable DNA-binding domains.
The DNA binding
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domains of a TALE are fused to the catalytic domain of a type ITS FokI
endonuclease to make a
targetable TALE nuclease. To induce site-specific mutation, two individual
TALEN arms, separated
by a 14-20 base pair spacer region, bring Fold monomers in close proximity to
dimerize and produce
a targeted double-strand break.
1009151 Several large, systematic studies utilizing various assembly methods
have indicated that
TALE repeats can be combined to recognize virtually any user-defined sequence.
Custom-designed
TALE arrays are also commercially available through Cellectis Bioresearch
(Paris, France),
Transposagen Biopharmaceuticals (Lexington, KY, USA), and Life Technologies
(Grand Island, NY,
USA). TALE and TALEN methods suitable for use in the present invention are
described in U.S.
Patent Application Publication Nos. US 2011/0201118 Al; US 2013/0117869 Al; US
2013/0315884
Al; US 2015/0203871 Al and US 2016/0120906 Al, the disclosures of each of
which are
incorporated by reference herein.
1009161 Non-limiting examples of genes that may be silenced or inhibited by
permanently gene-
editing TILs via a TALE method include PD-1, CTLA-4, LAG-3, HAVCR2 (TIM-3),
Cish, TGF13,
PKA, CBL-B, PPP2CA, PPP2CB, PTPN6, PTPN22, PDCD1, BTLA, CD160, TIGIT, TET2,
CD96,
CRTAM, LAIR1, SIGLEC7, SIGLEC9, CD244, TNFRSF10B, TNERSF10A, CASP8, CASP10,
CASP3, CASP6, CASP7, FADD, FAS, SMAD2, SMAD3, SMAD4, SMAD10, SKI, SKIL, TGIF1,
IL1 ORA, TL1ORB, HMOX2, IL6R, IL6ST, EIF2AK4, CSK, PAG1, STT1, FOXP3, PRDM1,
BATF,
GUCY1A2, GUCY1A3, GUCY1B2, GUCY1B3, TOX, SOCS1, ANKRD11, and BCOR.
[00917] Non-limiting examples of genes that may be enhanced by permanently
gene-editing TILs
via a TALE method include CCR2, CCR4, CCR5, CXCR2, CXCR3, CX3CR1, IL-2, IL12,
IL-15, and
IL-21.
[00918] Examples of systems, methods, and compositions for altering the
expression of a target
gene sequence by a TALE method, and which may be used in accordance with
embodiments of the
present invention, are described in U.S. Patent No. 8,586,526, which is
incorporated by reference
herein.
[00919] A method for expanding TILs into a therapeutic population may be
carried out in
accordance with any embodiment of the methods described herein or as described
in U.S. Patent
Application Publication Nos. US 2020/0299644 Al and US 2020/0121719 Al and
U.S. Patent No.
10,925,900, the disclosures of which are incorporated by reference herein,
wherein the method further
comprises gene-editing at least a portion of the TILs by a zinc finger or zinc
finger nuclease method.
According to particular embodiments, the use of a zinc finger method during
the TIL expansion
process causes expression of one or more immune checkpoint genes to be
silenced or reduced in at
least a portion of the therapeutic population of TILs. Alternatively, the use
of a zinc finger method
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during the TIL expansion process causes expression of one or more immune
checkpoint genes to be
enhanced in at least a portion of the therapeutic population of TILs.
[00920] An individual zinc finger contains approximately 30 amino acids in a
conserved 1313a
configuration. Several amino acids on the surface of the a-helix typically
contact 3 bp in the major
groove of DNA, with varying levels of selectivity. Zinc fingers have two
protein domains. The first
domain is the DNA binding domain, which includes eukaryotic transcription
factors and contain the
zinc finger. The second domain is the nuclease domain, which includes the FokI
restriction enzyme
and is responsible for the catalytic cleavage of DNA.
[00921] The DNA-binding domains of individual ZFNs typically contain between
three and six
individual zinc finger repeats and can each recognize between 9 and 18 base
pairs. If the zinc finger
domains are specific for their intended target site then even a pair of 3-
finger ZFNs that recognize a
total of 18 base pairs can, in theory, target a single locus in a mammalian
genome. One method to
generate new zinc-finger arrays is to combine smaller zinc-finger "modules" of
known specificity.
The most common modular assembly process involves combining three separate
zinc fingers that can
each recognize a 3 base pair DNA sequence to generate a 3-finger array that
can recognize a 9 base
pair target site. Alternatively, selection-based approaches, such as
oligomerized pool engineering
(OPEN) can be used to select for new zinc-finger arrays from randomized
libraries that take into
consideration context-dependent interactions between neighboring fingers.
Engineered zinc fingers
are available commercially from Sangamo Biosciences (Richmond, CA, USA) and
Sigma-Aldrich
(St. Louis, MO, USA).
[00922] Non-limiting examples of genes that may be silenced or inhibited by
permanently gene-
editing TILs via a zinc finger method include PD-1, CTLA-4, LAG-3, HAVCR2 (TIM-
3), Cish,
TGFI3, PKA, CBL-B, PPP2CA, PPP2CB, PTPN6, PTPN22, PDCD1, BTLA, CD160, TIGIT,
TET2,
CD96, CRTAM, LAIR', SIGLEC7, SIGLEC9, CD244, TNFRSF10B, TNFRSF1OA, CASP8,
CASP10, CASP3, CASP6, CASP7, FADD, FAS, SMAD2, SMAD3, SMAD4, SMAD10, SKI,
SKIL,
TGIF1, ILlORA, ILlORB, HMOX2, IL6R, IL6ST, EIF2AK4, CSK, PAG1, SIT1, FOXP3,
PRDM1,
BATF, GUCY1A2, GUCY1A3, GUCY1B2, GUCY1B3, TOX, SOCS1, ANKRD11, and BCOR.
[00923] Non-limiting examples of genes that may be enhanced by penuanently
gene-editing TILs
via a zinc finger method include CCR2, CCR4, CCR5, CXCR2, CXCR3, CX3CR1, 1L-2,
1L12, IL-
15, and IL-21.
[00924] Examples of systems, methods, and compositions for altering the
expression of a target
gene sequence by a zinc finger method, which may be used in accordance with
embodiments of the
present invention, are described in U.S. Patent Nos. 6,534,261, 6,607,882,
6,746,838, 6,794,136,
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6,824,978, 6,866,997, 6,933,113, 6,979,539, 7,013,219, 7,030,215, 7,220,719,
7,241,573, 7,241,574,
7,585,849, 7,595,376, 6,903,185, and 6,479,626, each of which are incorporated
by reference herein.
[00925] Other examples of systems, methods, and compositions for altering the
expression of a
target gene sequence by a zinc finger method, which may be used in accordance
with embodiments of
the present invention, are described in Beane, etal., Mol. Therapy, 2015, 23,
1380-1390, the
disclosure of which is incorporated by reference herein.
[00926] In some embodiments, the TILs are optionally genetically engineered to
include additional
functionalities, including, but not limited to, a high-affinity TCR_, e.g., a
TCR targeted at a tumor-
associated antigen such as MAGE-1, HER2, or NY-ESO-1, or a chimeric antigen
receptor (CAR)
which binds to a tumor-associated cell surface molecule (e.g., mesothelin) or
lineage-restricted cell
surface molecule (e.g., CD19). In some embodiments, the method comprises
genetically engineering a
population of TILs to include a high-affinity TCR, e.g., a TCR targeted at a
tumor-associated antigen
such as MAGE-1, HER2, or NY-ESO-1, or a chimeric antigen receptor (CAR) which
binds to a
tumor-associated cell surface molecule (e.g., mesothelin) or lineage-
restricted cell surface molecule
(e.g., CD19). Aptly, the population of TILs may be a first population, a
second population and/or a
third population as described herein.
E. Closed Systems for TIL Manufacturing
[00927] The present invention provides for the use of closed systems during
the TIL culturing
process. Such closed systems allow for preventing and/or reducing microbial
contamination, allow for
the use of fewer flasks, and allow for cost reductions. In some embodiments,
the closed system uses
two containers.
[00928] Such closed systems are well-known in the art and can be found, for
example, at
http://www.fda.govicber/guidelines.htm and
https://www.fda.gov/BiologicsBloodVaccines/GuidanceComplianceRegulatoryInformat
ion/Guidance
s/Blood/ucm076779.htm.
[00929] Sterile connecting devices (STCDs) produce sterile welds between two
pieces of
compatible tubing. This procedure permits sterile connection of a variety of
containers and tube
diameters. In some embodiments, the closed systems include luer lock and heat
sealed systems as
described in the Examples. In some embodiments, the closed system is accessed
via syringes under
sterile conditions in order to maintain the sterility and closed nature of the
system. In some
embodiments, a closed system as described in Example 21 is employed. In some
embodiments, the
TILs are formulated into a final product formulation container according to
the methods described
herein in the Examples'''.
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[00930] In some embodiments, the closed system uses one container from the
time the tumor
fragments are obtained until the TILs are ready for administration to the
patient or cryopreserving. In
some embodiments when two containers are used, the first container is a closed
G-container and the
population of TILs is centrifuged and transferred to an infusion bag without
opening the first closed
G-container. In some embodiments, when two containers are used, the infusion
bag is a
HypoThermosol-containing infusion bag. A closed system or closed TIL cell
culture system is
characterized in that once the tumor sample and/or tumor fragments have been
added, the system is
tightly sealed from the outside to form a closed environment free from the
invasion of bacteria, fungi,
and/or any other microbial contamination.
[00931] In some embodiments, the reduction in microbial contamination is
between about 5% and
about 100%. hi sonic embodiments, the reduction in microbial contamination is
between about 5%
and about 95%. In some embodiments, the reduction in microbial contamination
is between about 5%
and about 90%. In some embodiments, the reduction in microbial contamination
is between about
10% and about 90%. In some embodiments, the reduction in microbial
contamination is between
about 15% and about 85%. In some embodiments, the reduction in microbial
contamination is about
5%, about 10%, about 15%, about 20%, about 25%, about 30%, about 35%, about
40%, about 45%,
about 50%, about 55%, about 60%, about 65%, about 70%, about 75%, about 80%,
about 85%, about
90%, about 95%, about 97%, about 98%, about 99%, or about 100%.
[00932] The closed system allows for TIL growth in the absence and/or with a
significant reduction
in microbial contamination.
[00933] Moreover, pH, carbon dioxide partial pressure and oxygen partial
pressure of the TIL cell
culture environment each vary as the cells are cultured. Consequently, even
though a medium
appropriate for cell culture is circulated, the closed environment still needs
to be constantly
maintained as an optimal environment for TIL proliferation. To this end, it is
desirable that the
physical factors of pH, carbon dioxide partial pressure and oxygen partial
pressure within the culture
liquid of the closed environment be monitored by means of a sensor, the signal
whereof is used to
control a gas exchanger installed at the inlet of the culture environment, and
the that gas partial
pressure of the closed environment be adjusted in real time according to
changes in the culture liquid
so as to optimize the cell culture environment. In some embodiments, the
present invention provides a
closed cell culture system which incorporates at the inlet to the closed
environment a gas exchanger
equipped with a monitoring device which measures the pH, carbon dioxide
partial pressure and
oxygen partial pressure of the closed environment, and optimizes the cell
culture environment by
automatically adjusting gas concentrations based on signals from the
monitoring device.
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[00934] In some embodiments, the pressure within the closed environment is
continuously or
intermittently controlled. That is, the pressure in the closed environment can
be varied by means of a
pressure maintenance device for example, thus ensuring that the space is
suitable for growth of TILs
in a positive pressure state, or promoting exudation of fluid in a negative
pressure state and thus
promoting cell proliferation. By applying negative pressure intermittently,
moreover, it is possible to
uniformly and efficiently replace the circulating liquid in the closed
environment by means of a
temporary shrinkage in the volume of the closed environment.
[00935] In some embodiments, optimal culture components for proliferation of
the TILs can be
substituted or added, and including factors such as IL-2 and/or OKT3, as well
as combination, can be
added.
F. Optional Cryopreservation of TILs
[00936] Either the bulk TIL population (for example the second population of
TILs) or the expanded
population of TILs (for example the third population of TILs) can be
optionally cryopreserved. hi
some embodiments, cryopreservation occurs on the therapeutic TIL population.
In some
embodiments, cryopreservation occurs on the TILs harvested after the second
expansion. In some
embodiments, cryopreservation occurs on the TILs in exemplary Step F of
Figures 1 and/or 8 (in
particular, e.g., Figure 8A and/or Figure 8B and/or Figure 8C and/or Figure
8D). In some
embodiments, the TILs are cryopreserved in the infusion bag. in some
embodiments, the TILs are
cryopreserved prior to placement in an infusion bag. In some embodiments, the
TILs are
cryopreserved and not placed in an infusion bag. In some embodiments,
cryopreservation is
performed using a cryopreservation medium. In some embodiments, the
cryopreservation media
contains dimethylsulfoxide (DMSO). This is generally accomplished by putting
the TIL population
into a freezing solution, e.g. 85% complement inactivated AB serum and 15%
dimethyl sulfoxide
(DMSO). The cells in solution are placed into cryogenic vials and stored for
24 hours at -80 C, with
optional transfer to gaseous nitrogen freezers for cryopreservation. See,
Sadeglii, et al., Ada
Oncologica 2013, 52. 978-986.
[00937] When appropriate, the cells are removed from the freezer and thawed in
a 37 C water bath
until approximately 4/5 of the solution is thawed. The cells are generally
resuspended in complete
media and optionally washed one or more times. In some embodiments, the thawed
TILs can be
counted and assessed for viability as is known in the art.
[00938] In some embodiments, a population of TILs is cryopreserved using CS10
cryopreservation
media (CryoStor 10, BioLife Solutions). In some embodiments, a population of
TILs is cryopreserved
using a cryopreservation media containing dimethylsulfoxide (DMSO). In some
embodiments, a
population of TILs is cryopreserved using a 1:1 (vol :vol) ratio of CS10 and
cell culture media. In
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some embodiments, a population of TILs is cryopreserved using about a 1:1
(vol:vol) ratio of CS10
and cell culture media, further comprising additional IL-2.
[00939] As discussed above, and exemplified in Steps A through E as provided
in Figures 1 and/or 8
(in particular, e.g., Figure 8A and/or Figure 8B and/or Figure 8C and/or
Figure 8D), cryopreservation
can occur at numerous points throughout the TIL expansion process. In some
embodiments, the
expanded population of TILs after the first expansion (as provided for
example, according to Step B
or the expanded population of TiLs after the one or more second expansions
according to Step D of
Figures 1 or 8 (in particular, e.g., Figure 8A and/or Figure 8B and/or Figure
8C and/or Figure 8D) can
be cryopreserved. Cryopreservation can be generally accomplished by placing
the TIL population into
a freezing solution, e.g., 85% complement inactivated AB serum and 15%
dimethyl sulfoxide
(DMSO). The cells in solution are placed into cryogenic vials and stored for
24 hours at -80 C, with
optional transfer to gaseous nitrogen freezers for cryopreservation. See
Sadeghi, et al., Acta
Oncologica 2013, 52. 978-986. In some embodiments, the TILs are cryopreserved
in 5% DMSO. In
some embodiments, the TILs are cryopreserved in cell culture media plus 5%
DMSO. In some
embodiments, the TILs are cryopreserved according to the methods provided in
Example 6.
[00940] When appropriate, the cells are removed from the freezer and thawed in
a 37 'V water bath
until approximately 4/5 of the solution is thawed. The cells are generally
resuspended in complete
media and optionally washed one or more times. In some embodiments, the thawed
TILs can be
counted and assessed for viability as is known in the art.
[00941] In some cases, the Step B from Figures 1 or 8, (in particular, e.g.,
Figure 8A and/or Figure
8B and/or Figure 8C and/or Figure 8D) TIL population can be cryopreserved
immediately, using the
protocols discussed below. Alternatively, the bulk TIL population can be
subjected to Step C and Step
D from Figures 1 or 8, (in particular, e.g., Figure 8A and/or Figure 8B and/or
Figure 8C and/or Figure
8D) and then cryopreserved after Step D from Figures 1 or 8, (in particular,
e.g., Figure 8A and/or
Figure 8B and/or Figure 8C and/or Figure 8D). Similarly, in the case where
genetically modified TILs
will be used in therapy, the Step B or Step D from Figures 1 or 8, (in
particular, e.g., Figure 8A and/or
Figure 8B and/or Figure 8C and/or Figure 8D) TIL populations can be subjected
to genetic
modifications for suitable treatments.
G. Phenotypic Characteristics of Expanded TILs
[00981] In some embodiment, the TILs are analyzed for expression of numerous
phenotype markers
after expansion, including those described herein and in the Examples. In some
embodiments,
expression of one or more phenotypic markers is examined. In some embodiments,
the phenotypic
characteristics of the TILs are analyzed after the first expansion in Step B
from Figures 1 or 8, (in
particular, e.g., Figure SA and/or Figure 8B and/or Figure 8C and/or Figure
8D). In some
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embodiments, the phenotypic characteristics of the TILs are analyzed during
the transition in Step C
from Figures 1 or 8, (in particular, e.g., Figure 8A and/or Figure 8B and/or
Figure 8C and/or Figure
8D). In some embodiments, the phenotypic characteristics of the TILs are
analyzed during the
transition according to Step C from Figures 1 or 8, (in particular, e.g.,
Figure 8A and/or Figure 8B
and/or Figure 8C and/or Figure 8D) and after cryopreservation. In some
embodiments, the phenotypic
characteristics of the T1Ls arc analyzed after the second expansion according
to Step 1) from Figures 1
or 8, (in particular, e.g., Figure 8A and/or Figure 8B and/or Figure 8C and/or
Figure 8D). In some
embodiments, the phenotypic characteristics of the TILs are analyzed after two
or more expansions
according to Step D from Figures 1 or 8, (in particular, e.g., Figure 8A
and/or Figure 8B and/or Figure
8C and/or Figure 8D).
[00982] In some embodiments, the marker is selected from the group consisting
of CD8 and CD28.
In some embodiments, expression of CD8 is examined. In some embodiments,
expression of CD28 is
examined. In some embodiments, the expression of CD8 and/or CD28 is higher on
TILs produced
according the current invention process, as compared to other processes (e.g.,
the Gen 3 process as
provided for example in Figure 8 (in particular, e.g., Figure 8A and/or Figure
8B and/or Figure 8C
and/or Figure 8D), as compared to the 2A process as provided for example in
Figure 8 (in particular,
e.g., Figure 8A and/or Figure 8B and/or Figure 8C and/or Figure 8D). In some
embodiments, the
expression of CD8 is higher on TILs produced according the current invention
process, as compared
to other processes (e.g., the Gen 3 process as provided for example in Figure
8 (in particular, e.g.,
Figure 8B), as compared to the 2A process as provided for example in Figure 8
(in particular, e.g.,
Figure 8A and/or Figure 8B and/or Figure 8C and/or Figure 8D). In some
embodiments, the
expression of CD28 is higher on TILs produced according the current invention
process, as compared
to other processes (e.g., the Gcn 3 process as provided for example in Figure
8 (in particular, e.g.,
Figure 8A and/or Figure 8B and/or Figure 8C and/or Figure 8D), as compared to
the 2A process as
provided for example in Figure 8 (in particular, e.g., Figure 8A)). In some
embodiments, high CD28
expression is indicative of a younger, more presisitent TIL phenotype. In some
embodiments,
expression of one or more regulatory markers is measured.
[00983] In some embodiments, no selection of the first population of TILs,
second population of
TILs, third population of TILs, or harvested TIL population based on CD8
and/or CD28 expression is
performed during any of the steps for the method for expanding tumor
infiltrating lymphocytes (TILs)
described herein.
[00984] In some embodiments, the percentage of central memory cells is higher
on TILs produced
according the current invention process, as compared to other processes (e.g.,
the Gen 3 process as
provided for example in Figure 8 (in particular, e.g., Figure 8A and/or Figure
8B and/or Figure 8C
and/or Figure 8D), as compared to the 2A process as provided for example in
Figure 8 (in particular,
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e.g., Figure 8A)). In some embodiments the memory marker for central memory
cells is selected from
the group consisting of CCR7 and CD62L.
[00985] In some embodiments, the CD4+ and/or CD8+ TIL Memory subsets can be
divided into
different memory subsets. In some embodiments, the CD4+ and/or CD8+ TILs
comprise the naive
(CD45RA+CD62L+) TILs. In some embodiments, the CD4+ and/or CD8+ TILs comprise
the central
memory (CM; CD45RA-CD62L+) TILs. In some embodiments, the CD4+ and/or CD8+
TILs
comprise the effector memory (EM; CD45RA-CD62L-) TILs. In some embodiments,
the CD4+
and/or CDR+ TII,s comprise the, RA+ effector memory/effector (TEMRA/TEFF;
CD45RA+CD62L+) TILs.
[00986] In some embodiments, the TILs express one more markers selected from
the group
consisting of granzyme B, perforin, and granulysin. In some embodiments, the
TILs express
granzyme B. In some embodiments, the TILs express perforin. In some
embodiments, the TILs
express granulysin.
[00987] In some embodiments, restimulated TILs can also be evaluated for
cytokine release, using
cytokine release assays. In some embodiments, TILs can be evaluated for
interferon-'y (IFN-y)
secretion. In some embodiments, the IFN-y secretion is measured by an ELTSA
assay. in some
embodiments, the IFN-y secretion is measured by an ELISA assay after the rapid
second expansion
step, after Step D as provided in for example, Figure 8 (in particular, e.g.,
Figure 8A and/or Figure 8B
and/or Figure 8C and/or Figure 8D). In some embodiments, TIL health is
measured by 1FN-gamma
(IFN-y) secretion. In some embodiments, IFN-y secretion is indicative of
active TILs. In some
embodiments, a potency assay for IFN-y production is employed. IFN-y
production is another
measure of cytotoxic potential. IFN-y production can be measured by
determining the levels of the
cytokine IFN-y in the media of TIL stimulated with antibodies to CD3, CD28,
and CD137/4-1BB.
IFN-y levels in media from these stimulated TIL can be determined using by
measuring IFN-y release.
In some embodiments, an increase in IFN-y production in for example Step D in
the Gen 3 process as
provided in Figure 8 (in particular, e.g., Figure 8A and/or Figure 8B and/or
Figure 8C and/or Figure
8D) TILs as compared to for example Step D in the 2A process as provided in
Figure 8 (in particular,
e.g., Figure 8A) is indicative of an increase in cytotoxic potential of the
Step D TILs. In some
embodiments, IFN-y secretion is increased one-fold, two-fold, three-fold, four-
fold, or five-fold or
more. In some embodiments, IFN-y secretion is increased one-fold. In some
embodiments, IFN-y
secretion is increased two-fold. In some embodiments, IFN-y secretion is
increased three-fold. In
some embodiments, IFN-y secretion is increased four-fold. In some embodiments,
IFN-y secretion is
increased five-fold. In some embodiments, IFN-y is measured using a Quantikine
ELISA kit. In some
embodiments, IFN-y is measured in TILs ex vivo. In some embodiments, IFN-y is
measured in TILs
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ex vivo, including TILs produced by the methods of the present invention,
including, for example
Figure 8B methods.
[00988] In some embodiments, TILs capable of at least one-fold, two-fold,
three-fold, four-fold, or
five-fold or more 1FN-y secretion are TILs produced by the expansion methods
of the present
invention, including, for example Figure 8A and/or Figure 8B and/or Figure 8C
and/or Figure 8D
methods. In some embodiments, TILs capable of at least one-fold more IFN-y
secretion are TILs
produced by the expansion methods of the present invention, including, for
example Figure 8A and/or
Figure RB and/or Figure RC and/or Figure RD methods In some embodiments, TIT,s
capable of at
least two-fold more IFN-y secretion are TILs produced by the expansion methods
of the present
invention, including, for example Figure 8A and/or Figure 8B and/or Figure 8C
and/or Figure 8D
methods. In some embodiments, TILs capable of at least three-fold more IFN-y
secretion are TILs
produced by the expansion methods of the present invention, including, for
example Figure 8A and/or
Figure 8B and/or Figure 8C and/or Figure 8D methods. In some embodiments, TiLs
capable of at
least four-fold more IFN-y secretion are TILs produced by the expansion
methods of the present
invention, including, for example Figure 8A and/or Figure 8B and/or Figure 8C
and/or Figure 8D
methods. In some embodiments, TILs capable of at least five-fold more IFN-y
secretion are TILs
produced by the expansion methods of the present invention, including, for
example Figure 8A and/or
Figure 8B and/or Figure 8C and/or Figure 8D methods.
[00989] In some embodiments, TILs capable of at least 100 pg/mL to about 1000
pg/mL or more
IFN-y secretion are TILs produced by the expansion methods of the present
invention, including, for
example Figure 8A and/or Figure 8B and/or Figure 8C and/or Figure 8D methods.
In some
embodiments, TILs capable of at least 200 pg/mL, at least 250 pg/mL, at least
300 pg/mL, at least 350
pg/mL, at least 400 pg/mL, at least 450 pg/mL, at least 500 pg/mL, at least
550 pg/mL, at least 600
pg/mL, at least 650 pg/mL, at least 700 pg/mL, at least 750 pg/mL, at least
800 pg/mL, at least 850
pg/mL, at least 900 pg/mL, at least 950 pg/mL, or at least 1000 pg/mL or more
IFN-y secretion are
TILs produced by the expansion methods of the present invention, including,
for example Figure 8A
and/or Figure 8B and/or Figure 8C and/or Figure 8D methods. In some
embodiments, TILs capable of
at least 200 pg/mL IFN-y secretion are TILs produced by the expansion methods
of the present
invention, including, for example Figure 8A and/or Figure 8B and/or Figure 8C
and/or Figure 8D
methods. In some embodiments, TILs capable of at least 200 pg/mL IFN-y
secretion are TILs
produced by the expansion methods of the present invention, including, for
example Figure 8A and/or
Figure 8B and/or Figure 8C and/or Figure 8D methods. In some embodiments, TILs
capable of at
least 300 pg/mL 1FN-y secretion are TILs produced by the expansion methods of
the present
invention, including, for example Figure 8A and/or Figure 8B and/or Figure 8C
and/or Figure 8D
methods. In some embodiments, TILs capable of at least 400 pg/mL IFN-y
secretion are TILs
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produced by the expansion methods of the present invention, including, for
example Figure 8A and/or
Figure 8B and/or Figure 8C and/or Figure 8D methods. In some embodiments, TILs
capable of at
least 500 pg/mL IFN-y secretion are TILs produced by the expansion methods of
the present
invention, including, for example Figure 8A and/or Figure 8B and/or Figure 8C
and/or Figure 8D
methods. In some embodiments, TILs capable of at least 600 pg/mL IFN-y
secretion are TILs
produced by the expansion methods of the present invention, including, for
example Figure 8A and/or
Figure 8B and/or Figure 8C and/or Figure 8D methods. In some embodiments, TILs
capable of at
least 700 pg/mL IFN-y secretion are TILs produced by the expansion methods of
the present
invention, including, for example Figure 8A and/or Figure 8B and/or Figure 8C
and/or Figure 8D
methods. In some embodiments, TILs capable of at least 800 pg/mL IFN-y
secretion are TILs
produced by the expansion methods of the present invention, including, for
example Figure 8A and/or
Figure 8B and/or Figure 8C and/or Figure 8D methods. In some embodiments, TILs
capable of at
least 900 pg/mL IFN-y secretion are TILs produced by the expansion methods of
the present
invention, including, for example Figure 8A and/or Figure 8B and/or Figure 8C
and/or Figure 8D
methods. In some embodiments, TILs capable of at least 1000 pg/mL IFN-y
secretion are TILs
produced by the expansion methods of the present invention, including, for
example Figure 8A and/or
Figure 8B and/or Figure 8C and/or Figure 8D methods. In some embodiments, TILs
capable of at
least 2000 pg/mL IFN-y secretion are TILs produced by the expansion methods of
the present
invention, including, for example Figure 8A and/or Figure 8B and/or Figure 8C
and/or Figure 8D
methods. In some embodiments, TILs capable of at least 3000 pg/mL IFN-y
secretion are TILs
produced by the expansion methods of the present invention, including, for
example Figure 8A and/or
Figure 8B and/or Figure 8C and/or Figure 8D methods. In some embodiments, TILs
capable of at
least 4000 pg/mL IFN-y secretion are TILs produced by the expansion methods of
the present
invention, including, for example Figure 8A and/or Figure 8B and/or Figure 8C
and/or Figure 8D
methods. In some embodiments, TILs capable of at least 5000 pg/mL IFN-y
secretion are TILs
produced by the expansion methods of the present invention, including, for
example Figure 8A and/or
Figure 8B and/or Figure 8C and/or Figure 8D methods. In some embodiments, TILs
capable of at
least 6000 pg/mL IFN-y secretion are TILs produced by the expansion methods of
the present
invention, including, for example Figure 8A and/or Figure 8B and/or Figure 8C
and/or Figure 8D
methods. In some embodiments, TILs capable of at least 7000 pg/mL IFN-y
secretion are TILs
produced by the expansion methods of the present invention, including, for
example Figure 8A and/or
Figure 8B and/or Figure 8C and/or Figure 8D methods. In some embodiments, TILs
capable of at
least 8000 pg/mL IFN-y secretion are TILs produced by the expansion methods of
the present
invention, including, for example Figure 8A and/or Figure 8B and/or Figure 8C
and/or Figure 8D
methods. In some embodiments, TILs capable of at least 9000 pg/mL IFN-y
secretion are TILs
produced by the expansion methods of the present invention, including, for
example Figure 8A and/or
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Figure 8B and/or Figure 8C and/or Figure 8D methods. In some embodiments, TILs
capable of at
least 10,000 pg/mL IFN-y secretion are TILs produced by the expansion methods
of the present
invention, including, for example Figure 8A and/or Figure 8B and/or Figure 8C
and/or Figure 8D
methods. In some embodiments, TILs capable of at least 15,000 pg/mL IFN-y
secretion are TILs
produced by the expansion methods of the present invention, including, for
example Figure 8A and/or
Figure 813 and/or Figure SC and/or Figure 8D methods. In some embodiments,
IlLs capable of at
least 20,000 pg/mL IFN-y secretion are TILs produced by the expansion methods
of the present
invention, including, for example Figure 8A and/or Figure 8B and/or Figure 8C
and/or Figure 8D
methods. In some embodiments, TILs capable of at least 25,000 pg/mL IFN-y
secretion are TILs
produced by the expansion methods of the present invention, including, for
example Figure 8A and/or
Figure 8B and/or Figure 8C and/or Figure 8D methods. In some embodiments, TILs
capable of at
least 30,000 pg/mL IFN-y secretion are TILs produced by the expansion methods
of the present
invention, including, for example Figure 8A and/or Figure 8B and/or Figure 8C
and/or Figure 8D
methods. In some embodiments, TILs capable of at least 35,000 pg/mL IFN-y
secretion are TILs
produced by the expansion methods of the present invention, including, for
example Figure SA and/or
Figure 8B and/or Figure 8C and/or Figure 8D methods. In some embodiments, TILs
capable of at
least 40,000 pg/mL IFN-y secretion are TILs produced by the expansion methods
of the present
invention, including, for example Figure 8A and/or Figure 8B and/or Figure 8C
and/or Figure 8D
methods. In some embodiments, TILs capable of at least 45,000 pg/mL IFN-y
secretion are TILs
produced by the expansion methods of the present invention, including, for
example Figure 8A and/or
Figure 8B and/or Figure 8C and/or Figure 8D methods. In some embodiments, TILs
capable of at
least 50,000 pg/mL IFN-y secretion are TILs produced by the expansion methods
of the present
invention, including, for example Figure 8A and/or Figure 8B and/or Figure 8C
and/or Figure 8D
methods.
[00990] In some embodiments, TILs capable of at least 100
pg/mL/5e5 cells to about 1000
pg/mL/5e5 cells or more IFN-7 secretion are TILs produced by the expansion
methods of the present
invention, including, for example Figure 8A and/or Figure 8B and/or Figure 8C
and/or Figure 8D
methods. In some embodiments, TILs capable of at least 200 pg/mL/5e5 cells, at
least 250 pg/mL/5e5
cells, at least 300 pg/mL/5e5 cells, at least 350 pg/mL/5e5 cells, at least
400 pg/mL/5e5 cells, at least
450 pg/mL/5e5 cells, at least 500 pg/mL/5e5 cells, at least 550 pg/mL/5e5
cells, at least 600
pg/mL/5e5 cells, at least 650 pg/mL/5e5 cells, at least 700 pg/mL/5e5 cells,
at least 750 pg/mL/5e5
cells, at least 800 pg/mL/5e5 cells, at least 850 pg/mL/5e5 cells, at least
900 pg/mL/5e5 cells, at least
950 pg/mL/5e5 cells, or at least 1000 pg/mL/5e5 cells or more IFN-y secretion
are TILs produced by
the expansion methods of the present invention, including, for example Figure
8A and/or Figure 8B
and/or Figure SC and/or Figure 8D methods. In some embodiments, TILs capable
of at least 200
pg/mL/5e5 cells IFN-y secretion are TILs produced by the expansion methods of
the present
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invention, including, for example Figure 8A and/or Figure 8B and/or Figure 8C
and/or Figure 8D
methods. In some embodiments, TILs capable of at least 200 pg/mL/5e5 cells IFN-
y secretion are
TILs produced by the expansion methods of the present invention, including,
for example Figure SA
and/or Figure 8B and/or Figure 8C and/or Figure 8D methods. In some
embodiments, TILs capable of
at least 300 pg/mL/5e5 cells IFN-y secretion are TILs produced by the
expansion methods of the
present invention, including, for example Figure 8A and/or Figure 8B and/or
Figure SC and/or Figure
8D methods. In some embodiments, TILs capable of at least 400 pg/mL/5e5 cells
IFN-y secretion are
TILs produced by the expansion methods of the present invention, including,
for example Figure 8A
and/or Figure 8B and/or Figure 8C and/or Figure 8D methods. In some
embodiments, TILs capable of
at least 500 pg/mL/5e5 cells IFN-y secretion are TILs produced by the
expansion methods of the
present invention, including, for example Figure 8A and/or Figure 8B and/or
Figure 8C and/or Figure
8D methods. In some embodiments, TILs capable of at least 600 pg/mL/5e5 cells
IFN-y secretion are
TILs produced by the expansion methods of the present invention, including,
for example Figure 8A
and/or Figure 8B and/or Figure 8C and/or Figure 8D methods. In some
embodiments, TILs capable of
at least 700 pg/mL/5e5 cells IFN-y secretion are TILs produced by the
expansion methods of the
present invention, including, for example Figure 8A and/or Figure 8B and/or
Figure 8C and/or Figure
8D methods. In some embodiments, TILs capable of at least 800 pg/mL/5e5 cells
IFN-y secretion are
TILs produced by the expansion methods of the present invention, including,
for example Figure 8A
and/or Figure 8B and/or Figure 8C and/or Figure 8D methods. In some
embodiments, TILs capable of
at least 900 pg/mL/5e5 cells IFN-y secretion are TILs produced by the
expansion methods of the
present invention, including, for example Figure 8A and/or Figure 8B and/or
Figure 8C and/or Figure
8D methods. In some embodiments, TILs capable of at least 1000 pg/mL/5e5 cells
IFN-y secretion are
TILs produced by the expansion methods of the present invention, including,
for example Figure 8A
and/or Figure 8B and/or Figure 8C and/or Figure 8D methods. In some
embodiments, TILs capable of
at least 2000 pg/mL/5e5 cells 1FN-y secretion are TILs produced by the
expansion methods of the
present invention, including, for example Figure 8A and/or Figure 8B and/or
Figure 8C and/or Figure
8D methods. In some embodiments, TILs capable of at least 3000 pg/mL/5e5 cells
IFN-y secretion are
TILs produced by the expansion methods of the present invention, including,
for example Figure 8A
and/or Figure 8B and/or Figure 8C and/or Figure 8D methods. In some
embodiments, TILs capable of
at least 4000 pg/mL/5e5 cells IFN-y secretion are TILs produced by the
expansion methods of the
present invention, including, for example Figure SA and/or Figure 8B and/or
Figure 8C and/or Figure
8D methods. In some embodiments, TILs capable of at least 5000 pg/mL/5e5 cells
IFN-y secretion are
TILs produced by the expansion methods of the present invention, including,
for example Figure 8A
and/or Figure 8B and/or Figure 8C and/or Figure 8D methods. In some
embodiments, TILs capable of
at least 6000 pg/mL/5e5 cells IFN-y secretion are TILs produced by the
expansion methods of the
present invention, including, for example Figure 8A and/or Figure 8B and/or
Figure 8C and/or Figure
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8D methods. In some embodiments, TILs capable of at least 7000 pg/mL/5e5 cells
IFN-y secretion are
TILs produced by the expansion methods of the present invention, including,
for example Figure 8A
and/or Figure 8B and/or Figure 8C and/or Figure 8D methods. In some
embodiments, TILs capable of
at least 8000 pg/mL/5e5 cells IFN-y secretion are TILs produced by the
expansion methods of the
present invention, including, for example Figure 8A and/or Figure 8B and/or
Figure 8C and/or Figure
8D methods. In some embodiments, IlLs capable of at least 9000 pg/mL/5e5 cells
IFN-y secretion are
TILs produced by the expansion methods of the present invention, including,
for example Figure 8A
and/or Figure 8B and/or Figure 8C and/or Figure 8D methods. In some
embodiments, TILs capable of
at least 10,000 pg/mL/5e5 cells IFN-y secretion are TILs produced by the
expansion methods of the
present invention, including, for example Figure 8A and/or Figure 8B and/or
Figure 8C and/or Figure
8D methods. In some embodiments, TILs capable of at least 15,000 pg/mL/5e5
cells IFN-y secretion
are TILs produced by the expansion methods of the present invention,
including, for example Figure
8A and/or Figure 8B and/or Figure 8C and/or Figure 8D methods. In some
embodiments, TILs
capable of at least 20,000 pg/mL/5e5 cells IFN-y secretion are TILs produced
by the expansion
methods of the present invention, including, for example Figure SA and/or
Figure 8B and/or Figure
8C and/or Figure 8D methods. In some embodiments, TILs capable of at least
25,000 pg/mL/5e5 cells
IFN-y secretion are TILs produced by the expansion methods of the present
invention, including, for
example Figure 8A and/or Figure 8B and/or Figure SC and/or Figure 8D methods.
In some
embodiments, TILs capable of at least 30,000 pg/mL/5e5 cells IFN-y secretion
are TILs produced by
the expansion methods of the present invention, including, for example Figure
8A and/or Figure 8B
and/or Figure 8C and/or Figure 8D methods. In some embodiments, TILs capable
of at least 35,000
pg/mL/5e5 cells IFN-y secretion are TILs produced by the expansion methods of
the present
invention, including, for example Figure 8A and/or Figure 8B and/or Figure 8C
and/or Figure 8D
methods. In some embodiments, TILs capable of at least 40,000 pg/mL/5e5 cells
IFN-y secretion are
TILs produced by the expansion methods of the present invention, including,
for example Figure 8A
and/or Figure 8B and/or Figure 8C and/or Figure 8D methods. In some
embodiments, TILs capable of
at least 45,000 pg/mL/5e5 cells IFN-y secretion are TILs produced by the
expansion methods of the
present invention, including, for example Figure 8A and/or Figure 8B and/or
Figure 8C and/or Figure
8D methods. In some embodiments, TILs capable of at least 50,000 pg/mL/5e5
cells IFN-y secretion
are TILs produced by the expansion methods of the present invention,
including, for example Figure
8A and/or Figure 8B and/or Figure 8C and/or Figure 8D methods.
[00991] The diverse antigen receptors of T and B lymphocytes are produced by
somatic
recombination of a limited, but large number of gene segments. These gene
segments: V (variable), D
(diversity), J (joining), and C (constant), determine the binding specificity
and downstream
applications of immunoglobulins and T-cell receptors (TCRs). The present
invention provides a
method for generating TILs which exhibit and increase the T-cell repertoire
diversity. In some
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embodiments, the TILs obtained by the present method exhibit an increase in
the T-cell repertoire
diversity. In some embodiments, the TILs obtained by the present method
exhibit an increase in the T-
eel] repertoire diversity as compared to freshly harvested TILs and/or TTLs
prepared using other
methods than those provide herein including, for example, methods other than
those embodied in
Figure 8 (in particular, e.g., Figure 8A and/or Figure 8B and/or Figure 8C
and/or Figure 8D). In some
embodiments, the TILs obtained by the present method exhibit an increase in
the T-cell repertoire
diversity as compared to freshly harvested TILs and/or TILs prepared using
methods referred to as
Gen 2, as exemplified in Figure 8 (in particular, e.g., Figure 8A). In some
embodiments, the TILs
obtained in the first expansion exhibit an increase in the T-cell repertoire
diversity. In some
embodiments, the increase in diversity is an increase in the immunoglobulin
diversity and/or the T-
cell receptor diversity. In some embodiments, the diversity is in the
immunoglobulin is in the
immunoglobulin heavy chain. In some embodiments, the diversity is in the
immunoglobulin is in the
immunoglobulin light chain. In some embodiments, the diversity is in the T-
cell receptor. In some
embodiments, the diversity is in one of the T-cell receptors selected from the
group consisting of
alpha, beta, gamma, and delta receptors. In some embodiments, there is an
increase in the expression
of T-cell receptor (TCR) alpha and/or beta. In some embodiments, there is an
increase in the
expression of T-cell receptor (TCR) alpha. In some embodiments, there is an
increase in the
expression of T-cell receptor (TCR) beta. In some embodiments, there is an
increase in the expression
of TCRafl (i.e., TCRa/13). In some embodiments, the process as described
herein (e.g., the Gen 3
process) shows higher clonal diversity as compared to other processes, for
example the process
referred to as the Gen 2 based on the number of unique peptide CDRs within the
sample.
1009921 In some embodiments, the activation and exhaustion of TILs can be
determined by
examining one or more markers. In some embodiments, the activation and
exhaustion can bc
determined using multicolor flow cytometry. In some embodiments, the
activation and exhaustion of
markers include but not limited to one or more markers selected from the group
consisting of CD3,
PD-1, 2B4/CD244, CD8, CD25, BTLA, KLRG, TIM-3, CD194/CCR4, CD4, TIGIT, CD183,
CD69,
CD95, CD127, CD103, and/or LAG-3). In some embodiments, the activation and
exhaustion of
markers include but not limited to one or more markers selected from the group
consisting of BTLA,
CTLA-4, ICOS, Ki67, LAG-3, PD-1, TIGIT, and/or TIM-3. In some embodiments, the
activation and
exhaustion of markers include but not limited to one or more markers selected
from the group
consisting of BTLA, CTLA-4, ICOS, Ki67, LAG-3, CD103+/CD69+, CD103+/CD69-, PD-
1, TIGIT,
and/or TIM-3. In some embodiments, the T-cell markers (including activation
and exhaustion
markers) can be determined and/or analyzed to examine T-cell activation,
inhibition, or function. In
some embodiments, the T-cell markers can include but are not limited to one or
more markers
selected from the group consisting of TIGIT, CD3, FoxP3, Tim-3, PD-1, CD103,
CTLA-4, LAG-3,
BTLA-4, ICOS, Ki67, CD8, CD25, CD45, CD4, and/or CD59.
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[00993] In some embodiments, TILs that exhibit greater than 3000 pg/106 TILs
to 300000 pg/106
TILs or more Granzyme B secretion are TILs produced by the expansion methods
of the present
invention, including for example Figure 8A and/or Figure 8B and/or Figure 8C
and/or Figure 8D. In
some embodiments, TILs that exhibit greater than 3000 pg/106 TILs greater than
5000 pg/106 TILs,
greater than 7000 pg/106 TILs, greater than 9000 pg/106 TILs, greater than
11000 pg/106 TILs, greater
than 13000 pg/106 TILs, greater than 15000 pg/106 TILs, greater than 17000
pg/106 TILs, greater than
19000 pg/106 TILs, greater than 20000 pg/106 TILs, greater than 40000 pg/106
TILs, greater than
60000 pg/106 TILs, greater than 80000 pg/106 TILs, greater than 100000 pg/106
TILs, greater than
120000 pg/106 TILs, greater than 140000 pg/106 TILs, greater than 160000
pg/106 TILs, greater than
180000 pg/106 TILs, greater than 200000 pg/106 TILs, greater than 220000
pg/106 TILs, greater than
240000 pg/106 TILs, greater than 260000 pg/106 TILs, greater than 280000
pg/106 TILs, greater than
300000 pg/106 TILsor more Granzyme B secretion are TILs produced by the
expansion methods of
the present invention, including for example Figure 8A and/or Figure 8B and/or
Figure 8C and/or
Figure 8D. In some embodiments, TILs that exhibit greater than 3000 pg/106
TILs Granzyme B
secretion are TILs produced by the expansion methods of the present invention,
including for example
Figure 8A and/or Figure 8B and/or Figure 8C and/or Figure 8D. In some
embodiments, TILs that
exhibit greater than 5000 pg/106 TILs Granzyme B secretion are TILs produced
by the expansion
methods of the present invention, including for example Figure 8A and/or
Figure 8B and/or Figure 8C
and/or Figure 8D. In some embodiments, TILs that exhibit greater than 7000
pg/106 TILs Granzyme
B secretion are TILs produced by the expansion methods of the present
invention, including for
example Figure 8A and/or Figure 8B and/or Figure 8C and/or Figure 8D. In some
embodiments, TILs
that exhibit greater than 9000 pg/106 TILs Granzyme B secretion are TILs
produced by the expansion
methods of the present invention, including for example Figure 8A and/or
Figure 8B and/or Figure 8C
and/or Figure 8D. In some embodiments, TILs that exhibit greater than 11000
pg/106 TILs Granzyme
B secretion are TiLs produced by the expansion methods of the present
invention, including for
example Figure 8A and/or Figure 8B and/or Figure 8C and/or Figure 8D. In some
embodiments. TILs
that exhibit greater than 13000 pg/106 TILs Granzyme B secretion are TILs
produced by the
expansion methods of the present invention, including for example Figure 8A
and/or Figure 8B and/or
Figure 8C and/or Figure 8D. In some embodiments, TILs that exhibit greater
than 15000 pg/106 TILs
Granzyme B secretion are TILs produced by the expansion methods of the present
invention,
including for example Figure 8A and/or Figure 8B and/or Figure 8C and/or
Figure 8D. In some
embodiments, TILs that exhibit greater than 17000 pg/106 TILs Granzyme B
secretion are TILs
produced by the expansion methods of the present invention, including for
example Figure 8A and/or
Figure 8B and/or Figure 8C and/or Figure 8D. In some embodiments, TILs that
exhibit greater than
19000 pg/106 TILs Granzyme B secretion are TILs produced by the expansion
methods of the present
invention, including for example Figure 8A and/or Figure 8B and/or Figure 8C
and/or Figure 8D. In
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some embodiments, TILs that exhibit greater than 20000 pg/106 TILs Granzyme B
secretion are TILs
produced by the expansion methods of the present invention, including for
example Figure 8A and/or
Figure 8B and/or Figure 8C and/or Figure 8D. In some embodiments, TILs that
exhibit greater than
40000 pg/106 TILs Granzyme B secretion are TILs produced by the expansion
methods of the present
invention, including for example Figure 8A and/or Figure 8B and/or Figure 8C
and/or Figure 8D. In
some embodiments, IlLs that exhibit greater than 60000 pg/106 TILs Granzyme B
secretion are TILs
produced by the expansion methods of the present invention, including for
example Figure 8A and/or
Figure 8B and/or Figure 8C and/or Figure 8D. In some embodiments, TILs that
exhibit greater than
80000 pg/106 TILs Granzyme B secretion are TILs produced by the expansion
methods of the present
invention, including for example Figure 8A and/or Figure 8B and/or Figure 8C
and/or Figure 8D. In
some embodiments, TILs that exhibit greater than 100000 pg/106 TILs Granzyme B
secretion are
TILs produced by the expansion methods of the present invention, including for
example Figure 8A
and/or Figure 8B and/or Figure 8C and/or Figure 8D. In some embodiments, TILs
that exhibit greater
than 120000 pg/I06TILs Granzyme B secretion are TILs produced by the expansion
methods of the
present invention, including for example Figure 8A and/or Figure 8B and/or
Figure 8C and/or Figure
8D. In some embodiments, TILs that exhibit greater than 140000 pg/106 TILs
Granzyme B secretion
are TILs produced by the expansion methods of the present invention, including
for example Figure
8A and/or Figure 8B and/or Figure 8C and/or Figure 8D. In some embodiments,
TILs that exhibit
greater than 160000 pg/106 TILs Granzyme B secretion are TILs produced by the
expansion methods
of the present invention, including for example Figure 8A and/or Figure 8B
and/or Figure 8C and/or
Figure 8D. In some embodiments, TILs that exhibit greater than 180000 pg/106
TILs Granzyme B
secretion are TILs produced by the expansion methods of the present invention,
including for example
Figure 8A and/or Figure 8B and/or Figure 8C and/or Figure 8D. In some
embodiments, TILs that
exhibit greater than 200000 pg/106 TILs Granzyme B secretion are TILs produced
by the expansion
methods of the present invention, including for example Figure 8A and/or
Figure 8B and/or Figure 8C
and/or Figure 8D. In some embodiments, TILs that exhibit greater than 220000
pg/106 TILs
Granzyme B secretion are TILs produced by the expansion methods of the present
invention,
including for example Figure 8A and/or Figure 8B and/or Figure 8C and/or
Figure 8D. In some
embodiments, TILs that exhibit greater than 240000 pg/106 TILs Granzyme B
secretion are TILs
produced by the expansion methods of the present invention, including for
example Figure 8A and/or
Figure 8B and/or Figure 8C and/or Figure 8D. In some embodiments, TILs that
exhibit greater than
260000 pg/106 TILs Granzyme B secretion are TILs produced by the expansion
methods of the
present invention, including for example Figure 8A and/or Figure 8B and/or
Figure 8C and/or Figure
8D. In some embodiments, TILs that exhibit greater than 280000 pg/106 TILs
Granzyme B secretion
are TILs produced by the expansion methods of the present invention, including
for example Figure
8A and/or Figure 8B and/or Figure 8C and/or Figure 8D. In some embodiments,
TILs that exhibit
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greater than 300000 pg/106 TILs Granzyme B secretion are TILs produced by the
expansion methods
of the present invention, including for example Figure 8A and/or Figure 8B
and/or Figure 8C and/or
Figure 8D. in some embodiments, TILs that exhibit greater than 3000 pg/106
TiLs to 300000 pg/106
TILs or more Granzyme B secretion are TILs produced by the expansion methods
of the present
invention, including for example Figure 8A and/or Figure 8B and/or Figure 8C
and/or Figure 8D. In
some embodiments, IlLs that exhibit greater than 3000 pg/106 TILs greater than
5000 pg/106 TILs,
greater than 7000 pg/106 TILs, greater than 9000 pg/106 TILs, greater than
11000 pg/106 TILs, greater
than 13000 pg/106 TILs, greater than 15000 pg/106 TILs, greater than 17000
pg/106 TILs, greater than
19000 pg/106 TILs, greater than 20000 pg/106 TILs, greater than 40000 pg/106
TILs, greater than
60000 pg/106 TILs, greater than 80000 pg/106 TILs, greater than 100000 pg/106
TILs, greater than
120000 pg/106 TILs, greater than 140000 pg/106 TILs, greater than 160000
pg/106 TILs, greater than
180000 pg/106 TILs, greater than 200000 pg/106 TILs, greater than 220000
pg/106 TILs, greater than
240000 pg/106 TILs, greater than 260000 pg/106 TILs, greater than 280000
pg/106 TILs, greater than
300000 pg/106 TILsor more Granzyme B secretion are TILs produced by the
expansion methods of
the present invention, including for example Figure 8A and/or Figure 8B and/or
Figure 8C and/or
Figure 8D. In some embodiments, TILs that exhibit greater than 3000 pg/106
TILs Granzyme B
secretion are TILs produced by the expansion methods of the present invention,
including for example
Figure 8A and/or Figure 8B and/or Figure 8C and/or Figure 8D. In some
embodiments, TILs that
exhibit greater than 5000 pg/106 TILs Granzyme B secretion are TILs produced
by the expansion
methods of the present invention, including for example Figure 8A and/or
Figure 8B and/or Figure 8C
and/or Figure 8D. In some embodiments, TILs that exhibit greater than 7000
pg/106 TILs Granzyme
B secretion are TILs produced by the expansion methods of the present
invention, including for
example Figure 8A and/or Figure 8B and/or Figure 8C and/or Figure 8D. In some
embodiments. TILs
that exhibit greater than 9000 pg/106 TILs Granzyme B secretion are TILs
produced by the expansion
methods of the present invention, including for example Figure 8A and/or
Figure 8B and/or Figure 8C
and/or Figure 8D. In some embodiments, TILs that exhibit greater than 11000
pg/106 TILs Granzyme
B secretion are TILs produced by the expansion methods of the present
invention, including for
example Figure 8A and/or Figure 8B and/or Figure 8C and/or Figure 8D. In some
embodiments, TILs
that exhibit greater than 13000 pg/106 TILs Granzyme B secretion are TILs
produced by the
expansion methods of the present invention, including for example Figure 8A
and/or Figure 8B and/or
Figure 8C and/or Figure 8D. In some embodiments, TILs that exhibit greater
than 15000 pg/106 TILs
Granzyme B secretion are TILs produced by the expansion methods of the present
invention,
including for example Figure 8A and/or Figure 8B and/or Figure 8C and/or
Figure 8D. In some
embodiments, TILs that exhibit greater than 17000 pg/106 TiLs Granzyme B
secretion are TILs
produced by the expansion methods of the present invention, including for
example Figure 8A and/or
Figure 8B and/or Figure 8C and/or Figure 8D. In some embodiments, TILs that
exhibit greater than
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19000 pg/106 TILs Granzyme B secretion are TILs produced by the expansion
methods of the present
invention, including for example Figure 8A and/or Figure 8B and/or Figure 8C
and/or Figure 8D. In
some embodiments, TILs that exhibit greater than 20000 pg/106TiLs Granzyme B
secretion are 'TILs
produced by the expansion methods of the present invention, including for
example Figure 8A and/or
Figure 8B and/or Figure 8C and/or Figure 8D. In some embodiments, TILs that
exhibit greater than
40000 pg/106 TILs Granzyme B secretion are TILs produced by the expansion
methods of the present
invention, including for example Figure 8A and/or Figure 8B and/or Figure 8C
and/or Figure 8D. In
some embodiments, TILs that exhibit greater than 60000 pg/106 TILs Granzyme B
secretion are TILs
produced by the expansion methods of the present invention, including for
example Figure 8A and/or
Figure 8B and/or Figure 8C and/or Figure 8D. In some embodiments, TILs that
exhibit greater than
80000 pg/106 TILs Granzyme B secretion are TILs produced by the expansion
methods of the present
invention, including for example Figure 8A and/or Figure 8B and/or Figure 8C
and/or Figure 8D. In
some embodiments, TILs that exhibit greater than 100000 pg/106 TILs Granzyme B
secretion are
TILs produced by the expansion methods of the present invention, including for
example Figure 8A
and/or Figure 8B and/or Figure 8C and/or Figure 8D. In some embodiments, TILs
that exhibit greater
than 120000 pg/106 TILs Granzyme B secretion are TILs produced by the
expansion methods of the
present invention, including for example Figure 8A and/or Figure 8B and/or
Figure 8C and/or Figure
8D. In some embodiments. TILs that exhibit greater than 140000 pg/106 TILs
Granzyme B secretion
are TILs produced by the expansion methods of the present invention, including
for example Figure
8A and/or Figure 8B and/or Figure 8C and/or Figure 8D. In some embodiments,
TILs that exhibit
greater than 160000 pg/106 TILs Granzyme B secretion are TILs produced by the
expansion methods
of the present invention, including for example Figure 8A and/or Figure 8B
and/or Figure 8C and/or
Figure 8D. In some embodiments, TILs that exhibit greater than 180000 pg/106
TILs Granzyme B
secretion are TILs produced by the expansion methods of the present invention,
including for example
Figure 8A and/or Figure 8B and/or Figure 8C and/or Figure 8D. In some
embodiments, TILs that
exhibit greater than 200000 pg/106 TILs Granzyme B secretion are TILs produced
by the expansion
methods of the present invention, including for example Figure 8A and/or
Figure 8B and/or Figure 8C
and/or Figure 8D. In some embodiments, TILs that exhibit greater than 220000
pg/106 TILs
Granzyme B secretion are TILs produced by the expansion methods of the present
invention,
including for example Figure 8A and/or Figure 8B and/or Figure 8C and/or
Figure 8D. In some
embodiments, TILs that exhibit greater than 240000 pg/106 TILs Granzyme B
secretion are TILs
produced by the expansion methods of the present invention, including for
example Figure 8A and/or
Figure 8B and/or Figure 8C and/or Figure 8D. In some embodiments, TILs that
exhibit greater than
260000 pg/106 TILs Granzyme B secretion are TiLs produced by the expansion
methods of the
present invention, including for example Figure 8A and/or Figure 8B and/or
Figure 8C and/or Figure
8D. In some embodiments, TILs that exhibit greater than 280000 pg/106 TILs
Granzyme B secretion
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are TILs produced by the expansion methods of the present invention, including
for example Figure
8A and/or Figure 8B and/or Figure 8C and/or Figure 8D. In some embodiments,
TILs that exhibit
greater than 300000 pg/l 06 TILs Granzyme B secretion are TILs produced by the
expansion methods
of the present invention, including for example Figure 8A and/or Figure 8B
and/or Figure 8C and/or
Figure 8D.
1009941 In some embodiments, TILs that exhibit greater than 1000
pg/mL to 300000 pg/mL or
more Granzyme B secretion are TILs produced by the expansion methods of the
present invention,
including for example Figure RA and/or Figure R13 and/or Figure RC and/or
Figure RD. In some
embodiments, TILs that exhibit greater than 1000 pg/mL, greater than 2000
pg/mL, greater than 3000
pg/mL, greater than 4000 pg/mL, greater than 5000 pg/mL, greater than 6000
pg/mL, greater than
7000 pg/mL, greater than 8000 pg/mL, greater than 9000 pg/mL, greater than
10000 pg/mL, greater
than 20000 pg/mL, greater than 30000 pg/mL, greater than 40000 pg/mL, greater
than 50000 pg/mL,
greater than 60000 pg/mL, greater than 70000 pg/mL, greater than 80000 pg/mL,
greater than 90000
pg/mL, greater than 100000 pg/mL or more Granzyme B secretion are TILs
produced by the
expansion methods of the present invention, including for example Figure 8A
and/or Figure 8B and/or
Figure 8C and/or Figure 8D. In some embodiments, TILs that exhibit greater
than 1000 pg/mL
Granzyme B are TILs produced by the expansion methods of the present
invention, including for
example Figure 8A and/or Figure 8B and/or Figure 8C and/or Figure 8D. In some
embodiments, TILs
that exhibit greater than 2000 pg/mL Granzyme B are TILs produced by the
expansion methods of the
present invention, including for example Figure 8A and/or Figure 8B and/or
Figure 8C and/or Figure
8D. In some embodiments, TILs that exhibit greater than 3000 pg/mL Granzyme B
are TILs produced
by the expansion methods of the present invention, including for example
Figure 8A and/or Figure 8B
and/or Figure 8C and/or Figure 8D. In some embodiments, TILs that exhibit
greater than 4000 pg/mL
Granzyme B are TILs produced by the expansion methods of the present
invention, including for
example Figure 8A and/or Figure 8B and/or Figure 8C and/or Figure 8D. In some
embodiments, TILs
that exhibit greater than 5000 pg/mL Granzyme B are TILs produced by the
expansion methods of the
present invention, including for example Figure 8A and/or Figure 8B and/or
Figure 8C and/or Figure
8D. In some embodiments, TILs that exhibit greater than 6000 pg/mL Granzyme B
are TILs produced
by the expansion methods of the present invention, including for example
Figure 8A and/or Figure 8B
and/or Figure 8C and/or Figure 8D. In some embodiments, TILs that exhibit
greater than 7000 pg/mL
Granzyme B are TILs produced by the expansion methods of the present
invention, including for
example Figure 8A and/or Figure 8B and/or Figure 8C and/or Figure 8D. In some
embodiments, TILs
that exhibit greater than 8000 pg/mL Granzyme B are TILs produced by the
expansion methods of the
present invention, including for example Figure 8A and/or Figure 8B and/or
Figure 8C and/or Figure
8D. In some embodiments, TILs that exhibit greater than 9000 pg/mL Granzyme B
arc TILs produced
by the expansion methods of the present invention, including for example
Figure 8A and/or Figure 8B
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and/or Figure 8C and/or Figure 8D. In some embodiments, TILs that exhibit
greater than 10000
pg/mL Granzyme B are TILs produced by the expansion methods of the present
invention, including
for example Figure SA and/or Figure 8B and/or Figure 8C and/or Figure RD. In
some embodiments,
TILs that exhibit greater than 20000 pg/mL Granzyme B are TILs produced by the
expansion methods
of the present invention, including for example Figure 8A and/or Figure 8B
and/or Figure 8C and/or
Figure 8D. In some embodiments, IlLs that exhibit greater than 30000 pg/mL
Granzyme B arc IlLs
produced by the expansion methods of the present invention, including for
example Figure 8A and/or
Figure 8B and/or Figure 8C and/or Figure 8D. In some embodiments, TILs that
exhibit greater than
40000 pg/mL Granzyme B are TILs produced by the expansion methods of the
present invention,
including for example Figure 8A and/or Figure 8B and/or Figure 8C and/or
Figure 8D. In some
embodiments, TILs that exhibit greater than 50000 pg/mL Granzyme B are TILs
produced by the
expansion methods of the present invention, including for example Figure 8A
and/or Figure 8B and/or
Figure 8C and/or Figure 8D. In some embodiments, TILs that exhibit greater
than 60000 pg/mL
Granzyme B are TILs produced by the expansion methods of the present
invention, including for
example Figure 8A and/or Figure 8B and/or Figure 8C and/or Figure 8D. In some
embodiments, TILs
that exhibit greater than 70000 pg/mL Granzyme B are TILs produced by the
expansion methods of
the present invention, including for example Figure 8A and/or Figure 8B and/or
Figure 8C and/or
Figure 8D. In some embodiments, TILs that exhibit greater than 80000 pg/mL
Granzyme B are TILs
produced by the expansion methods of the present invention, including for
example Figure 8A and/or
Figure 8B and/or Figure 8C and/or Figure 8D. In some embodiments, TILs that
exhibit greater than
90000 pg/mL Granzyme B are TILs produced by the expansion methods of the
present invention,
including for example Figure 8A and/or Figure 8B and/or Figure 8C and/or
Figure 8D. In some
embodiments, TILs that exhibit greater than 100000 pg/mL Granzyme B are TILs
produced by the
expansion methods of the present invention, including for example Figure 8A
and/or Figure 8B and/or
Figure 8C and/or Figure 8D. In some embodiments, TILs that exhibit greater
than 120000 pg/mL
Granzyme B secretion are TILs produced by the expansion methods of the present
invention,
including for example Figure 8A and/or Figure 8B and/or Figure 8C and/or
Figure 8D. In some
embodiments, TILs that exhibit greater than 140000 pg/mL Granzyme B are TILs
Granzyme B
secretion are TILs produced by the expansion methods of the present invention,
including for example
Figure 8A and/or Figure 8B and/or Figure 8C and/or Figure 8D. In some
embodiments, TILs that
exhibit greater than 160000 pg/mL Granzyme B secretion are TILs produced by
the expansion
methods of the present invention, including for example Figure 8A and/or
Figure 8B and/or Figure 8C
and/or Figure 8D. In some embodiments, TILs that exhibit greater than 180000
pg/mL Granzyme B
secretion are TILs produced by the expansion methods of the present invention,
including for example
Figure 8A and/or Figure 8B and/or Figure 8C and/or Figure 8D. In some
embodiments, TILs that
exhibit greater than 200000 pg/mL Granzyme B secretion are TILs produced by
the expansion
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methods of the present invention, including for example Figure 8A and/or
Figure 8B and/or Figure 8C
and/or Figure 8D. In some embodiments, TILs that exhibit greater than 220000
pg/mL Granzyme B
secretion are TILs produced by the expansion methods of the present invention,
including for example
Figure 8A and/or Figure 8B and/or Figure 8C and/or Figure 8D. In some
embodiments, TILs that
exhibit greater than 240000 pg/mL Granzyme B secretion are TILs produced by
the expansion
methods of the present invention, including for example Figure 8A and/or
Figure 8B and/or Figure 8C
and/or Figure 8D. In some embodiments, TILs that exhibit greater than 260000
pg/mL Granzyme B
secretion are TILs produced by the expansion methods of the present invention,
including for example
Figure 8A and/or Figure 8B and/or Figure 8C and/or Figure 8D. In some
embodiments, TILs that
exhibit greater than 280000 pg/mL Granzyme B secretion are TILs produced by
the expansion
methods of the present invention, including for example Figure 8A and/or
Figure 8B and/or Figure 8C
and/or Figure 8D. In some embodiments, TILs that exhibit greater than 300000
pg/mL Granzyme B
secretion are TILs produced by the expansion methods of the present invention,
including for example
Figure 8A and/or Figure 8B and/or Figure 8C and/or Figure 8D.
[00995] In some embodiments, the expansion methods of the present
invention produce an
expanded population of TILs that exhibits increased Granzyme B secretion in
vitro including for
example TILs as provided in Figure 8A and/or Figure 8B and/or Figure 8C and/or
Figure 8D, as
compared to non-expanded population of TILs. In some embodiments, Granzyme B
secretion of the
expanded population of TILs of the present invention is increased by at least
one-fold to fifty-fold or
more as compared to non-expanded population or Tits. hi some embodiments, IFN-
7 secretion is
increased by at least one-fold, at least two-fold, at least three-fold, at
least four-fold, at least five-fold,
at least six-fold, at least seven-fold, at least eight-fold, at least nine-
fold, at least ten-fold, at least
twenty-fold, at least thirty-fold, at least forty-fold, at least fifty-fold or
more as compared to non-
expanded population of TILs. In some embodiments, Granzyme B secretion of the
expanded
population of TILs of the present invention is increased by at least one-fold
as compared to non-
expanded population of TILs. In some embodiments, Granzyme B secretion of the
expanded
population of TILs of the present invention is increased by at least two-fold
as compared to non-
expanded population of TILs. In some embodiments, Granzyme B secretion of the
expanded
population of TILs of the present invention is increased by at least three-
fold as compared to non-
expanded population of TILs. In some embodiments, Granzyme B secretion of the
expanded
population of TILs of the present invention is increased by at least four-fold
as compared to non-
expanded population of TILs. in some embodiments, Granzyme B secretion of the
expanded
population of TILs of the present invention is increased by at least five-fold
as compared to non-
expanded population of TILs. In some embodiments, Granzyme B secretion of the
expanded
population of TILs of the present invention is increased by at least six-fold
as compared to non-
expanded population of TILs. In some embodiments, Granzyme B secretion of the
expanded
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population of TILs of the present invention is increased by at least seven-
fold as compared to non-
expanded population of TILs. In some embodiments, Granzyme B secretion of the
expanded
population of TILs of the present invention is increased by at least eight-
fold as compared to non-
expanded population of TILs. In some embodiments, Granzyme B secretion of the
expanded
population of TILs of the present invention is increased by at least nine-fold
as compared to non-
expanded population of IlLs. In some embodiments, Granzyme B secretion of the
expanded
population of TILs of the present invention is increased by at least ten-fold
as compared to non-
expanded population of TILs. In some embodiments, Granzyme B secretion of the
expanded
population of TILs of the present invention is increased by at least twenty-
fold as compared to non-
expanded population of TILs. In some embodiments, Granzyme B secretion of the
expanded
population of TILs of the present invention is increased by at least thirty-
fold as compared to non-
expanded population of TILs. In some embodiments, Granzyme B secretion of the
expanded
population of TILs of the present invention is increased by at least forty-
fold as compared to non-
expanded population of TILs. In some embodiments, Granzyme B secretion of the
expanded
population of TILs of the present invention is increased by at least fifty-
fold as compared to non-
expanded population of TILs.
[00996]
In some embodiments, TILs capable of at least one-fold, two-fold, three-
fold, four-
fold, or five-fold or more lower levels of TNr-a (i.e., TNF-alpha) secretion
as compared to IFN-y
secretion are TILs produced by the expansion methods of the present invention,
including, for
example Figure SA and/or Figure 8B and/or Figure 8C and/or Figure 8D methods.
In some
embodiments, TILs capable of at least one-fold lower levels of TNF-a secretion
as compared to IFN-y
secretion are TILs produced by the expansion methods of the present invention,
including, for
example Figure 8A and/or Figure 8B and/or Figure 8C and/or Figure 8D methods.
In some
embodiments, TILs capable of at least two-fold lower levels of TNF-a secretion
as compared to IFN-y
secretion are TILs produced by the expansion methods of the present invention,
including, for
example Figure 8A and/or Figure 8B and/or Figure 8C and/or Figure 8D methods.
In some
embodiments, TILs capable of at least three-fold lower levels of TNF-a
secretion as compared to IFN-
y secretion are TILs produced by the expansion methods of the present
invention, including, for
example Figure 8A and/or Figure 8B and/or Figure 8C and/or Figure 8D methods.
In some
embodiments, TILs capable of at least four-fold lower levels of TNF-a
secretion as compared to IFN-
y secretion are TILs produced by the expansion methods of the present
invention, including, for
example Figure 8A and/or Figure 8B and/or Figure 8C and/or Figure 8D methods.
In some
embodiments, TILs capable of at least five-fold lower levels of TNF-a
secretion as compared to IFN-y
secretion are TILs produced by the expansion methods of the present invention,
including, for
example Figure 8A and/or Figure 8B and/or Figure 8C and/or Figure 8D methods.
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[00997]
In some embodiments, TILs capable of at least 200 pg/mL/5e5 cells to about
10,000
pg/mL/5e5 cells or more TNF-a (i.e., TNF-alpha) secretion are TILs produced by
the expansion
methods of the present invention, including, for example Figure 8A and/or
Figure 8B and/or Figure
8C and/or Figure 8D methods. In some embodiments, TILs capable of at least 500
pg/mL/5e5 cells to
about 10,000 pg/mL/5e5 cells or more TNF-a secretion are TILs produced by the
expansion methods
of the present invention, including, for example Figure 8A and/or Figure 8B
and/or Figure 8C and/or
Figure 8D methods. In some embodiments, TILs capable of at least 1000
pg/mL/5e5 cells to about
10,000 pg/mL/5e5 cells or more TNF-a secretion are TILs produced by the
expansion methods of the
present invention, including, for example Figure 8A and/or Figure 8B and/or
Figure 8C and/or Figure
8D methods. In some embodiments, TILs capable of at least 2000 pg/mL/5e5 cells
to about 10,000
pg/mL/5e5 cells or more TNF-a secretion are TILs produced by the expansion
methods of the present
invention, including, for example Figure 8A and/or Figure 8B and/or Figure 8C
and/or Figure 8D
methods. In some embodiments, TILs capable of at least 3000 pg/mL/5e5 cells to
about 10,000
pg/mL/5e5 cells or more TNF-a secretion are TILs produced by the expansion
methods of the present
invention, including, for example Figure 8A and/or Figure 8B and/or Figure 8C
and/or Figure 8D
methods. In some embodiments, TILs capable of at least 4000 pg/mL/5e5 cells to
about 10,000
pg/mL/5e5 cells or more TNF-a secretion are TILs produced by the expansion
methods of the present
invention, including, for example Figure 8A and/or Figure 8B and/or Figure 8C
and/or Figure 8D
methods. In some embodiments, TILs capable of at least 5000 pg/mL/5e5 cells to
about 10,000
pg/mL/5e5 cells or more TNF-a secretion are TILs produced by the expansion
methods of the present
invention, including, for example Figure 8A and/or Figure 8B and/or Figure 8C
and/or Figure 8D
methods. In some embodiments, TILs capable of at least 6000 pg/mL/5e5 cells to
about 10,000
pg/mL/5e5 cells or more TNF-a secretion are TILs produced by the expansion
methods of the present
invention, including, for example Figure 8A and/or Figure 8B and/or Figure 8C
and/or Figure 8D
methods. In some embodiments, TILs capable of at least 7000 pg/mL/5e5 cells to
about 10,000
pg/mL/5e5 cells or more TNF-a secretion are TILs produced by the expansion
methods of the present
invention, including, for example Figure 8A and/or Figure 8B and/or Figure 8C
and/or Figure 8D
methods. In some embodiments, TILs capable of at least 8000 pg/mL/5e5 cells to
about 10,000
pg/mL/5e5 cells or more TNF-a secretion are TILs produced by the expansion
methods of the present
invention, including, for example Figure 8A and/or Figure 8B and/or Figure 8C
and/or Figure 8D
methods. In some embodiments, TILs capable of at least 9000 pg/mL/5e5 cells to
about 10,000
pg/mL/5e5 cells or more TNF-a secretion are TILs produced by the expansion
methods of the present
invention, including, for example Figure 8A and/or Figure 8B and/or Figure 8C
and/or Figure 8D
methods.
[00998]
In some embodiments, ITN-7 and granzyrne B levels are measured to
determine the
phenotypic characteristics of the TILs produced by the expansion methods of
the present invention,
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including, for example Figure 8A and/or Figure 8B and/or Figure 8C and/or
Figure 8D methods. In
some embodiments, IFN-y and TNF-a levels are measured to determine the
phenotypic characteristics
of the TILs produced by the expansion methods of the present invention,
including, for example
Figure 8A and/or Figure 8B and/or Figure 8C and/or Figure 8D methods. In some
embodiments,
granzyme B and TNF-a levels are measured to determine the phenotypic
characteristics of the TILs
produced by the expansion methods of the present invention, including, for
example Figure 8A and/or
Figure 8B and/or Figure 8C and/or Figure 8D methods. In some embodiments, IFN-
y, granzyme B
and TNF-a levels are measured to determine the phenotypic characteristics of
the TILs produced by
the expansion methods of the present invention, including, for example Figure
8A and/or Figure 8B
and/or Figure 8C and/or Figure 8D methods.
[00999] In some embodiments, the phenotypic characterization is examined after
cryopreservation.
H. Additional Process Embodiments
[0010001M some embodiments, the invention provides a method for expanding
tumor infiltrating
lymphocytes (TILs) into a therapeutic population of TILs comprising: (a)
obtaining a first population
of TILs from a tumor resected from a subject by processing a tumor sample
obtained from the subject
into multiple tumor fragments; (b) performing a priming first expansion by
culturing the first
population of TILs in a cell culture medium comprising IL-2 and OKT-3, wherein
the priming first
expansion is performed for about 1 to 7 days or about about 1 to 8 days to
obtain the second
population of TILs, wherein the second population of TILs is greater in number
than the first
population of TILs, (c) performing a rapid second expansion by contacting the
second population of
TILs with a cell culture medium comprising IL-2, OKT-3 and exogenous antigen
presenting cells
(APCs) to produce a third population of TILs, wherein the rapid second
expansion is performed for
about 1 to 11 days or about 1 to 10 days to obtain the third population of
TILs, wherein the third
population of TILs is a therapeutic population of TILs; and (d) harvesting the
therapeutic population
of TILs obtained from step (c). In some embodiments, the step of rapid second
expansion is split into
a plurality of steps to achieve a scaling up of the culture by: (1) performing
the rapid second
expansion by culturing the second population of TILs in a small scale culture
in a first container, e.g.,
a G-REX 100MCS container, for a period of about 3 to 4 days, or about 2 to 4
days, and then (2)
effecting the transfer of the second population of TILs from the small scale
culture to a second
container larger than the first container, e.g., a G-REX 500MCS container,
wherein in the second
container the second population of TILs from the small scale culture is
cultured in a larger scale
culture for a period of about 4 to 7 days, or about 4 to 8 days. In some
embodiments, the step of rapid
expansion is split into a plurality of steps to achieve a scaling out of the
culture by: (1) performing the
rapid second expansion by culturing the second population of TILs in a first
small scale culture in a
first container, e.g., a G-REX 100MCS container, for a period of about 3 to 4
days, and then (2)
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effecting the transfer and apportioning of the second population of TILs from
the first small scale
culture into and amongst at least 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14,
15, 16, 17, 18, 19, or 20
second containers that are equal in size to the first container, wherein in
each second container the
portion of the second population of TILs from the first small scale culture
transferred to such second
container is cultured in a second small scale culture for a period of about 4
to 7 days, or about about 4
to 8 days. In some embodiments, the step of rapid expansion is split into a
plurality of steps to achieve
a scaling out and scaling up of the culture by: (1) performing the rapid
second expansion by culturing
the second population of TILs in a small scale culture in a first container,
e.g., a G-REX 100MCS
container, for a period of about 3 to 4 days, or about 2 to 4 days, and then
(2) effecting the transfer
and apportioning of the second population of TILs from the first small scale
culture into and amongst
at least 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14,15, 16, 17, 18, 19, or 20
second containers that are
larger in size than the first container, e.g., G-REX 500MCS containers,
wherein in each second
container the portion of the second population of TILs transferred from the
small scale culture to such
second container is cultured in a larger scale culture for a period of about 4
to 7 days, or about 4 to 8
days. in some embodiments, the step of rapid expansion is split into a
plurality of steps to achieve a
scaling out and scaling up of the culture by: (1) performing the rapid second
expansion by culturing
the second population of TILs in a small scale culture in a first container,
e.g., a G-REX 100MCS
container, for a period of about 3 to 4 days, and then (2) effecting the
transfer and apportioning of the
second population of TILs from the first small scale culture into and amongst
2, 3 or 4 second
containers that are larger in size than the first container, e.g., G-REX
500MCS containers, wherein in
each second container the portion of the second population of TILs transferred
from the small scale
culture to such second container is cultured in a larger scale culture for a
period of about 5 to 7 days.
10010011In some embodiments, the invention provides a method for expanding
tumor infiltrating
lymphocytes (TILs) into a therapeutic population of TILs comprising: (a)
obtaining a first population
of TILs from a tumor resected from a subject by processing a tumor sample
obtained from the subject
into multiple tumor fragments; (b) performing a priming first expansion by
culturing the first
population of TILs in a cell culture medium comprising IL-2 and OKT-3, wherein
the priming first
expansion is performed for about 1 to 8 days to obtain the second population
of TILs, wherein the
second population of TILs is greater in number than the first population of
TILs; (c) performing a
rapid second expansion by contacting the second population of TILs with a cell
culture medium
comprising IL-2, OKT-3 and exogenous antigen presenting cells (APCs) to
produce a third population
of TILs, wherein the rapid second expansion is performed for about 1 to 8 days
to obtain the third
population of TILs, wherein the third population of TILs is a therapeutic
population of TILs; and (d)
harvesting the therapeutic population of TILs obtained from step (c). In some
embodiments, the step
of rapid second expansion is split into a plurality of steps to achieve a
scaling up of the culture by: (1)
performing the rapid second expansion by culturing the second population of
TILs in a small scale
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culture in a first container, e.g., a G-REX 100MCS container, for a period of
about 2 to 4 days, and
then (2) effecting the transfer of the second population of TILs from the
small scale culture to a
second container larger than the first container, e.g., a G-REX 500MCS
container, wherein in the
second container the second population of TILs from the small scale culture is
cultured in a larger
scale culture for a period of about 4 to 8 days. In some embodiments, the step
of rapid expansion is
split into a plurality of steps to achieve a scaling out of the culture by:
(1) performing the rapid second
expansion by culturing the second population of TILs in a first small scale
culture in a first container,
e.g., a G-REX 100MCS container, for a period of about 2 to 4 days, and then
(2) effecting the transfer
and apportioning of the second population of TILs from the first small scale
culture into and amongst
at least 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14,15, 16, 17, 18, 19, or 20
second containers that are
equal in size to the first container, wherein in each second container the
portion of the second
population of TILs from the first small scale culture transferred to such
second container is cultured in
a second small scale culture for a period of about 4 to 6 days. In some
embodiments, the step of rapid
expansion is split into a plurality of steps to achieve a scaling out and
scaling up of the culture by: (1)
performing the rapid second expansion by culturing the second population of
TILs in a small scale
culture in a first container, e.g., a G-REX 100MCS container, for a period of
about 2 to 4 days, and
then (2) effecting the transfer and apportioning of the second population of
TILs from the first small
scale culture into and amongst at least 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12,
13, 14, 15, 16, 17, 18, 19, or 20
second containers that are larger in size than the first container, e.g., G-
REX 500MCS containers,
wherein in each second container the portion of the second population of TILs
transferred from the
small scale culture to such second container is cultured in a larger scale
culture for a period of about 4
to 6 days. In some embodiments, the step of rapid expansion is split into a
plurality of steps to achieve
a scaling out and scaling up of the culture by: (1) performing the rapid
second expansion by culturing
the second population of TILs in a small scale culture in a first container,
e.g., a G-REX 100MCS
container, for a period of about 3 to 4 days, and then (2) effecting the
transfer and apportioning of the
second population of TILs from the first small scale culture into and amongst
2, 3 or 4 second
containers that are larger in size than the first container, e.g., G-REX
500MCS containers, wherein in
each second container the portion of the second population of TILs transferred
from the small scale
culture to such second container is cultured in a larger scale culture for a
period of about 4 to 5 days.
10010021 In some embodiments, the invention provides a method for expanding
tumor infiltrating
lymphocytes (TILs) into a therapeutic population of TILs comprising: (a)
obtaining a first population
of TILs from a tumor resected from a subject by processing a tumor sample
obtained from the subject
into multiple tumor fragments; (b) performing a priming first expansion by
culturing the first
population of TILs in a cell culture medium comprising IL-2 and OKT-3, wherein
the priming first
expansion is performed for about 1 to 7 days to obtain the second population
of TILs, wherein the
second population of TILs is greater in number than the first population of
TILs; (c) performing a
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rapid second expansion by contacting the second population of TILs with a cell
culture medium
comprising IL-2, OKT-3 and exogenous antigen presenting cells (APCs) to
produce a third population
of TILs, wherein the rapid second expansion is performed for about 1 to 11
days to obtain the third
population of TILs, wherein the third population of TILs is a therapeutic
population of TILs; and (d)
harvesting the therapeutic population of TILs obtained from step (c). In some
embodiments, the step
of rapid second expansion is split into a plurality of steps to achieve a
scaling up of the culture by: (1)
performing the rapid second expansion by culturing the second population of
TILs in a small scale
culture in a first container, e.g., a G-REX 100MCS container, for a period of
about 3 to 4 days, and
then (2) effecting the transfer of the second population of TILs from the
small scale culture to a
second container larger than the first container, e.g., a G-REX 500MCS
container, wherein in the
second container the second population of TILs from the small scale culture is
cultured in a larger
scale culture for a period of about 4 to 7 days. In some embodiments, the step
of rapid expansion is
split into a plurality of steps to achieve a scaling out of the culture by:
(1) performing the rapid second
expansion by culturing the second population of TILs in a first small scale
culture in a first container,
e.g., a G-REX 100MCS container, for a period of about 3 to 4 days, and then
(2) effecting the transfer
and apportioning of the second population of TILs from the first small scale
culture into and amongst
at least 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, or 20
second containers that are
equal in size to the first container, wherein in each second container the
portion of the second
population of TILs from the first small scale culture transferred to such
second container is cultured in
a second small scale culture for a period of about 4 to 7 days. In some
embodiments, the step of rapid
expansion is split into a plurality of steps to achieve a scaling out and
scaling up of the culture by: (1)
performing the rapid second expansion by culturing the second population of
TILs in a small scale
culture in a first container, e.g., a G-REX 100MCS container, for a period of
about 3 to 4 days, and
then (2) effecting the transfer and apportioning of the second population of
TILs from the first small
scale culture into and amongst at least 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12,
13, 14, 15, 16, 17, 18, 19, or 20
second containers that are larger in size than the first container, e.g., G-
REX 500MCS containers,
wherein in each second container the portion of the second population of TILs
transferred from the
small scale culture to such second container is cultured in a larger scale
culture for a period of about 4
to 7 days. In some embodiments, the step of rapid expansion is split into a
plurality of steps to achieve
a scaling out and scaling up of the culture by: (1) performing the rapid
second expansion by culturing
the second population of TILs in a small scale culture in a first container,
e.g., a G-REX 100MCS
container, for a period of about 4 days, and then (2) effecting the transfer
and apportioning of the
second population of TILs from the first small scale culture into and amongst
2, 3 or 4 second
containers that are larger in size than the first container, e.g., G-REX
500MCS containers, wherein in
each second container the portion of the second population of TILs transferred
from the small scale
culture to such second container is cultured in a larger scale culture for a
period of about 5 days.
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[0010031M other embodiments, the invention provides the method described in
any of the preceding
paragraphs as applicable above modified such that in step (b) the primary
first expansion is performed
by contacting the first population of TILs with a culture medium which further
comprises exogenous
antigen-presenting cells (APCs), wherein the number of APCs in the culture
medium in step (c) is
greater than the number of APCs in the culture medium in step (b).
10010041ln other embodiments, the invention provides the method described in
any of the preceding
paragraphs as applicable above modified such that in step (c) the culture
medium is supplemented
with additional exogenous APCs.
10010051ln other embodiments, the invention provides the method described in
any of the preceding
paragraphs as applicable above modified such that the ratio of number of APCs
added in the rapid
second expansion to the number of APCs added in step (b) is selected from a
range of from at or
about 1.1:1 to at or about 20:1.
10010061 In other embodiments, the invention provides the method described in
any of the preceding
paragraphs as applicable above modified such that the ratio of number of APCs
added in the rapid
second expansion to the number of APCs added in step (b) is selected from a
range of from at or
about 1.1:1 to at or about 10:1.
10010071in other embodiments, the invention provides the method described in
any of the preceding
paragraphs as applicable above modified such that the ratio of number of APCs
added in the rapid
second expansion to the number of APCs added in step (b) is selected from a
range of from at or
about 1.1:1 to at or about 9:1.
10010081ln other embodiments, the invention provides the method described in
any of the preceding
paragraphs as applicable above modified such that the ratio of number of APCs
added in the rapid
second expansion to the number of APCs added in step (b) is selected from a
range of from at or
about 1.1:1 to at or about 8:1.
10010091ln other embodiments, the invention provides the method described in
any of the preceding
paragraphs as applicable above modified such that the ratio of number of APCs
added in the rapid
second expansion to the number of APCs added in step (b) is selected from a
range of from at or
about 1.1:1 to at or about 7:1.
10010101 In other embodiments, the invention provides the method described in
any of the preceding
paragraphs as applicable above modified such that the ratio of number of APCs
added in the rapid
second expansion to the number of APCs added in step (b) is selected from a
range of from at or
about 1.1:1 to at or about 6:1.
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10010111 In other embodiments, the invention provides the method described in
any of the preceding
paragraphs as applicable above modified such that the ratio of number of APCs
added in the rapid
second expansion to the number of APCs added in step (b) is selected from a
range of from at or
about 1.1:1 to at or about 5:1.
100101211n other embodiments, the invention provides the method described in
any of the preceding
paragraphs as applicable above modified such that the ratio of number of APCs
added in the rapid
second expansion to the number of APCs added in step (b) is selected from a
range of from at or
about 1.1:1 to at or about 4:1.
100101311n other embodiments, the invention provides the method described in
any of the preceding
paragraphs as applicable above modified such that the ratio of number of APCs
added in the rapid
second expansion to the number of APCs added in step (b) is selected from a
range of from at or
about 1.1:1 to at or about 3:1.
10010141111 other embodiments, the invention provides the method described in
any of the preceding
paragraphs as applicable above modified such that the ratio of number of APCs
added in the rapid
second expansion to the number of APCs added in step (b) is selected from a
range of from at or
about 1.1:1 to at or about 2.9:1.
[001015] In other embodiments, the invention provides the method described in
any of the preceding
paragraphs as applicable above modified such that the ratio of number of APCs
added in the rapid
second expansion to the number of APCs added in step (b) is selected from a
range of from at or
about 1.1:1 to at or about 2.8:1.
1001016] In other embodiments, the invention provides the method described in
any of the preceding
paragraphs as applicable above modified such that the ratio of number of APCs
added in the rapid
second expansion to the number of APCs added in step (b) is selected from a
range of from at or
about 1.1:1 to at or about 2.7:1.
100101711n other embodiments, the invention provides the method described in
any of the preceding
paragraphs as applicable above modified such that the ratio of number of APCs
added in the rapid
second expansion to the number of APCs added in step (b) is selected from a
range of from at or
about 1.1:1 to at or about 2.6:1.
10010181111 other embodiments, the invention provides the method described in
any of the preceding
paragraphs as applicable above modified such that the ratio of number of APCs
added in the rapid
second expansion to the number of APCs added in step (b) is selected from a
range of from at or
about 1.1:1 to at or about 2.5: 1.
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1001019] In other embodiments, the invention provides the method described in
any of the preceding
paragraphs as applicable above modified such that the ratio of number of APCs
added in the rapid
second expansion to the number of APCs added in step (b) is selected from a
range of from at or
about 1.1:1 to at or about 2.4:1.
10010201 In other embodiments, the invention provides the method described in
any of the preceding
paragraphs as applicable above modified such that the ratio of number of APCs
added in the rapid
second expansion to the number of APCs added in step (b) is selected from a
range of from at or
about 1.1:1 to at or about 2.3:1.
10010211 In other embodiments, the invention provides the method described in
any of the preceding
paragraphs as applicable above modified such that the ratio of number of APCs
added in the rapid
second expansion to the number of APCs added in step (b) is selected from a
range of from at or
about 1.1:1 to at or about 2.2:1.
10010221 In other embodiments, the invention provides the method described in
any of the preceding
paragraphs as applicable above modified such that the ratio of number of APCs
added in the rapid
second expansion to the number of APCs added in step (b) is selected from a
range of from at or
about 1.1:1 to at or about 2.1:1.
[001023] In other embodiments, the invention provides the method described in
any of the preceding
paragraphs as applicable above modified such that the ratio of number of APCs
added in the rapid
second expansion to the number of APCs added in step (b) is selected from a
range of from at or
about 1.1:1 to at or about 2:1.
10010241 In other embodiments, the invention provides the method described in
any of the preceding
paragraphs as applicable above modified such that the ratio of number of APCs
added in the rapid
second expansion to the number of APCs added in step (b) is selected from a
range of from at or
about 2:1 to at or about 10:1.
10010251 In other embodiments, the invention provides the method described in
any of the preceding
paragraphs as applicable above modified such that the ratio of number of APCs
added in the rapid
second expansion to the number of APCs added in step (b) is selected from a
range of from at or
about 2:1 to at or about 5:1.
10010261 In other embodiments, the invention provides the method described in
any of the preceding
paragraphs as applicable above modified such that the ratio of number of APCs
added in the rapid
second expansion to the number of APCs added in step (b) is selected from a
range of from at or
about 2:1 to at or about 4:1.
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[0010271M other embodiments, the invention provides the method described in
any of the preceding
paragraphs as applicable above modified such that the ratio of number of APCs
added in the rapid
second expansion to the number of APCs added in step (b) is selected from a
range of from at or
about 2:1 to at or about 3:1.
10010281ln other embodiments, the invention provides the method described in
any of the preceding
paragraphs as applicable above modified such that the ratio of number of APCs
added in the rapid
second expansion to the number of APCs added in step (b) is selected from a
range of from at or
about 2:1 to at or about 2.9:1.
10010291ln other embodiments, the invention provides the method described in
any of the preceding
paragraphs as applicable above modified such that the ratio of number of APCs
added in the rapid
second expansion to the number of APCs added in step (b) is selected from a
range of from at or
about 2:1 to at or about 2.8:1.
10010301 In other embodiments, the invention provides the method described in
any of the preceding
paragraphs as applicable above modified such that the ratio of number of APCs
added in the rapid
second expansion to the number of APCs added in step (b) is selected from a
range of from at or
about 2:1 to at or about 2.7:1.
10010311in other embodiments, the invention provides the method described in
any of the preceding
paragraphs as applicable above modified such that the ratio of number of APCs
added in the rapid
second expansion to the number of APCs added in step (b) is selected from a
range of from at or
about 2:1 to at or about 2.6:1.
10010321ln other embodiments, the invention provides the method described in
any of the preceding
paragraphs as applicable above modified such that the ratio of number of APCs
added in the rapid
second expansion to the number of APCs added in step (b) is selected from a
range of from at or
about 2:1 to at or about 2.5:1.
10010331ln other embodiments, the invention provides the method described in
any of the preceding
paragraphs as applicable above modified such that the ratio of number of APCs
added in the rapid
second expansion to the number of APCs added in step (b) is selected from a
range of from at or
about 2:1 to at or about 2.4:1.
10010341 In other embodiments, the invention provides the method described in
any of the preceding
paragraphs as applicable above modified such that the ratio of number of APCs
added in the rapid
second expansion to the number of APCs added in step (b) is selected from a
range of from at or
about 2:1 to at or about 2.3:1.
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1001035] In other embodiments, the invention provides the method described in
any of the preceding
paragraphs as applicable above modified such that the ratio of number of APCs
added in the rapid
second expansion to the number of APCs added in step (b) is selected from a
range of from at or
about 2:1 to at or about 2.2:1.
10010361ln other embodiments, the invention provides the method described in
any of the preceding
paragraphs as applicable above modified such that the ratio of number of APCs
added in the rapid
second expansion to the number of APCs added in step (b) is selected from a
range of from at or
about 2:1 to at or about 2.1:1.
100103711n other embodiments, the invention provides the method described in
any of the preceding
paragraphs as applicable above modified such that the ratio of number of APCs
added in the rapid
second expansion to the number of APCs added in step (b) is at or about 2:1.
100103811n other embodiments, the invention provides the method described in
any of the preceding
paragraphs as applicable above modified such that the ratio of number of APCs
added in the rapid
second expansion to the number of APCs added in step (b) is at or about 1.1:1,
1.2:1, 1.3:1, 1.4:1,
1.5:1, 1.6:1, 1.7:1, 1.8:1, 1.9:1, 2:1, 2.1:1, 2.2:1, 2.3:1, 2.4:1, 2.5:1,
2.6:1, 2.7:1. 2.8:1, 2.9:1, 3:1,
3.1:1, 3.2:1, 3.3:1, 3.4:1, 3.5:1, 3.6:1, 3.7:1, 3.8:1, 3.9:1, 4:1, 4.1:1,
4.2:1, 4.3:1, 4.4:1, 4.5:1, 4.6:1,
4.7:1, 4.8:1, 4.9:1, or 5:1.
10010391ln other embodiments, the invention provides the method described in
any of the preceding
paragraphs as applicable above modified such that the number of APCs added in
the primary first
expansion is at or about 1x108, 1.1x108, 1.2x108, 1.3 x108, 1.4x 108, 1.5x
108, 1.6x108, 1.7x108,
1.8x108, 1.9x108, 2x 108, 2.1x 108, 2.2x 108, 2.3x108, 2.4x108, 2.5><108,
2.6x108, 2.7x108, 2.8x108,
2.9<108, 3 x108, 3.1x 108, 3.2x 108, 3.3 x108, 3.4x108 or 35x 108 APCs, and
such that the number of
APCs added in the rapid second expansion is at or about 3.5<108, 3.6x 108,
3.7>< 108, 3.8 x108, 3.9>< 108,
4x108, 4.1x108, 4.2x108, 4.3x 108, 4.4x 108, 4.5x108, 4.6x108, 4.7x108,
4.8x108, 4.9x108, 5x108,
5.1x108, 5.2x108, 5.3x 108, 5.4x 108, 5.5x108, 5.6x108, 5.7x108, 5.8x108,
5.9x108, 6x108, 6.1x108,
6.2x108, 6.3x108, 6.4x108, 6.5x108, 6.6x 108, 6.7x 108, 6.8x108, 6.9x108,
7x108, 7.1x108, 7.2x108,
7.3x108, 7.4x108, 7.5x 108, 7.6x 108, 7.7x 108, 7.8>108, 7.9x108, 8x108,
8.1x108, 8.2x108, 8.3><10,
8.4x108, 8.5x108, 8.6x108, 8.7x108, 8.8x10, 8.9x108, 9x108, 9.1x108, 9.2x108,
9.3x108, 9.4x108,
9.5x108, 9.6x108, 9.7x108, 9.8x108, 9.9x108 or 1 x109 APCs.
10010401In other embodiments, the invention provides the method described in
any of the preceding
paragraphs as applicable above modified such that the number of APCs added in
the primary first
expansion is selected from the range of at or about lx 108 APCs to at or about
3.5 x108 APCs, and
wherein the number of APCs added in the rapid second expansion is selected
from the range of at or
about 3.5 x108 APCs to at or about 1x109APCs.
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10010411111 other embodiments, the invention provides the method described in
any of the preceding
paragraphs as applicable above modified such that the number of APCs added in
the primary first
expansion is selected from the range of at or about 1.5 A108 APCs to at or
about 3 x 108 APCs, and
wherein the number of APCs added in the rapid second expansion is selected
from the range of at or
about 4x 108 APCs to at or about 7.5 x108 APCs.
10010421 In other embodiments, the invention provides the method described in
any of the preceding
paragraphs as applicable above modified such that the number of APCs added in
the primary first
expansion is selected from the range of at or about 2x 108 APCs to at or about
2.5 x108 APCs, and
wherein the number of APCs added in the rapid second expansion is selected
from the range of at or
about 4.5 x108 APCs to at or about 5.5 x108 APCs.
10010431 In other embodiments, the invention provides the method described in
any of the preceding
paragraphs as applicable above modified such that at or about 2.5 x 108 APCs
are added to the primary
first expansion and at or about 5>< 108 APCs are added to the rapid second
expansion.
10010441In other embodiments, the invention provides the method described in
any of the preceding
paragraphs as applicable above modified such that the antigen-presenting cells
are peripheral blood
mononuclear cells (PBMCs).
10010451 In other embodiments, the invention provides the method described in
any of the preceding
paragraphs as applicable above modified such that the multiple tumor fragments
arc distributed into a
plurality of separate containers, in each of which separate containers the
first population of TILs is
obtained in step (a), the second population of TILs is obtained in step (b),
and the third population of
TILs is obtained in step (c), and the therapeutic populations of TILs from the
plurality of containers in
step (c) are combined to yield the harvested TIL population from step (d).
10010461 In other embodiments, the invention provides the method described in
any of the preceding
paragraphs as applicable above modified such that the multiple tumors are
evenly distributed into the
plurality of separate containers.
10010471 In other embodiments, the invention provides the method described in
any of the preceding
paragraphs as applicable above modified such that the plurality of separate
containers comprises at
least two separate containers.
10010481 In other embodiments, the invention provides the method described in
any of the preceding
paragraphs as applicable above modified such that the plurality of separate
containers comprises from
two to twenty separate containers.
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1001049] In other embodiments, the invention provides the method described in
any of the preceding
paragraphs as applicable above modified such that the plurality of separate
containers comprises from
two to fifteen separate containers.
10010501 In other embodiments, the invention provides the method described in
any of the preceding
paragraphs as applicable above modified such that the plurality of separate
containers comprises from
two to ten separate containers.
10010511 In other embodiments, the invention provides the method described in
any of the preceding
paragraphs as applicable above modified such that the plurality of separate
containers comprises from
two to five separate containers.
10010521 In other embodiments, the invention provides the method described in
any of the preceding
paragraphs as applicable above modified such that the plurality of separate
containers comprises 2, 3,
4, 5, 6, 7, 8,9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, or 20 separate
containers.
10010531 In other embodiments, the invention provides the method described in
any of the preceding
paragraphs as applicable above modified such that for each container in which
the priming first
expansion is performed on a first population of TILs in step (b) the rapid
second expansion in step (c)
is performed in the same container on the second population of TILs produced
from such first
population of TILs.
10010541 In other embodiments, the invention provides the method described in
any of the preceding
paragraphs as applicable above modified such that each of the separate
containers comprises a first
gas-permeable surface area.
10010551 In other embodiments, the invention provides the method described in
any of the preceding
paragraphs as applicable above modified such that the multiple tumor fragments
are distributed in a
single container.
10010561 In other embodiments, the invention provides the method described in
any of the preceding
paragraphs as applicable above modified such that the single container
comprises a first gas-
permeable surface area.
10010571 In other embodiments, the invention provides the method described in
any of the preceding
paragraphs as applicable above modified such that in step (b) the primary
first expansion is perfornied
by supplementing the cell culture medium of the first population of TILs with
additional antigen-
presenting cells (APCs), wherein the number of APCs added in step (c) is
greater than the number of
APCs added in step (b), and wherein in step (b) the APCs are layered onto the
first gas-permeable
surface area at an average thickness of at or about one cell layer to at or
about three cell layers.
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1001058] In other embodiments, the invention provides the method described in
any of the preceding
paragraphs as applicable above modified such that in step (b) the APCs are
layered onto the first gas-
permeable surface area at an average thickness of at or about 1.5 cell layers
to at or about 2.5 cell
layers.
10010591ln other embodiments, the invention provides the method described in
any of the preceding
paragraphs as applicable above modified such that in step (b) the APCs are
layered onto the first gas-
permeable surface area at an average thickness of at or about 2 cell layers.
10010601ln other embodiments, the invention provides the method described in
any of the preceding
paragraphs as applicable above modified such that in step (b) the APCs are
layered onto the first gas-
permeable surface area at an average thickness of at or about 1, 1.1, 1.2,
1.3, 1.4, 1.5, 1.6, 1.7, 1.8,
1.9, 2, 2.1, 2.2, 2.3, 2.4, 2.5, 2.6, 2.7, 2.8, 2.9 or 3 cell layers.
1001061] In other embodiments, the invention provides the method described in
any of the preceding
paragraphs as applicable above modified such that in step (c) the APCs are
layered onto the first gas-
permeable surface area at an average thickness of at or about 3 cell layers to
at or about 10 cell layers.
10010621 In other embodiments, the invention provides the method described in
any of the preceding
paragraphs as applicable above modified such that in step (c) the APCs are
layered onto the first gas-
permeable surface area at an average thickness of at or about 4 cell layers to
at or about 8 cell layers.
10010631 In other embodiments, the invention provides the method described in
any of the preceding
paragraphs as applicable above modified such that in step (c) the APCs are
layered onto the first gas-
permeable surface area at an average thickness of at or about 3, 4, 5, 6, 7,
8, 9 or 10 cell layers.
10010641 In other embodiments, the invention provides the method described in
any of the preceding
paragraphs as applicable above modified such that in step (c) the APCs are
layered onto the first gas-
permeable surface area at an average thickness of at or about 4, 4.1, 4.2,
4.3, 4.4, 4.5, 4.6, 4.7, 4.8,
4.9, 5, 5.1, 5.2, 5.3, 5.4, 5.5, 5.6, 5.7, 5.8, 5.9, 6, 6.1, 6.2, 6.3, 6.4,
6.5, 6.6, 6.7, 6.8, 6.9, 7, 7.1, 7.2,
7.3, 7.4, 7.5, 7.6, 7.7, 7.8, 7.9 or 8 cell layers.
10010651 In other embodiments, the invention provides the method described in
any of the preceding
paragraphs as applicable above modified such that in step (b) the priming
first expansion is performed
in a first container comprising a first gas-permeable surface area and in step
(c) the rapid second
expansion is performed in a second container comprising a second gas-permeable
surface area.
10010661 In other embodiments, the invention provides the method described in
any of the preceding
paragraphs as applicable above modified such that the second container is
larger than the first
container.
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10010671111 other embodiments, the invention provides the method described in
any of the preceding
paragraphs as applicable above modified such that in step (b) the primary
first expansion is performed
by supplementing the cell culture medium of the first population of TILs with
additional antigen-
presenting cells (APCs), wherein the number of APCs added in step (c) is
greater than the number of
APCs added in step (b), and wherein in step (b) the APCs are layered onto the
first gas-permeable
surface area at an average thickness of at or about one cell layer to at or
about three cell layers.
10010681In other embodiments, the invention provides the method described in
any of the preceding
paragraphs as applicable above modified such that in step (b) the APCs are
layered onto the first gas-
permeable surface area at an average thickness of at or about 1.5 cell layers
to at or about 2.5 cell
layers.
10010691In other embodiments, the invention provides the method described in
any of the preceding
paragraphs as applicable above modified such that in step (b) the APCs arc
layered onto the first gas-
permeable surface area at an average thickness of at or about 2 cell layers.
10010701In other embodiments, the invention provides the method described in
any of the preceding
paragraphs as applicable modified such that in step (b) the APCs are layered
onto the first gas-
permeable surface area at an average thickness of at or about 1, 1.1, 1.2,
1.3, 1.4, 1.5, 1.6, 1.7, 1.8,
1.9, 2, 2.1, 2.2, 2.3, 2.4, 2.5, 2.6, 2.7, 2.8, 2.9 or 3 cell layers.
10010711In other embodiments, the invention provides the method described in
any of the preceding
paragraphs as applicable above modified such that in step (c) the APCs are
layered onto the second
gas-permeable surface area at an average thickness of at or about 3 cell
layers to at or about 10 cell
layers.
10010721In other embodiments, the invention provides the method described in
any of the preceding
paragraphs as applicable above modified such that in step (c) the APCs are
layered onto the second
gas-permeable surface area at an average thickness of at or about 4 cell
layers to at or about 8 cell
layers.
10010731In other embodiments, the invention provides the method described in
any of the preceding
paragraphs as applicable above modified such that in step (c) the APCs are
layered onto the second
gas-permeable surface area at an average thickness of at or about 3, 4, 5, 6,
7, 8, 9 or 10 cell layers.
10010741 In other embodiments, the invention provides the method described in
any of the preceding
paragraphs as applicable modified such that in step (c) the APCs are layered
onto the second gas-
permeable surface area at an average thickness of at or about 4, 4.1, 4.2,
4.3, 4.4,4.5, 4.6, 4.7, 4.8,
4.9, 5, 5.1, 5.2, 5.3, 5.4, 5.5, 5.6, 5.7, 5.8, 5.9, 6, 6.1, 6.2, 6.3, 6.4,
6.5, 6.6, 6.7, 6.8, 6.9, 7, 7.1, 7.2,
7.3, 7.4, 7.5, 7.6, 7.7, 7.8, 7.9 or 8 cell layers.
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10010751111 other embodiments, the invention provides the method described in
any of the preceding
paragraphs as applicable above modified such that in step (b) the priming
first expansion is performed
in a first container comprising a first gas-permeable surface area and in step
(c) the rapid second
expansion is performed in the first container.
10010761In other embodiments, the invention provides the method described in
any of the preceding
paragraphs as applicable above modified such that in step (b) the primary
first expansion is performed
by supplementing the cell culture medium of the first population of TILs with
additional antigen-
presenting cells (APCs), wherein the number of APCs added in step (c) is
greater than the number of
APCs added in step (b), and wherein in step (b) the APCs are layered onto the
first gas-permeable
surface area at an average thickness of at or about one cell layer to at or
about three cell layers.
10010771In other embodiments, the invention provides the method described in
any of the preceding
paragraphs as applicable above modified such that in step (b) the APCs are
layered onto the first gas-
permeable surface area at an average thickness of at or about 1.5 cell layers
to at or about 2.5 cell
layers.
10010781In other embodiments, the invention provides the method described in
any of the preceding
paragraphs as applicable above modified such that in step (b) the APCs are
layered onto the first gas-
permeable surface area at an average thickness of at or about 2 cell layers.
10010791In other embodiments, the invention provides the method described any
of the preceding
paragraphs as applicable above modified such that in step (b) the APCs are
layered onto the first gas-
permeable surface area at an average thickness of at or about 1, 1.1, 1.2,
1.3, 1.4, 1.5, 1.6, 1.7, 1.8,
1.9, 2, 2.1, 2.2, 2.3, 2.4, 2.5, 2.6, 2.7, 2.8, 2.9 or 3 cell layers.
10010801In other embodiments, the invention provides the method described in
any of the preceding
paragraphs as applicable above modified such that in step (c) the APCs are
layered onto the first gas-
permeable surface area at an average thickness of at or about 3 cell layers to
at or about 10 cell layers.
10010811In other embodiments, the invention provides the method described in
any of the preceding
paragraphs as applicable above modified such that in step (c) the APCs are
layered onto the first gas-
permeable surface area at an average thickness of at or about 4 cell layers to
at or about 8 cell layers.
10010821In other embodiments, the invention provides the method described in
any of the preceding
paragraphs as applicable above modified such that in step (c) the APCs are
layered onto the first gas-
perrneable surface area at an average thickness of at or about 3, 4, 5, 6, 7,
8, 9 or 10 cell layers.
100108311n other embodiments, the invention provides the method described in
any of the preceding
paragraphs as applicable above modified such that in step (c) the APCs are
layered onto the first gas-
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permeable surface area at an average thickness of at or about 4, 4.1, 4.2,
4.3, 4.4, 4.5, 4.6, 4.7, 4.8,
4.9, 5, 5.1, 5.2, 5.3, 5.4, 5.5, 5.6, 5.7, 5.8, 5.9, 6, 6.1, 6.2, 6.3, 6.4,
6.5, 6.6, 6.7, 6.8, 6.9, 7, 7.1, 7.2,
7.3, 7.4, 7.5, 7.6, 7.7, 7.8, 7.9 or 8 cell layers.
10010841 In other embodiments, the invention provides the method described in
any of the preceding
paragraphs as applicable above modified such that in step (b) the primary
first expansion is performed
by supplementing the cell culture medium of the first population of TILs with
additional antigen-
presenting cells (APCs), wherein the number of APCs added in step (c) is
greater than the number of
APCs added in step (b), and wherein the ratio of the average number oflayers
of APCs layered in step
(b) to the average number of layers of APCs layered in step (c) is selected
from the range of at or
about 1:1.1 to at or about 1:10.
10010851 In other embodiments, the invention provides the method described in
any of the preceding
paragraphs as applicable above modified such that in step (b) the primary
first expansion is performed
by supplementing the cell culture medium of the first population of TILs with
additional antigen-
presenting cells (APCs), wherein the number of APCs added in step (c) is
greater than the number of
APCs added in step (b), and wherein the ratio of the average number of layers
of APCs layered in step
(b) to the average number of layers of APCs layered in step (c) is selected
from the range of at or
about 1:1.1 to at or about 1:9.
10010861 In other embodiments, the invention provides the method described in
any of the preceding
paragraphs as applicable above modified such that in step (b) the primary
first expansion is performed
by supplementing the cell culture medium of the first population of TILs with
additional antigen-
presenting cells (APCs), wherein the number of APCs added in step (c) is
greater than the number of
APCs added in step (b), and wherein the ratio of the average number of layers
of APCs layered in step
(b) to the average number of layers of APCs layered in step (c) is selected
from the range of at or
about 1:1.1 to at or about 1:8.
10010871 In other embodiments, the invention provides the method described in
any of the preceding
paragraphs as applicable above modified such that in step (b) the primary
first expansion is performed
by supplementing the cell culture medium of the first population of TILs with
additional antigen-
presenting cells (APCs), wherein the number of APCs added in step (c) is
greater than the number of
APCs added in step (b), and wherein the ratio of the average number of layers
of APCs layered in step
(b) to the average number of layers of APCs layered in step (c) is selected
from the range of at or
about 1:1.1 to at or about 1:7.
10010881 In other embodiments, the invention provides the method described in
any of the preceding
paragraphs as applicable above modified such that in step (b) the primary
first expansion is performed
by supplementing the cell culture medium of the first population of TILs with
additional antigen-
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presenting cells (APCs), wherein the number of APCs added in step (c) is
greater than the number of
APCs added in step (b), and wherein the ratio of the average number of layers
of APCs layered in step
(b) to the average number of layers of APCs layered in step (c) is selected
from the range of at or
about 1:1.1 to at or about 1:6.
1001089] In other embodiments, the invention provides the method described in
any of the preceding
paragraphs as applicable above modified such that in step (b) the primary
first expansion is performed
by supplementing the cell culture medium of the first population of TILs with
additional antigen-
presenting cells (APCs), wherein the number of APCs added in step (c) is
greater than the number of
APCs added in step (b), and wherein the ratio of the average number of layers
of APCs layered in step
(b) to the average number of layers of APCs layered in step (c) is selected
from the range of at or
about 1:1.1 to at or about 1:5.
1001090] In other embodiments, the invention provides the method described in
any of the preceding
paragraphs as applicable above modified such that in step (b) the primary
first expansion is performed
by supplementing the cell culture medium of the first population of TILs with
additional antigen-
presenting cells (APCs), wherein the number of APCs added in step (c) is
greater than the number of
APCs added in step (b), and wherein the ratio of the average number of layers
of APCs layered in step
(b) to the average number of layers of APCs layered in step (c) is selected
from the range of at or
about 1 : 1 .1 to at or about 1:4.
1001091] In other embodiments, the invention provides the method described in
any of the preceding
paragraphs as applicable above modified such that in step (b) the primary
first expansion is performed
by supplementing the cell culture medium of the first population of TILs with
additional antigen-
presenting cells (APCs), wherein the number of APCs added in step (c) is
greater than the number of
APCs added in step (b), and wherein the ratio of the average number of layers
of APCs layered in step
(b) to the average number of layers of APCs layered in step (c) is selected
from the range of at or
about 1:1.1 to at or about 1:3.
100109211n other embodiments, the invention provides the method described in
any of the preceding
paragraphs as applicable above modified such that in step (b) the primary
first expansion is performed
by supplementing the cell culture medium of the first population of TILs with
additional antigen-
presenting cells (APCs), wherein the number of APCs added in step (c) is
greater than the number of
APCs added in step (b), and wherein the ratio of the average number of layers
of APCs layered in step
(b) to the average number of layers of APCs layered in step (c) is selected
from the range of at or
about 1:1.1 to at or about 1:2.
10010931 In other embodiments, the invention provides the method described in
any of the preceding
paragraphs as applicable above modified such that in step (b) the primary
first expansion is performed
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by supplementing the cell culture medium of the first population of TILs with
additional antigen-
presenting cells (APCs), wherein the number of APCs added in step (c) is
greater than the number of
APCs added in step (b), and wherein the ratio of the average number of layers
of APCs layered in step
(b) to the average number of layers of APCs layered in step (c) is selected
from the range of at or
about 1:1.2 to at or about 1:8.
10010941111 other embodiments, the invention provides the method described in
any of the preceding
paragraphs as applicable above modified such that in step (b) the primary
first expansion is performed
by supplementing the cell culture medium of the first population of TILs with
additional antigen-
presenting cells (APCs), wherein the number of APCs added in step (c) is
greater than the number of
APCs added in step (b), and wherein the ratio of the average number of layers
of APCs layered in step
(b) to the average number of layers of APCs layered in step (c) is selected
from the range of at or
about 1:1.3 to at or about 1:7.
1001095] In other embodiments, the invention provides the method described in
any of the preceding
paragraphs as applicable above modified such that in step (b) the primary
first expansion is performed
by supplementing the cell culture medium of the first population of TILs with
additional antigen-
presenting cells (APCs), wherein the number of APCs added in step (c) is
greater than the number of
APCs added in step (b), and wherein the ratio of the average number of layers
of APCs layered in step
(b) to the average number of layers of APCs layered in step (c) is selected
from the range of at or
about 1:1.4 to at or about 1:6.
10010961 In other embodiments, the invention provides the method described in
any of the preceding
paragraphs as applicable above modified such that in step (b) the primary
first expansion is performed
by supplementing the cell culture medium of the first population of TILs with
additional antigen-
presenting cells (APCs), wherein the number of APCs added in step (c) is
greater than the number of
APCs added in step (b), and wherein the ratio of the average number of layers
of APCs layered in step
(b) to the average number of layers of APCs layered in step (c) is selected
from the range of at or
about 1:1.5 to at or about 1:5.
10010971 In other embodiments, the invention provides the method described in
any of the preceding
paragraphs as applicable above modified such that in step (b) the primary
first expansion is performed
by supplementing the cell culture medium of the first population of TILs with
additional antigen-
presenting cells (APCs), wherein the number of APCs added in step (c) is
greater than the number of
APCs added in step (b), and wherein the ratio of the average number of layers
of APCs layered in step
(b) to the average number of layers of APCs layered in step (c) is selected
from the range of at or
about 1:1.6 to at or about 1:4.
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[0010981M other embodiments, the invention provides the method described in
any of the preceding
paragraphs as applicable above modified such that in step (b) the primary
first expansion is performed
by supplementing the cell culture medium of the first population of TILs with
additional antigen-
presenting cells (APCs), wherein the number of APCs added in step (c) is
greater than the number of
APCs added in step (b), and wherein the ratio of the average number of layers
of APCs layered in step
(b) to the average number of layers of APCs layered in step (c) is selected
from the range of at or
about 1:1.7 to at or about 1:3.5.
10010991ln other embodiments, the invention provides the method described in
any of the preceding
paragraphs as applicable above modified such that in step (b) the primary
first expansion is performed
by supplementing the cell culture medium of the first population of TILs with
additional antigen-
presenting cells (APCs), wherein the number of APCs added in step (c) is
greater than the number of
APCs added in step (b), and wherein the ratio of the average number of layers
of APCs layered in step
(b) to the average number of layers of APCs layered in step (c) is selected
from the range of at or
about 1:1.8 to at or about 1:3.
[0011001in other embodiments, the invention provides the method described in
any of the preceding
paragraphs as applicable above modified such that in step (b) the primary
first expansion is performed
by supplementing the cell culture medium of the first population of TILs with
additional antigen-
presenting cells (APCs), wherein the number of APCs added in step (c) is
greater than the number of
APCs added in step (b), and wherein the ratio of the average number of layers
of APCs layered in step
(b) to the average number of layers of APCs layered in step (c) is selected
from the range of at or
about 1:1.9 to at or about 1:2.5.
10011011In other embodiments, the invention provides the method described in
any of the preceding
paragraphs as applicable above modified such that in step (b) the primary
first expansion is performed
by supplementing the cell culture medium of the first population of TILs with
additional antigen-
presenting cells (APCs), wherein the number of APCs added in step (c) is
greater than the number of
APCs added in step (b), and wherein the ratio of the average number of layers
of APCs layered in step
(b) to the average number of layers of APCs layered in step (c) is selected
from the range of at or
about 1:2.
10011021In other embodiments, the invention provides the method described in
any of the preceding
paragraphs as applicable above modified such that in step (b) the primary
first expansion is performed
by supplementing the cell culture medium of the first population of TILs with
additional antigen-
presenting cells (APCs), wherein the number of APCs added in step (c) is
greater than the number of
APCs added in step (b), and wherein the ratio of the average number of layers
of APCs layered in step
(b) to the average number of layers of APCs layered in step (c) is selected
from at or about 1:1.1,
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1:1.2, 1:1.3, 1:1.4, 1:1.5, 1:1.6, 1:1.7, 1:1.8, 1:1.9, 1:2, 1:2.1, 1:2.2,
1:2.3, 1:2.4, 1:2.5, 1:2.6, 1:2.7,
1:2.8, 1:2.9, 1:3, 1:3.1, 1:3.2, 1:3.3, 1:3.4, 1:3.5, 1:3.6, 1:3.7, 1:3.8,
1:3.9, 1:4, 1:4.1, 1:4.2, 1:4.3,
1:4.4, 1:4.5, 1:4.6, 1:4.7, 1:4.8, 1:4.9, 1:5, 1:5.1, 1:5.2, 1:5.3, 1:5.4,
1:5.5, 1:5.6, 1:5.7, 1:5.8, 1:5.9,
1:6, 1:6.1. 1:6.2, 1:6.3, 1:6.4, 1:6.5, 1:6.6, 1:6.7, 1:6.8, 1:6.9, 1:7,
1:7.1, 1:7.2, 1:7.3, 1:7.4, 1:7.5,
1:7.6, 1:7.7, 1:7.8, 1:7.9, 1:8, 1:8.1, 1:8.2, 1:8.3, 1:8.4, 1:8.5, 1:8.6,
1:8.7, 1:8.8, 1:8.9, 1:9, 1:9.1,
1:9.2, 1:9.3, 1:9.4, 1:9.5, 1:9.6, 1:9.7, 1:9.8, 1:9.9 or 1:10.
1001103] In other embodiments, the invention provides the method described in
any of the preceding
paragraphs as applicable above modified such that the ratio of the number of
TILs in the second
population of TILs to the number of TILs in the first population of TILs is at
or about 1.5:1 to at or
about 100:1.
10011041 In other embodiments, the invention provides the method described in
any of the preceding
paragraphs as applicable above modified such that the ratio of the number of
TILs in the second
population of TILs to the number of TILs in the first population of TILs is at
or about 50:1.
100H051I11 other embodiments, the invention provides the method described in
any of the preceding
paragraphs as applicable above modified such that the ratio of the number of
TILs in the second
population of TILs to the number of TILs in the first population of TILs is at
or about 25:1_
10011061 In other embodiments, thc invention provides the method described in
any of thc preceding
paragraphs as applicable above modified such that the ratio of the number of
TILs in the second
population of TILs to the number of TILs in the first population of TILs is at
or about 20:1.
10011071ln other embodiments, the invention provides the method described in
any of the preceding
paragraphs as applicable above modified such that the ratio of the number of
TILs in the second
population of TILs to the number of TILs in the first population of TILs is at
or about 10:1.
10011081 In other embodiments, the invention provides the method described in
any of the preceding
paragraphs as applicable above modified such that the second population of
TILs is at least at or about
50-fold greater in number than the first population of TILs.
10011091 In other embodiments, the invention provides the method described in
any of the preceding
paragraphs as applicable above modified such that the second population of
TILs is at least at or about
1-, 2-, 3-, 4-, 5-, 6-, 7-, 8-, 9-, 10-, 11-, 12-, 13-, 14-, 15-, 16-, 17-, 18-
, 19-, 20-, 21-, 22-, 23-, 24-, 25-
,26-, 27-, 28-, 29-, 30-, 31-, 32-, 33-, 34-, 35-, 36-, 37-, 38-, 39-, 40-, 41-
, 42-, 43-, 44-, 45-, 46-, 47-,
48-, 49- or 50-fold greater in number than thc first population of TILs.
10011101 In other embodiments, the invention provides the method described in
any of the preceding
paragraphs as applicable above modified such that at or about 2 days or at or
about 3 days after the
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commencement of the second period in step (c), the cell culture medium is
supplemented with
additional IL-2.
10011111 In other embodiments, the invention provides the method described in
any of the preceding
paragraphs as applicable above modified to further comprise the step of
cryopre serving the harvested
TIL population in step (d) using a cryopreservation process.
100111211n other embodiments, the invention provides the method described in
any of the preceding
paragraphs as applicable above modified to comprise performing after step (d)
the additional step of
(e) transferring the harvested TIL population from step (d) to an infusion bag
that optionally contains
Hypo'Thermosol.
10011131 In other embodiments, the invention provides the method described in
any of the preceding
paragraphs as applicable above modified to comprise the step of cryopreserving
the infusion bag
comprising the harvested TIL population in step (e) using a cryopreservation
process.
10011141 In other embodiments, the invention provides the method described in
any of the preceding
paragraphs as applicable above modified such that the cryopreservation process
is perfon-ned using a
1: 1 ratio of harvested TIL population to cryopreservation media.
[001115] In other embodiments, the invention provides the method described in
any of the preceding
paragraphs as applicable above modified such that the antigen-presenting cells
are peripheral blood
mononuclear cells (PBMCs).
10011161 In other embodiments, the invention provides the method described in
any of the preceding
paragraphs as applicable above modified such that the PBMCs are irradiated and
allogeneic.
10011171 In other embodiments, the invention provides the method described in
any of the preceding
paragraphs as applicable above modified such that the total number of APCs
added to the cell culture
in step (b) is 2.5 x 108.
10011181 In other embodiments, the invention provides the method described in
any of the preceding
paragraphs as applicable above modified such that the total number of APCs
added to the cell culture
in step (c) is 5 X 108.
10011191 In other embodiments, the invention provides the method described in
any of the preceding
paragraphs as applicable above modified such that the APCs are PBMCs.
10011201 In other embodiments, the invention provides the method described in
any of the preceding
paragraphs as applicable above modified such that the PBMCs are irradiated and
allogeneic.
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1001121] In other embodiments, the invention provides the method described in
any of the preceding
paragraphs as applicable above modified such that the antigen-presenting cells
are artificial antigen-
presenting cells.
1001122] In other embodiments, the invention provides the method described in
any of the preceding
paragraphs as applicable above modified such that the harvesting in step (d)
is performed using a
membrane-based cell processing system.
10011231 In other embodiments, the invention provides the method described in
any of the preceding
paragraphs as applicable above modified such that the harvesting in step (d)
is performed using a
LOVO cell processing system.
100112411n other embodiments, the invention provides the method described in
any of the preceding
paragraphs as applicable above modified such that the multiple fragments
comprise at or about 5 to at
or about 60 fragments per container in step (b).
10011251111 other embodiments, the invention provides the method described in
any of the preceding
paragraphs as applicable above modified such that the multiple fragments
comprise at or about 10 to
at or about 60 fragments per container in step (b).
10011261 In other embodiments, the invention provides the method described in
any of the preceding
paragraphs as applicable above modified such that the multiple fragments
comprise at or about 15 to
at or about 60 fragments per container in step (b).
10011271 In other embodiments, the invention provides the method described in
any of the preceding
paragraphs as applicable above modified such that the multiple fragments
comprise at or about 20 to
at or about 60 fragments per container in step (b).
10011281 In other embodiments, the invention provides the method described in
any of the preceding
paragraphs as applicable above modified such that the multiple fragments
comprise at or about 25 to
at or about 60 fragments per container in step (b).
10011291 In other embodiments, the invention provides the method described in
any of the preceding
paragraphs as applicable above modified such that the multiple fragments
comprise at or about 30 to
at or about 60 fragments per container in step (b).
1001130] In other embodiments, the invention provides the method described in
any of the preceding
paragraphs as applicable above modified such that the multiple fragments
comprise at or about 35 to
at or about 60 fragments per container in step (b).
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1001131] In other embodiments, the invention provides the method described in
any of the preceding
paragraphs as applicable above modified such that the multiple fragments
comprise at or about 40 to
at or about 60 fragments per container in step (b).
1001132] In other embodiments, the invention provides the method described in
any of the preceding
paragraphs as applicable above modified such that the multiple fragments
comprise at or about 45 to
at or about 60 fragments per container in step (b).
10011331 In other embodiments, the invention provides the method described in
any of the preceding
paragraphs as applicable above modified such that the multiple fragments
comprise at or about 50 to
at or about 60 fragments per container in step (b).
100113411n other embodiments, the invention provides the method described in
any of the preceding
paragraphs as applicable above modified such that the multiple fragments
comprise at or about 2, 3, 4,
5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25,
26, 27, 28, 29, 30, 31, 32, 33,
34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50, 51, 52,
53, 54, 55, 56, 57, 58, 59 or 60
fragment(s) per container in step (b).
1001135] In other embodiments, the invention provides the method described in
any of the preceding
paragraphs as applicable above modified such that each fragment has a volume
of at or about 27 min3.
100113611n other embodiments, the invention provides the method described in
any of the preceding
paragraphs as applicable above modified such that each fragment has a volume
of at or about 20 min3
to at or about 50 mm3.
1001137] In other embodiments, the invention provides the method described in
any of the preceding
paragraphs as applicable above modified such that each fragment has a volume
of at or about 21 min3
to at or about 30 min3.
100113811n other embodiments, the invention provides the method described in
any of the preceding
paragraphs as applicable above modified such that each fragment has a volume
of at or about 22 min3
to at or about 29.5 min3.
10011391111 other embodiments, the invention provides the method described in
any of the preceding
paragraphs as applicable above modified such that each fragment has a volume
of at or about 23 min3
to at or about 29 nim3.
100114011n other embodiments, the invention provides the method described in
any of the preceding
paragraphs as applicable above modified such that each fragment has a volume
of at or about 24 min3
to at or about 28.5 min3.
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1001141] In other embodiments, the invention provides the method described in
any of the preceding
paragraphs as applicable above modified such that each fragment has a volume
of at or about 25 mm3
to at or about 28 mm3.
100114211n other embodiments, the invention provides the method described in
any of the preceding
paragraphs as applicable above modified such that each fragment has a volume
of at or about 26.5
mm3 to at or about 27.5 mm3.
10011431 In other embodiments, the invention provides the method described in
any of the preceding
paragraphs as applicable above modified such that each fragment has a volume
of at or about 21, 22,
23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41,
42, 43, 44, 45, 46, 47, 48, 49
or 50 mm3.
1001144] In other embodiments, the invention provides the method described in
any of the preceding
paragraphs as applicable above modified such that the multiple fragments
comprise at or about 30 to
at or about 60 fragments with a total volume of at or about 1300 mm3 to at or
about 1500 mm3.
10011451111 other embodiments, the invention provides the method described in
any of the preceding
paragraphs as applicable above modified such that the multiple fragments
comprise at or about 50
fragments with a total volume of at or about 1350 mm3.
100114611n other embodiments, the invention provides the method described in
any of the preceding
paragraphs as applicable above modified such that the multiple fragments
comprise at or about 50
fragments with a total mass of at or about 1 gram to at or about 1.5 grams.
1001147] In other embodiments, the invention provides the method described in
any of the preceding
paragraphs as applicable above modified such that the cell culture medium is
provided in a container
that is a G-container or a Xuri cellbag.
1001148] In other embodiments, the invention provides the method described in
any of the preceding
paragraphs as applicable above modified such that the IL-2 concentration in
the cell culture medium is
about 10,000 IU/mL to about 5,000 IU/mL.
100114911n other embodiments, the invention provides the method described in
any of the preceding
paragraphs as applicable above modified such that the IL-2 concentration in
the cell culture medium is
about 6,000 IU/mL.
100115011n other embodiments, the invention provides the method described in
any of the preceding
paragraphs as applicable above modified such that the cryopreservation media
comprises
dimethlysulfoxide (DMSO).
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10011511 In other embodiments, the invention provides the method described in
any of the preceding
paragraphs as applicable above modified such that the cryopreservation media
comprises 7% to 10%
DMSO.
10011521 In other embodiments, the invention provides the method described in
any of the preceding
paragraphs as applicable above modified such that the first period in step (b)
is performed within a
period of at or about 1 day, 2 days, 3 days, 4 days, 5 days, 6 days, or 7
days.
10011531 In other embodiments, the invention provides the method described in
any of the preceding
paragraphs as applicable above modified such that the second period in step
(c) is performed within a
period of at or about 1 day, 2 days, 3 days, 4 days, 5 days, 6 days, 7 days, 8
days, 9 days, 10 days or
11 days.
1001154] In other embodiments, the invention provides the method described in
any of the preceding
paragraphs as applicable above modified such that the first period in step (b)
and the second period in
step (c) are each individually performed within a period of at or about 1 day,
2 days, 3 days, 4 days, 5
days, 6 days, or 7 days.
10011551 In other embodiments, the invention provides the method described in
any of the preceding
paragraphs as applicable above modified such that the first period in step (b)
and the second period in
step (c) are each individually performed within a period of at or about 5
days, 6 days, or 7 days.
10011561 In other embodiments, the invention provides the method described in
any of the preceding
paragraphs as applicable above modified such that the first period in step (b)
and the second period in
step (c) are each individually performed within a period of at or about 7
days.
10011571 In other embodiments, the invention provides the method described in
any of the preceding
paragraphs as applicable above modified such that steps (a) through (d) are
performed in a total of at
or about 14 days to at or about 18 days.
10011581ln other embodiments, the invention provides the method described in
any of the preceding
paragraphs as applicable above modified such that steps (a) through (d) are
performed in a total of at
or about 15 days to at or about 18 days.
10011591 In other embodiments, the invention provides the method described in
any of the preceding
paragraphs as applicable above modified such that steps (a) through (d) are
performed in a total of at
or about 16 days to at or about 18 days.
10011601 In other embodiments, the invention provides the method described in
any of the preceding
paragraphs as applicable above modified such that steps (a) through (d) are
performed in a total of at
or about 17 days to at or about 18 days.
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1001161] In other embodiments, the invention provides the method described in
any of the preceding
paragraphs as applicable above modified such that steps (a) through (d) are
performed in a total of at
or about 14 days to at or about 17 days.
100116211n other embodiments, the invention provides the method described in
any of the preceding
paragraphs as applicable above modified such that steps (a) through (d) are
performed in a total of at
or about 15 days to at or about 17 days.
10011631 In other embodiments, the invention provides the method described in
any of the preceding
paragraphs as applicable above modified such that steps (a) through (d) are
performed in a total of at
or about 16 days to at or about 17 days.
100116411n other embodiments, the invention provides the method described in
any of the preceding
paragraphs as applicable above modified such that steps (a) through (d) are
performed in a total of at
or about 14 days to at or about 16 days.
100116511n other embodiments, the invention provides the method described in
any of the preceding
paragraphs as applicable above modified such that steps (a) through (d) are
performed in a total of at
or about 15 days to at or about 16 days.
100116611n other embodiments, the invention provides the method described in
any of the preceding
paragraphs as applicable above modified such that steps (a) through (d) are
performed in a total of at
or about 14 days.
10011671 In other embodiments, the invention provides the method described in
any of the preceding
paragraphs as applicable above modified such that steps (a) through (d) are
performed in a total of at
or about 15 days.
100116811n other embodiments, the invention provides the method described in
any of the preceding
paragraphs as applicable above modified such that steps (a) through (d) are
performed in a total of at
or about 16 days.
10011691 In other embodiments, the invention provides the method described in
any of the preceding
paragraphs as applicable above modified such that steps (a) through (d) are
performed in a total of at
or about 17 days.
1001170] In other embodiments, the invention provides the method described in
any of the preceding
paragraphs as applicable above modified such that steps (a) through (d) are
performed in a total of at
or about 18 days.
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10011711111 other embodiments, the invention provides the method described in
any of the preceding
paragraphs as applicable above modified such that steps (a) through (d) are
performed in a total of at
or about 14 days or less.
10011721In other embodiments, the invention provides the method described in
any of the preceding
paragraphs as applicable above modified such that steps (a) through (d) are
performed in a total of at
or about 15 days or less.
10011731In other embodiments, the invention provides the method described in
any of the preceding
paragraphs as applicable above modified such that steps (a) through (d) are
performed in a total of at
or about 16 days or less.
100117411n other embodiments, the invention provides the method described in
any of the preceding
paragraphs as applicable above modified such that steps (a) through (d) are
performed in a total of at
or about 18 days or less.
10011751111 other embodiments, the invention provides the method described in
any of the preceding
paragraphs as applicable above modified such that the therapeutic population
of TILs harvested in
step (d) comprises sufficient TILs for a therapeutically effective dosage of
the TILs.
10011761In other embodiments, the invention provides the method described in
any of the preceding
paragraphs as applicable above modified such that the number of TILs
sufficient for a therapeutically
effective dosage is from at or about 2.3 x101 to at or about 13.7x 101 .
10011771In other embodiments, the invention provides the method described in
any of the preceding
paragraphs as applicable above modified such that the third population of TILs
in step (c) provides for
increased efficacy, increased interferon-gamma production, and/or increased
polyclonality.
100117811n other embodiments, the invention provides the method described in
any of the preceding
paragraphs as applicable above modified such that the third population of TILs
in step (c) provides for
at least a one-fold to five-fold or more interferon-gamma production as
compared to TILs prepared by
a process longer than 16 days.
10011791In other embodiments, the invention provides the method described in
any of the preceding
paragraphs as applicable above modified such that the third population of TILs
in step (c) provides for
at least a one-fold to five-fold or more interferon-gamma production as
compared to TILs prepared by
a process longer than 17 days.
10011801In other embodiments, the invention provides the method described in
any of the preceding
paragraphs as applicable above modified such that the third population of TILs
in step (c) provides for
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at least a one-fold to five-fold or more interferon-gamma production as
compared to TILs prepared by
a process longer than 18 days.
1001181[In other embodiments, the invention provides the method described in
any of the preceding
paragraphs as applicable above modified such that the effector T cells and/or
central memory T cells
obtained from the third population of TILs step (c) exhibit increased CD8 and
CD28 expression
relative to effector T cells and/or central memory T cells obtained from the
second population of cells
step (b).
100118211n other embodiments, the invention provides the method described in
any of the preceding
paragraphs as applicable above modified such that each container recited in
the method is a closed
container.
100118311n other embodiments, the invention provides the method described in
any of the preceding
paragraphs as applicable above modified such that each container recited in
the method is a G-
container.
[001184] In other embodiments, the invention provides the method described in
any of the preceding
paragraphs as applicable above modified such that each container recited in
the method is a GREX-
10.
100118511n other embodiments, the invention provides the method described in
any of the preceding
paragraphs as applicable above modified such that each container recited in
the method is a GREX-
100.
[0011861ln other embodiments, the invention provides the method described in
any of the preceding
paragraphs as applicable above modified such that each container recited in
the method is a GREX-
500.
[00118711n other embodiments, the invention provides the therapeutic
population of tumor
infiltrating lymphocytes (TILs) made by the method described in any of the
preceding paragraphs as
applicable above.
[0011881M other embodiments, the invention provides a therapeutic population
of tumor infiltrating
lymphocytes (TILs) prepared from tumor tissue of a patient, wherein the
therapeutic population of
TILs provides for increased efficacy, increased interferon-gamma production,
and/or increased
polyclonality compared to TILs prepared by a process in which the first
expansion of TILs is
performed without any added antigen-presenting cells (APCs) or OKT3.
[001189] In other embodiments, the invention provides a therapeutic population
of tumor infiltrating
lymphocytes (TILs) prepared from tumor tissue of a patient, wherein the
therapeutic population of
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TILs provides for increased efficacy, increased interferon-gamma production,
and/or increased
polyclonality compared to TILs prepared by a process in which the first
expansion of TILs is
performed without any added antigen-presenting cells (APCs).
100119011n other embodiments, the invention provides a therapeutic population
of tumor infiltrating
lymphocytes (TILs) prepared from tumor tissue of a patient, wherein the
therapeutic population of
TILs provides for increased efficacy, increased interferon-gamma production,
and/or increased
polyclonality compared to TILs prepared by a process in which the first
expansion of TILs is
performed without any added OKT3.
10011911In other embodiments, the invention provides a therapeutic population
of tumor infiltrating
lymphocytes (TILs) prepared from tumor tissue of a patient, wherein the
therapeutic population of
TILs provides for increased efficacy, increased interferon-gamma production,
and/or increased
polyclonality compared to TILs prepared by a process in which the first
expansion of TILs is
performed with no added antigen-presenting cells (APCs) and no added OKT3.
10011921In other embodiments, the invention provides a therapeutic population
of tumor infiltrating
lymphocytes (TILs) prepared from tumor tissue of a patient, wherein the
therapeutic population of
TILs provides for increased efficacy, increased interferon-gamma production,
and/or increased
polyclonality compared to TILs prepared by a process by a process longer than
16 days.
10011931In other embodiments, the invention provides a therapeutic population
of tumor infiltrating
lymphocytes (TILs) prepared from tumor tissue of a patient, wherein the
therapeutic population of
TILs provides for increased efficacy, increased interferon-gamma production,
and/or increased
polyclonality compared to TILs prepared by a process by a process longer than
17 days.
10011941 In other embodiments, the invention provides a therapeutic population
of tumor infiltrating
lymphocytes (TILs) prepared from tumor tissue of a patient, wherein the
therapeutic population of
TILs provides for increased efficacy, increased interferon-gamma production,
and/or increased
polyclonality compared to TILs prepared by a process by a process longer than
18 days.
10011951In other embodiments, the invention provides for the therapeutic
population of TILs
described in any of the preceding paragraphs as applicable above that provides
for increased
interferon-gamma production.
10011961In other embodiments, the invention provides for the therapeutic
population of TILs
described in any of the preceding paragraphs as applicable above that provides
for increased
polyclonality.
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1001197] In other embodiments, the invention provides for the therapeutic
population of TILs
described in any of the preceding paragraphs as applicable above that provides
for increased efficacy.
1001198] In other embodiments, the invention provides for the therapeutic
population of TILs
described in any of the preceding paragraphs as applicable above modified such
that the therapeutic
population of TILs is capable of at least one-fold more interferon-gamma
production as compared to
TILs prepared by a process longer than 16 days. In other embodiments, the
invention provides for the
therapeutic population of TILs described in any of the preceding paragraphs as
applicable above
modified such that the therapeutic population of TILs is capable of at least
one-fold more interferon-
gamma production as compared to TILs prepared by a process longer than 17
days. In other
embodiments, the invention provides for the therapeutic population of TILs
described in any of the
preceding paragraphs as applicable above modified such that the therapeutic
population of TILs is
capable of at least one-fold more interferon-gamma production as compared to
TILs prepared by a
process longer than 18 days. In other embodiments, the TILs are rendered
capable of the at least one-
fold more interferon-gamma production due to the expansion process described
herein, for example as
described in Steps A through F above or according to Steps A through F above
(also as shown, for
example, in Figure 8 (in particular, e.g., Figure 8A and/or Figure 8B and/or
Figure 8C and/or Figure
8D).
100119911n other embodiments, the invention provides for the therapeutic
population of TILs
described in any of the preceding paragraphs as applicable above modified such
that the therapeutic
population of TILs is capable of at least two-fold more interferon-gamma
production as compared to
TILs prepared by a process longer than 16 days. In other embodiments, the
invention provides for the
therapeutic population of TILs described in any of the preceding paragraphs as
applicable above
modified such that the therapeutic population of TILs is capable of at least
two-fold more interferon-
gamma production as compared to TILs prepared by a process longer than 17
days. In other
embodiments, the invention provides for the therapeutic population of TILs
described in any of the
preceding paragraphs as applicable above modified such that the therapeutic
population of TILs is
capable of at least two-fold more interferon-gamma production as compared to
TILs prepared by a
process longer than 18 days. In other embodiments, the TILs are rendered
capable of the at least two-
fold more interferon-gamma production due to the expansion process described
herein, for example as
described in Steps A through F above or according to Steps A through F above
(also as shown, for
example, in Figure 8 (in particular, e.g., Figure 8A and/or Figure 8B and/or
Figure 8C and/or Figure
8D).
10012001 In other embodiments, the invention provides for the therapeutic
population of TILs
described in any of the preceding paragraphs as applicable above modified such
that the therapeutic
population of TILs is capable of at least three-fold more interferon-gamma
production as compared to
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TILs prepared by a process longer than 16 days. In other embodiments, the
invention provides for the
therapeutic population of TILs described in any of the preceding paragraphs as
applicable above
modified such that the therapeutic population of TILs is capable of at least
three-fold more interferon-
gamma production as compared to TILs prepared by a process longer than 17
days. In other
embodiments, the invention provides for the therapeutic population of TILs
described in any of the
preceding paragraphs as applicable above modified such that the therapeutic
population of TILs is
capable of at least three-fold more interferon-gamma production as compared to
TILs prepared by a
process longer than 18 days. In other embodiments, the TILs are rendered
capable of the at least three-
fold more interferon-gamma production due to the expansion process described
herein, for example as
described in Steps A through F above or according to Steps A through F above
(also as shown, for
example, in Figure 8 (in particular, e.g., Figure 8A and/or Figure 8B and/or
Figure 8C and/or Figure
8D).
10012011In other embodiments, the invention provides for a therapeutic
population of tumor
infiltrating lymphocytes (TILs) that is capable of at least one-fold more
interferon-gamma production
as compared to TILs prepared by a process in which the first expansion of TILs
is performed without
any added antigen-presenting cells (APCs). In other embodiments, the TILs are
rendered capable of
the at least one-fold more interferon-gamma production due to the expansion
process described
herein, for example as described in Steps A through F above or according to
Steps A through F above
(also as shown, for example, in Figure 8 (in particular, e.g. Figure 8A and/or
Figure 8B and/or Figure
8C and/or Figure 8D).
[001202] hi other embodiments, the invention provides for a therapeutic
population of tumor
infiltrating lymphocytes (TILs) that is capable of at least one-fold more
interferon-gamma production
as compared to TILs prepared by a process in which the first expansion of TILs
is performed without
any added OKT3. In other embodiments, the TILs are rendered capable of the at
least one-fold more
interferon-gamma production due to the expansion process described herein, for
example as described
in Steps A through F above or according to Steps A through F above (also as
shown, for example, in
Figure 8 (in particular, e.g., Figure 8A and/or Figure 8B and/or Figure 8C
and/or Figure 8D).
[001203] In other embodiments, the invention provides for a therapeutic
population of TILs that is
capable of at least two-fold more interferon-gamma production as compared to
TILs prepared by a
process in which the first expansion of TILs is performed without any added
APCs. In other
embodiments, the TILs are rendered capable of the at least two-fold more
interferon-gamma
production due to the expansion process described herein, for example as
described in Steps A
through F above or according to Steps A through F above (also as shown, for
example, in Figure 8 (in
particular, e.g., Figure 8A and/or Figure 8B and/or Figure 8C and/or Figure
8D).
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10012041 In other embodiments, the invention provides for a therapeutic
population of TILs that is
capable of at least two-fold more interferon-gamma production as compared to
TILs prepared by a
process in which the first expansion of TILs is performed without any added
OKT3. In other
embodiments, the TILs are rendered capable of the at least two-fold more
interferon-gamma
production due to the expansion process described herein, for example as
described in Steps A
through F above or according to Steps A through F above (also as shown, for
example, in Figure 8 (in
particular, e.g., Figure 8A and/or Figure 8B and/or Figure 8C and/or Figure
8D).
10012051ln other embodiments, the invention provides for a therapeutic
population of TILs that is
capable of at least three-fold more interferon-gamma production as compared to
TILs prepared by a
process in which the first expansion of TILs is performed without any added
APCs. In other
embodiments, the TILs are rendered capable of the at least one-fold more
interferon-gamma
production due to the expansion process described herein, for example as
described in Steps A
through F above or according to Steps A through F above (also as shown, for
example, in Figure 8 (in
particular, e.g. Figure 8A and/or Figure 8B and/or Figure 8C and/or Figure
8D).
[001206] In other embodiments, the invention provides for a therapeutic
population of TILs that is
capable of at least three-fold more interferon-gamma production as compared to
TILs prepared by a
process in which the first expansion of TILs is performed without any added
OKT3. In other
embodiments, the TILs are rendered capable of the at least three-fold more
interferon-gamma
production due to the expansion process described herein, for example as
described in Steps A
through F above or according to Steps A through F above (also as shown, for
example, in Figure 8 (in
particular, e.g., Figure 8A and/or Figure 8B and/or Figure 8C and/or Figure
8D).
10012071 In other embodiments, the invention provides the method
described in any of the
preceding paragraphs as applicable above modified such that the tumor
fragments are small biopsies
(including, for example, a punch biopsy), core biopsies, core needle biopsies
or fine needle aspirates.
10012081 In other embodiments, the invention provides the method
described in any of the
preceding paragraphs as applicable above modified such that the tumor
fragments are core biopsies.
10012091 In other embodiments, the invention provides the method
described in any of the
preceding paragraphs as applicable above modified such that the tumor
fragments are fine needle
aspirates.
10012101 In other embodiments, the invention provides the method
described in any of the
preceding paragraphs as applicable above modified such that the tumor
fragments are small biopsies
(including, for example, a punch biopsy).
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10012111 In other embodiments, the invention provides the method
described in any of the
preceding paragraphs as applicable above modified such that the tumor
fragments are core needle
biopsies.
10012121 In other embodiments, the invention provides the method
described in any of the
preceding paragraphs as applicable above modified such that (i) the method
comprises obtaining the
first population of TILs from one or more small biopsies (including, for
example, a punch biopsy),
core biopsies, core needle biopsies or fine needle aspirates of tumor tissue
from the subject, (ii) the
method comprises performing the step of culturing the first population of TILs
in a cell culture
medium comprising IL-2 for a period of about 3 days prior to performing the
step of the priming first
expansion, (iii) the method comprises performing the priming first expansion
for a period of about 8
days, and (iv) the method comprises performing the rapid second expansion for
a period of about 11
days. In some of the foregoing embodiments, the steps of the method are
completed in about 22 days.
10012131 In other embodiments, the invention provides the method
described in any of the
preceding paragraphs as applicable above modified such that (i) the method
comprises obtaining the
first population of TILs from one or more small biopsies (including, for
example, a punch biopsy),
core biopsies, core needle biopsies or fine needle aspirates of tumor tissue
from the subject, (ii) the
method comprises performing the step of culturing the first population of TILs
in a cell culture
medium comprising IL-2 for a period of about 3 days prior to performing the
step of the priming first
expansion, (iii) the method comprises performing the priming first expansion
for a period of about 8
days, and (iv) the method comprises performing the rapid second expansion by
culturing the culture
of the second population of TILs for about 5 days, splitting the culture into
up to 5 subcultures and
culturing the subcultures for about 6 days. In some of the foregoing
embodiments, the up to 5
subcultures are each cultured in a container that is the same size or larger
than the container in which
the culture of the second population of TILs is commenced in the rapid second
expansion. In some of
the foregoing embodiments, the culture of the second population of TILs is
equally divided amongst
the up to 5 subcultures. In some of the foregoing embodiments, the steps of
the method are completed
in about 22 days.
10012141 In other embodiments, the invention provides the method
described in any of the
preceding paragraphs as applicable above modified such that the first
population of TILs is obtained
from 1 to about 20 small biopsies (including, for example, a punch biopsy),
core biopsies, core needle
biopsies or fine needle aspirates of tumor tissue from the subject.
10012151 In other embodiments, the invention provides the method
described in any of the
preceding paragraphs as applicable above modified such that the first
population of TILs is obtained
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from 1 to about 10 small biopsies (including, for example, a punch biopsy),
core biopsies, core needle
biopsies or fine needle aspirates of tumor tissue from the subject.
10012161 In other embodiments, the invention provides the method
described in any of the
preceding paragraphs as applicable above modified such that the first
population of TILs is obtained
from 1, 2,3, 4, 5, 6, 7, 8,9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19 or 20
small biopsies (including, for
example, a punch biopsy), core biopsies, core needle biopsies or fine needle
aspirates of tumor tissue
from the subject.
10012171 In other embodiments, the invention provides the method
described in any of the
preceding paragraphs as applicable above modified such that the first
population of TILs is obtained
from 1, 2, 3, 4, 5, 6, 7, 8, 9 or 10 small biopsies (including, for example, a
punch biopsy), core
biopsies, core needle biopsies or fine needle aspirates of tumor tissue from
the subject.
10012181 In other embodiments, the invention provides the method
described in any of the
preceding paragraphs as applicable above modified such that the first
population of TILs is obtained
from 1 to about 20 core biopsies of tumor tissue from the subject.
10012191 In other embodiments, the invention provides the method
described in any of the
preceding paragraphs as applicable above modified such that the first
population of TILs is obtained
from 1 to about 10 core biopsies of tumor tissue from the subject.
10012201 In other embodiments, the invention provides the method
described in any of the
preceding paragraphs as applicable above modified such that the first
population of TILs is obtained
from 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19 or 20
core biopsies of tumor tissue
from the subject.
10012211 In other embodiments, the invention provides the method
described in any of the
preceding paragraphs as applicable above modified such that the first
population of TILs is obtained
from 1, 2, 3, 4, 5, 6, 7, 8, 9 or 10 core biopsies of tumor tissue from the
subject.
10012221 In other embodiments, the invention provides the method
described in any of the
preceding paragraphs as applicable above modified such that the first
population of TILs is obtained
from 1 to about 20 fine needle aspirates of tumor tissue from the subject.
10012231 In other embodiments, the invention provides the method
described in any of the
preceding paragraphs as applicable above modified such that the first
population of TILs is obtained
from 1 to about 10 fine needle aspirates of tumor tissue from the subject.
10012241 In other embodiments, the invention provides the method
described in any of the
preceding paragraphs as applicable above modified such that the first
population of TILs is obtained
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from 1, 2,3, 4, 5, 6, 7, 8,9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19 or 20
fine needle aspirates of tumor
tissue from the subject.
10012251 In other embodiments, the invention provides the method
described in any of the
preceding paragraphs as applicable above modified such that the first
population of TILs is obtained
from 1, 2, 3, 4, 5, 6, 7, 8, 9 or 10 fine needle aspirates of tumor tissue
from the subject.
10012261 In other embodiments, the invention provides the method
described in any of the
preceding paragraphs as applicable above modified such that the first
population of TILs is obtained
from 1 to about 20 core needle biopsies of tumor tissue from the subject.
10012271 In other embodiments, the invention provides the method
described in any of the
preceding paragraphs as applicable above modified such that the first
population of TILs is obtained
from 1 to about 10 core needle biopsies of tumor tissue from the subject.
10012281 In other embodiments, the invention provides the method
described in any of the
preceding paragraphs as applicable above modified such that the first
population of TILs is obtained
from 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19 or 20
core needle biopsies of tumor
tissue from the subject.
10012291 In other embodiments, the invention provides the method
described in any of the
preceding paragraphs as applicable above modified such that the first
population of TILs is obtained
from 1, 2, 3, 4, 5, 6, 7, 8, 9 or 10 core needle biopsies of tumor tissue from
the subject.
10012301 In other embodiments, the invention provides the method
described in any of the
preceding paragraphs as applicable above modified such that the first
population of TILs is obtained
from 1 to about 20 small biopsies (including, for example, a punch biopsy) of
tumor tissue from the
subj ect.
10012311 In other embodiments, the invention provides the method
described in any of the
preceding paragraphs as applicable above modified such that the first
population of TILs is obtained
from 1 to about 10 small biopsies (including, for example, a punch biopsy) of
tumor tissue from the
subj ect.
10012321 In other embodiments, the invention provides the method
described in any of the
preceding paragraphs as applicable above modified such that the first
population of TILs is obtained
from 1, 2,3, 4, 5, 6, 7, 8,9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19 or 20
small biopsies (including, for
example, a punch biopsy) of tumor tissue from the subject.
10012331 In other embodiments, the invention provides the method
described in any of the
preceding paragraphs as applicable above modified such that the first
population of TILs is obtained
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from 1, 2, 3, 4, 5, 6, 7, 8, 9 or 10 small biopsies (including, for example, a
punch biopsy) of tumor
tissue from the subject.
10012341 In some embodiments, the invention provides the method
described in any of the
preceding paragraphs as applicable above modified such that (i) the method
comprises obtaining the
first population of TILs from 1 to about 10 core biopsies of tumor tissue from
the subject, (ii) the
method comprises performing the step of culturing the first population of TILs
in a cell culture
medium comprising IL-2 for a period of about 3 days prior to performing the
step of the priming first
expansion, (iii) the method comprises performing the priming first expansion
step by culturing the
first population of TILs in a culture medium comprising IL-2, OKT-3 and
antigen presenting cells
(APCs) for a period of about 8 days to obtain the second population of TILs,
and (iv) the method
comprises performing the rapid second expansion step by culturing the second
population of TILs in a
culture medium comprising IL-2, OKT-3 and APCs for a period of about 11 days.
In some of the
foregoing embodiments, the steps of the method are completed in about 22 days.
10012351 In some embodiments, the invention provides the method
described in any of the
preceding paragraphs as applicable above modified such that (i) the method
comprises obtaining the
first population of TILs from 1 to about 10 core biopsies of tumor tissue from
the subject, (ii) the
method comprises performing the step of culturing the first population of TILs
in a cell culture
medium comprising TL-2 for a period of about 3 days prior to performing the
step of the priming first
expansion, (iii) the method comprises performing the priming first expansion
step by culturing the
first population of TILs in a culture medium comprising IL-2, OKT-3 and
antigen presenting cells
(APCs) for a period of about 8 days to obtain the second population of TILs,
and (iv) the method
comprises performing the rapid second expansion by culturing the culture of
the second population of
TILs in a culture medium comprising IL-2, OKT-3 and APCs for about 5 days,
splitting the culture
into up to 5 subcultures and culturing each of the subcultures in a culture
medium comprising IL-2 for
about 6 days. In some of the foregoing embodiments, the up to 5 subcultures
are each cultured in a
container that is the same size or larger than the container in which the
culture of the second
population of TILs is commenced in the rapid second expansion. In some of the
foregoing
embodiments, the culture of the second population of TILs is equally divided
amongst the up to 5
subcultures. In some of the foregoing embodiments, the steps of the method are
completed in about 22
days.
10012361 In some embodiments, the invention provides the method
described in any of the
preceding paragraphs as applicable above modified such that (i) the method
comprises obtaining the
first population of TILs from 1 to about 10 core biopsies of tumor tissue from
the subject, (ii) the
method comprises performing the step of culturing the first population of TILs
in a cell culture
medium comprising 6000 IU IL-2/mL in 0.5 L of CM1 culture medium in a G-REX
100M flask for a
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period of about 3 days prior to performing the step of the priming first
expansion, (iii) the method
comprises performing the priming first expansion by adding 0.5 L of CM1
culture medium containing
6000 IU/mL 1L-2, 30 ng/mL OKT-3, and about 10x feeder cells and culturing for
a period of about 8
days, and (iv) the method comprises performing the rapid second expansion by
(a) transferring the
second population of TILs to a G-REX 500MCS flask containing 5 L of CM2
culture medium with
3000 IU/mL 1L-2, 30 ng/mL OK1-3, and 5x109 feeder cells and culturing for
about 5 days (b)
splitting the culture into up to 5 subcultures by transferring 109 TILs into
each of up to 5 G-REX
500MCS flasks containing 5 L of AIM-V medium with 3000 IU/mL IL-2, and
culturing the
subcultures for about 6 days. In some of the foregoing embodiments, the steps
of the method are
completed in about 22 days.
10012371 In some embodiments, the invention provides a method of expanding T
cells comprising:
(a) performing a priming first expansion of a first population of T cells
obtained from a donor by
culturing the first population of T cells to effect growth and to prime an
activation of the first
population of T cells; (b) after the activation of the first population of T
cells primed in step (a) begins
to decay, performing a rapid second expansion of the first population of T
cells by culturing the first
population of T cells to effect growth and to boost the activation of the
first population of T cells to
obtain a second population of T cells; and (c) harvesting the second
population of T cells. In some
embodiments, the step of rapid second expansion is split into a plurality of
steps to achieve a scaling
up of the culture by: (a) performing the rapid second expansion by culturing
the first population of T
cells in a small scale culture in a first container, e.g., a G-REX 100MCS
container, for a period of
about 3 to 4 days, and then (b) effecting the transfer of the first population
of T cells from the small
scale culture to a second container larger than the first container, e.g., a G-
REX 500MCS container,
and culturing the first population of T cells from the small scale culture in
a larger scale culture in the
second container for a period of about 4 to 7 days. In some embodiments, the
step of rapid expansion
is split into a plurality of steps to achieve a scaling out of the culture by:
(a) performing the rapid
second expansion by culturing the first population of T cells in a first small
scale culture in a first
container, e.g., a G-REX 100MCS container, for a period of about 3 to 4 days,
and then (b) effecting
the transfer and apportioning of the first population of T cells from the
first small scale culture into
and amongst at least 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17,
18, 19, or 20 second containers
that are equal in size to the first container, wherein in each second
container the portion of the first
population of T cells from first small scale culture transferred to such
second container is cultured in a
second small scale culture for a period of about 4 to 7 days. In some
embodiments, the step of rapid
expansion is split into a plurality of steps to achieve a scaling out and
scaling up of the culture by: (a)
performing the rapid second expansion by culturing the first population of T
cells in a small scale
culture in a first container, e.g., a G-REX 100MCS container, for a period of
about 3 to 4 days, and
then (b) effecting the transfer and apportioning of the first population of T
cells from the small scale
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culture into and amongst at least 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14,
15, 16, 17, 18, 19, or 20
second containers that are larger in size than the first container, e.g., G-
REX 500MCS containers,
wherein in each second container the portion of the first population of T
cells from the small scale
culture transferred to such second container is cultured in a larger scale
culture for a period of about 4
to 7 days. In some embodiments, the step of rapid expansion is split into a
plurality of steps to achieve
a scaling out and scaling up of the culture by: (a) performing the rapid
second expansion by culturing
the first population of T cells in a small scale culture in a first container,
e.g., a G-REX 100MCS
container, for a period of about 4 days, and then (b) effecting the transfer
and apportioning of the first
population of T cells from the small scale culture into and amongst 2, 3 or 4
second containers that are
larger in size than the first container, e.g., G-REX 500MCS containers,
wherein in each second
container the portion of the first population of T cells from the small scale
culture transferred to such
second container is cultured in a larger scale culture for a period of about 5
days.
10012381 In some embodiments, the invention provides the method described in
any of the preceding
paragraphs as applicable above modified such that the step of rapid second
expansion is split into a
plurality of steps to achieve a scaling up of the culture by: (a) performing
the rapid second expansion
by culturing the first population of T cells in a small scale culture in a
first container, e.g., a G-REX
100MCS container, for a period of about 2 to 4 days, and then (b) effecting
the transfer of the first
population of T cells from the small scale culture to a second container
larger than the first container,
e.g., a G-REX 500MCS container, and culturing the first population of T cells
from the small scale
culture in a larger scale culture in the second container for a period of
about 5 to 7 days.
10012391 In some embodiments, the invention provides the method described in
any of the preceding
paragraphs as applicable above modified such that the step of rapid expansion
is split into a plurality
of steps to achieve a scaling out of the culture by: (a) performing the rapid
second expansion by
culturing the first population of T cells in a first small scale culture in a
first container, e.g., a G-REX
100MCS container, for a period of about 2 to 4 days, and then (b) effecting
the transfer and
apportioning of the first population of T cells from the first small scale
culture into and amongst at
least 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, or 20
second containers that are equal
in size to the first container, wherein in each second container the portion
of the first population of T
cells from first small scale culture transferred to such second container is
cultured in a second small
scale culture for a period of about 5 to 7 days.
10012401 In some embodiments, the invention provides the method described in
any of the preceding
paragraphs as applicable above modified such that the step of rapid expansion
is split into a plurality
of steps to achieve a scaling out and scaling up of the culture by: (a)
performing the rapid second
expansion by culturing the first population of T cells in a small scale
culture in a first container, e.g., a
G-REX 100MCS container, for a period of about 2 to 4 days, and then (b)
effecting the transfer and
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apportioning of the first population of T cells from the small scale culture
into and amongst at least 2,
3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, or 20 second
containers that are larger in size
than the first container, e.g., G-REX 500MCS containers, wherein in each
second container the
portion of the first population of T cells from the small scale culture
transferred to such second
container is cultured in a larger scale culture for a period of about 5 to 7
days.
10012411111 some embodiments, the invention provides the method described in
any of the preceding
paragraphs as applicable above modified such that the step of rapid expansion
is split into a plurality
of steps to achieve a scaling out and scaling up of the culture by: (a)
performing the rapid second
expansion by culturing the first population of T cells in a small scale
culture in a first container, e.g., a
G-REX 100MCS container, for a period of about 3 to 4 days, and then (b)
effecting the transfer and
apportioning of the first population of T cells from the small scale culture
into and amongst 2, 3 or 4
second containers that are larger in size than the first container, e.g., G-
REX 500MCS containers,
wherein in each second container the portion of the first population of T
cells from the small scale
culture transferred to such second container is cultured in a larger scale
culture for a period of about 5
to 6 days.
10012421in some embodiments, the invention provides the method described in
any of the preceding
paragraphs as applicable above modified such that the step of rapid expansion
is split into a plurality
of steps to achieve a scaling out and scaling up of the culture by: (a)
performing the rapid second
expansion by culturing the first population of T cells in a small scale
culture in a first container, e.g., a
G-REX 100MCS container, for a period of about 3 to 4 days, and then (b)
effecting the transfer and
apportioning of the first population of T cells from the small scale culture
into and amongst 2, 3 or 4
second containers that are larger in size than the first container, e.g., G-
REX 500MCS containers,
wherein in each second container the portion of the first population of T
cells from the small scale
culture transferred to such second container is cultured in a larger scale
culture for a period of about 5
days.
10012431 In some embodiments, the invention provides the method described in
any of the preceding
paragraphs as applicable above modified such that the step of rapid expansion
is split into a plurality
of steps to achieve a scaling out and scaling up of the culture by: (a)
performing the rapid second
expansion by culturing the first population of T cells in a small scale
culture in a first container, e.g., a
G-REX 100MCS container, for a period of about 3 to 4 days, and then (b)
effecting the transfer and
apportioning of the first population of T cells from the small scale culture
into and amongst 2, 3 or 4
second containers that are larger in size than the first container, e.g., G-
REX 500MCS containers,
wherein in each second container the portion of the first population of T
cells from the small scale
culture transferred to such second container is cultured in a larger scale
culture for a period of about 6
days.
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1001244] In some embodiments, the invention provides the method described in
any of the preceding
paragraphs as applicable above modified such that the step of rapid expansion
is split into a plurality
of steps to achieve a scaling out and scaling up of the culture by: (a)
performing the rapid second
expansion by culturing the first population of T cells in a small scale
culture in a first container, e.g., a
G-REX 100MCS container, for a period of about 3 to 4 days, and then (b)
effecting the transfer and
apportioning of the first population of T cells from the small scale culture
into and amongst 2, 3 or 4
second containers that are larger in size than the first container, e.g., G-
REX 500MCS containers,
wherein in each second container the portion of the first population of T
cells from the small scale
culture transferred to such second container is cultured in a larger scale
culture for a period of about 7
days.
10012451 In some embodiments, the invention provides the method described in
any of the preceding
paragraphs as applicable above modified such that the priming first expansion
of step (a) is performed
during a period of up to 7 days.
19012461In some embodiments, the invention provides the method described in
any of the preceding
paragraphs as applicable above modified such that the rapid second expansion
of step (b) is performed
during a period of up to 8 days.
10012471 In some embodiments, the invention provides the method described in
any of the preceding
paragraphs as applicable above modified such that the rapid second expansion
of step (b) is performed
during a period of up to 9 days.
10012481 In some embodiments, the invention provides the method described in
any of the preceding
paragraphs as applicable above modified such that the rapid second expansion
of step (b) is performed
during a period of up to 10 days.
10012491 In some embodiments, the invention provides the method described in
any of the preceding
paragraphs as applicable above modified such that the rapid second expansion
of step (b) is performed
during a period of up to 11 days.
10012501 In some embodiments, the invention provides the method described in
any of the preceding
paragraphs as applicable above modified such that the priming first expansion
in step (a) is performed
during a period of 7 days and the rapid second expansion of step (b) is
performed during a period of
up to 9 days.
1001251] In some embodiments, the invention provides the method described in
any of the preceding
paragraphs as applicable above modified such that the priming first expansion
in step (a) is performed
during a period of 7 days and the rapid second expansion of step (b) is
performed during a period of
up to 10 days.
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[0012521M some embodiments, the invention provides the method described in any
of the preceding
paragraphs as applicable above modified such that the priming first expansion
in step (a) is performed
during a period of 7 days or 8 days and the rapid second expansion of step (b)
is performed during a
period of up to 9 days.
10012531ln some embodiments, the invention provides the method described in
any of the preceding
paragraphs as applicable above modified such that the priming first expansion
in step (a) is performed
during a period of 7 days or 8 days and the rapid second expansion of step (b)
is performed during a
period of up to 10 days.
10012541ln some embodiments, the invention provides the method described in
any of the preceding
paragraphs as applicable above modified such that the priming first expansion
in step (a) is performed
during a period of 8 days and the rapid second expansion of step (b) is
performed during a period of
up to 9 days.
10012551 In some embodiments, the invention provides the method described in
any of the preceding
paragraphs as applicable above modified such that the priming first expansion
in step (a) is performed
during a period of 8 days and the rapid second expansion of step (b) is
performed during a period of
up to 8 days.
10012561in some embodiments, the invention provides the method described in
any of the preceding
paragraphs as applicable above modified such that in step (a) the first
population of T cells is cultured
in a first culture medium comprising OKT-3 and IL-2.
10012571ln some embodiments, the invention provides the method described in
any of the preceding
paragraphs as applicable above modified such that the first culture medium
comprises 4-1BB agonist,
OKT-3 and IL-2.
100125811n some embodiments, the invention provides the method described in
any of the preceding
paragraphs as applicable above modified such that the first culture medium
comprises OKT-3, IL-2
and antigen-presenting cells (APCs).
10012591ln some embodiments, the invention provides the method described in
any of the preceding
paragraphs as applicable above modified such that the first culture medium
comprises 4-1BB agonist,
OKT-3, IL-2 and antigen-presenting cells (APCs).
10012601ln some embodiments, the invention provides the method described in
any of the preceding
paragraphs as applicable above modified such that in step (b) the first
population of T cells is cultured
in a second culture medium comprising OKT-3, 1L-2 and antigen-presenting cells
(APCs).
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1001261] In some embodiments, the invention provides the method described in
any of the preceding
paragraphs as applicable above modified such that the second culture medium
comprises 4-1BB
agonist, OKT-3, IL-2 and antigen-presenting cells (APCs).
10012621 In some embodiments, the invention provides the method described in
any of the preceding
paragraphs as applicable above modified such that in step (a) the first
population of T cells is cultured
in a first culture medium in a container comprising a first gas-permeable
surface, wherein the first
culture medium comprises OKT-3, IL-2 and a first population of antigen-
presenting cells (APCs),
wherein the first population of APCs is exogenous to the donor of the first
population of T cells and
the first population of APCs is layered onto the first gas-permeable surface,
wherein in step (b) the
first population of T cells is cultured in a second culture medium in the
container, wherein the second
culture medium comprises OKT-3, 1L-2 and a second population of APCs, wherein
the second
population of APCs is exogenous to the donor of the first population of T
cells and the second
population of APCs is layered onto the first gas-permeable surface, and
wherein the second
population of APCs is greater than the first population of APCs.
1001263] In some embodiments, the invention provides the method described in
any of the preceding
paragraphs as applicable above modified such that in step (a) the first
population of T cells is cultured
in a first culture medium in a container comprising a first gas-permeable
surface, wherein the first
culture medium comprises 4-1BB agonist, OKT-3, IL-2 and a first population of
antigen-presenting
cells (APCs), wherein the first population of APCs is exogenous to the donor
of the first population of
T cells and the first population of APCs is layered onto the first gas-
permeable surface, wherein in
step (b) the first population of T cells is cultured in a second culture
medium in the container, wherein
the second culture medium comprises OKT-3, IL-2 and a second population of
APCs, wherein the
second population of APCs is exogenous to the donor of the first population of
T cells and the second
population of APCs is layered onto the first gas-permeable surface, and
wherein the second
population of APCs is greater than the first population of APCs.
1001264] In some embodiments, the invention provides the method described in
any of the preceding
paragraphs as applicable above modified such that in step (a) the first
population of T cells is cultured
in a first culture medium in a container comprising a first gas-permeable
surface, wherein the first
culture medium comprises OKT-3, IL-2 and a first population of antigen-
presenting cells (APCs),
wherein the first population of APCs is exogenous to the donor of the first
population of T cells and
the first population of APCs is layered onto the first gas-permeable surface,
wherein in step (b) the
first population of T cells is cultured in a second culture medium in the
container, wherein the second
culture medium comprises 4-1BB agonist, OKT-3, IL-2 and a second population of
APCs, wherein
the second population of APCs is exogenous to the donor of the first
population of T cells and the
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second population of APCs is layered onto the first gas-permeable surface, and
wherein the second
population of APCs is greater than the first population of APCs.
10012651ln some embodiments, the invention provides the method described in
any of the preceding
paragraphs as applicable above modified such that in step (a) the first
population of T cells is cultured
in a first culture medium in a container comprising a first gas-permeable
surface, wherein the first
culture medium comprises 4-1BB agonist, OKT-3, IL-2 and a first population of
antigen-presenting
cells (APCs), wherein the first population of APCs is exogenous to the donor
of the first population of
T cells and the fi rst population of APCs is layered onto the first gas-
permeable surface, wherein in
step (b) the first population of T cells is cultured in a second culture
medium in the container, wherein
the second culture medium comprises 4-1BB agonist, OKT-3, IL-2 and a second
population of APCs,
wherein the second population of APCs is exogenous to the donor of the first
population of T cells
and the second population of APCs is layered onto the first gas-permeable
surface, and wherein the
second population of APCs is greater than the first population of APCs.
10012661ln some embodiments, the invention provides the method described in
any of the preceding
paragraphs as applicable above modified such that the ratio of the number of
APCs in the second
population of APCs to the number of APCs in the first population of APCs is
about 2:1.
10012671ln some embodiments, the invention provides the method described in
any of the preceding
paragraphs as applicable above modified such that the number of APCs in the
first population of
APCs is about 2.5 x 108 and the number of APCs in the second population of
APCs is about 5 x 108.
10012681ln some embodiments, the invention provides the method described in
any of the preceding
paragraphs as applicable above modified such that in step (a) the first
population of APCs is layered
onto the first gas-permeable surface at an average thickness of 2 layers of
APCs.
10012691ln some embodiments, the invention provides the method described in
any of the preceding
paragraphs as applicable above modified such that in step (b) the second
population of APCs is
layered onto the first gas-permeable surface at an average thickness selected
from the range of 4 to 8
layers of APCs.
10012701ln some embodiments, the invention provides the method described in
any of the preceding
paragraphs as applicable above modified such that the ratio of the average
number of layers of APCs
layered onto the first gas-permeable surface in step (b) to the average number
of layers of APCs
layered onto the first gas-permeable surface in step (a) is 2:1.
100127111n some embodiments, the invention provides the method described in
any of the preceding
paragraphs as applicable above modified such that in step (a) the first
population of APCs is seeded
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on the first gas permeable surface at a density selected from the range of at
or about 1.0 x 106
APCs/cm2 to at or about 4.5 x 106 APCs/cm2.
10012721 In some embodiments, the invention provides the method described in
any of the preceding
paragraphs as applicable above modified such that in step (a) the first
population of APCs is seeded
on the first gas permeable surface at a density selected from the range of at
or about 1.5 x106
APCs/cm2 to at or about 3.5 x 106 APCs/cm2.
10012731 In some embodiments, the invention provides the method described in
any of the preceding
paragraphs as applicable above modified such that in step (a) the first
population of APCs is seeded
on the first gas permeable surface at a density selected from the range of at
or about 2.0x 106
APCs/cm2 to at or about 3.0x 106 APCs/cm2.
100127411n some embodiments, the invention provides the method described in
any of the preceding
paragraphs as applicable above modified such that in step (a) the first
population of APCs is seeded
on the first gas permeable surface at a density of at or about 2.0x 106
APCs/cm2.
10012751 In some embodiments, the invention provides the method described in
any of the preceding
paragraphs as applicable above modified such that in step (b) the second
population of APCs is
seeded on the first gas permeable surface at a density selected from the range
of at or about 2.5 x106
APCs/cm2 to at or about 7.5 x 106 APCs/cm2.
10012761 In some embodiments, the invention provides the method described in
any of the preceding
paragraphs as applicable above modified such that in step (b) the second
population of APCs is
seeded on the first gas permeable surface at a density selected from the range
of at or about 3.5 x106
APCs/cm2 to at or about 6.0x 106 APCs/cm2.
10012771 In some embodiments, the invention provides the method described in
any of the preceding
paragraphs as applicable above modified such that in step (b) the second
population of APCs is
seeded on the first gas permeable surface at a density selected from the range
of at or about 4.0 x106
APCs/cm2 to at or about 5.5 x 106 APCs/cm2.
10012781 In some embodiments, the invention provides the method described in
any of the preceding
paragraphs as applicable above modified such that in step (b) the second
population of APCs is
seeded on the first gas permeable surface at a density of at or about 4.0x 106
APCs/cm2.
10012791 In some embodiments, the invention provides the method described in
any of the preceding
paragraphs as applicable above modified such that in step (a) the first
population of APCs is seeded
on the first gas permeable surface at a density selected from the range of at
or about 1.0 x 106
APCs/cm2 to at or about 4.5 x 106 APCs/cm2 and in step (b) the second
population of APCs is seeded
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on the first gas permeable surface at a density selected from the range of at
or about 2.5 x106
APCs/cm2 to at or about 7.5 x106 APCs/cm2.
1001280] In some embodiments, the invention provides the method described in
any of the preceding
paragraphs as applicable modified such that in step (a) the first population
of APCs is seeded on the
first gas permeable surface at a density selected from the range of at or
about 1.5 x106 APCs/cm2 to at
or about 3.5>< 106 APCs/cm2 and in step (b) the second population of APCs is
seeded on the first gas
permeable surface at a density selected from the range of at or about 3.5 x106
APCs/cm2 to at or about
6 0 x 106 APCs/cm2.
10012811 In some embodiments, the invention provides the method described in
any of the preceding
paragraphs as applicable above modified such that in step (a) the first
population of APCs is seeded
on the first gas permeable surface at a density selected from the range of at
or about 2.0>< 106
APCs/cm2 to at or about 3.0 x 106 APCs/cm2 and in step (b) the second
population of APCs is seeded
on the first gas permeable surface at a density selected from the range of at
or about 4.0 x 106
APCs/cm2 to at or about 5.5 x 106 APCs/cm2.
10012821 In some embodiments, the invention provides the method described in
any of the preceding
paragraphs as applicable above modified such that in step (a) the first
population of APCs is seeded
on the first gas permeable surface at a density of at or about 2.0x 106
APCs/cm2 and in step (b) the
second population of APCs is seeded on the first gas permeable surface at a
density of at or about
4.0 x106 APCs/cm2.
10012831 In some embodiments, the invention provides the method described in
any of the preceding
paragraphs as applicable above modified such that the APCs are peripheral
blood mononuclear cells
(PBMCs).
10012841 In some embodiments, the invention provides the method described in
any of the preceding
paragraphs as applicable above modified such that the PBMCs are irradiated and
exogenous to the
donor of the first population of T cells.
10012851 In some embodiments, the invention provides the method described in
any of the preceding
paragraphs as applicable above modified such that the T cells are tumor
infiltrating lymphocytes
(TILs).
10012861 In some embodiments, the invention provides the method described in
any of the preceding
paragraphs as applicable above modified such that the T cells arc marrow
infiltrating lymphocytes
(MILs).
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[001287] In some embodiments, the invention provides the method described in
any of the preceding
paragraphs as applicable above modified such that the T cells are peripheral
blood lymphocytes
(PBLs).
[001288] In some embodiments, the invention provides the method described in
any of the preceding
paragraphs as applicable above modified such that the first population of T
cells is obtained by
separation from the whole blood of the donor.
[001289] In some embodiments, the invention provides the method described in
any of the preceding
paragraphs as applicable above modified such that the first population of T
cells is obtained by
separation from the apheresis product of the donor.
[001290] In some embodiments, the invention provides the method described in
any of the preceding
paragraphs as applicable above modified such that the first population of T
cells is separated from the
whole blood or apheresis product of the donor by positive or negative
selection of a T cell phenotype.
10012911 In some embodiments, the invention provides the method described in
any of the preceding
paragraphs as applicable above modified such that the T cell phenotype is CD3+
and CD45+.
[001292] In some embodiments, the invention provides the method described in
any of the preceding
paragraphs as applicable above modified such that before performing the
priming first expansion of
the first population of T cells the T cells are separated from NK cells. In
some embodiments, the T
cells are separated from NK cells in the first population of T cells by
removal of CD3- CD56+ cells
from the first population of T cells. In some embodiments, the CD3- CD56+
cells are removed from
the first population of T cells by subjecting the first population of T cells
to cell sorting using a gating
strategy that removes the CD3- CD56+ cell fraction and recovers the negative
fraction. In some
embodiments, the foregoing method is utilized for the expansion of T cells in
a first population of T
cells characterized by a high percentage of NK cells. In some embodiments, the
foregoing method is
utilized for the expansion of T cells in a first population of T cells
characterized by a high percentage
of CD3- CD56+ cells. In some embodiments, the foregoing method is utilized for
the expansion of T
cells in tumor tissue characterized by the present of a high number of NK
cells. In some embodiments,
the foregoing method is utilized for the expansion of T cells in tumor tissue
characterized by a high
number of CD3- CD56+ cells. In some embodiments, the foregoing method is
utilized for the
expansion of T cells in tumor tissue obtained from a patient suffering from a
tumor characterized by
the presence of a high number of NK cells. In some embodiments, the foregoing
method is utilized for
the expansion of T cells in tumor tissue obtained from a patient suffering
from a tumor characterized
by the presence of a high number of CD3- CD56+ cells. In some embodiments, the
foregoing method
is utilized for the expansion of T cells in tumor tissue obtained from a
patient suffering from ovarian
cancer.
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10012931111 some embodiments, the invention provides the method described in
any of the preceding
paragraphs as applicable above modified such that at or about lx107T cells
from the first population
of T cells are seeded in a container to initiate the primary first expansion
culture in such container.
10012941111 some embodiments, the invention provides the method described in
any of the preceding
paragraphs as applicable above modified such that the first population of T
cells is distributed into a
plurality of containers, and in each container at or about lx107T cells from
the first population of T
cells are seeded to initiate the primary first expansion culture in such
container.
10012951In some embodiments, the invention provides the method described in
any of the preceding
paragraphs as applicable above modified such that the second population of T
cells harvested in step
(c) is a therapeutic population of TILs.
10012961 In some embodiments, the invention provides the method
described in any of the
preceding paragraphs as applicable above modified such that the first
population of T cells is obtained
from one or more small biopsies (including, for example, a punch biopsy), core
biopsies, core needle
biopsies or fine needle aspirates of tumor tissue from the donor.
10012971 In some embodiments, the invention provides the method
described in any of the
preceding paragraphs as applicable above modified such that the first
population of T cells is obtained
from 1 to 20 small biopsies (including, for example, a punch biopsy), core
biopsies, core needle
biopsies or fine needle aspirates of tumor tissue from the donor.
10012981 In some embodiments, the invention provides the method
described in any of the
preceding paragraphs as applicable above modified such that the first
population of T cells is obtained
from 1 to 10 small biopsies (including, for example, a punch biopsy), core
biopsies, core needle
biopsies or fine needle aspirates of tumor tissue from the donor.
10012991 In some embodiments, the invention provides the method
described in any of the
preceding paragraphs as applicable above modified such that the first
population of T cells is obtained
from 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19 or 20
small biopsies (including, for
example, a punch biopsy), core biopsies, core needle biopsies or fine needle
aspirates of tumor tissue
from the donor.
10013001 In some embodiments, the invention provides the method
described in any of the
preceding paragraphs as applicable above modified such that the first
population of T cells is obtained
from 1, 2, 3, 4, 5, 6, 7, 8, 9, or I 0 small biopsies (including, for example,
a punch biopsy), core
biopsies, core needle biopsies or fine needle aspirates of tumor tissue from
the donor.
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10013011 In some embodiments, the invention provides the method
described in any of the
preceding paragraphs as applicable above modified such that the first
population of T cells is obtained
from one or more core biopsies of tumor tissue from the donor.
10013021 In some embodiments, the invention provides the method
described in any of the
preceding paragraphs as applicable above modified such that the first
population of T cells is obtained
from 1 to 20 core biopsies of tumor tissue from the donor.
10013031 In some embodiments, the invention provides the method
described in any of the
preceding paragraphs as applicable above modified such that the first
population of T cells is obtained
from 1 to 10 core biopsies of tumor tissue from the donor.
10013041 In some embodiments, the invention provides the method
described in any of the
preceding paragraphs as applicable above modified such that the first
population of T cells is obtained
from 1, 2,3, 4, 5, 6, 7, 8,9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19 or 20
core biopsies of tumor tissue
from the donor.
10013051 In some embodiments, the invention provides the method
described in any of the
preceding paragraphs as applicable above modified such that the first
population of T cells is obtained
from 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 core biopsies of tumor tissue from the
donor.
10013061 In some embodiments, the invention provides the method
described in any of the
preceding paragraphs as applicable above modified such that the first
population of T cells is obtained
from one or more fine needle aspirates of tumor tissue from the donor.
10013071 In some embodiments, the invention provides the method
described in any of the
preceding paragraphs as applicable above modified such that the first
population of T cells is obtained
from 1 to 20 fine needle aspirates of tumor tissue from the donor.
10013081 In some embodiments, the invention provides the method
described in any of the
preceding paragraphs as applicable above modified such that the first
population of T cells is obtained
from 1 to 10 fine needle aspirates of tumor tissue from the donor.
10013091 In some embodiments, the invention provides the method
described in any of the
preceding paragraphs as applicable above modified such that the first
population of T cells is obtained
from 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19 or 20
fine needle aspirates of tumor
tissue from the donor.
10013101 In some embodiments, the invention provides the method
described in any of the
preceding paragraphs as applicable above modified such that the first
population of T cells is obtained
from 1, 2,3, 4, 5, 6, 7, 8,9, or 10 fine needle aspirates of tumor tissue from
the donor.
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10013111 In some embodiments, the invention provides the method
described in any of the
preceding paragraphs as applicable above modified such that the first
population of T cells is obtained
from one or more small biopsies (including, for example, a punch biopsy) of
tumor tissue from the
donor.
10013121 In some embodiments, the invention provides the method
described in any of the
preceding paragraphs as applicable above modified such that the first
population of T cells is obtained
from 1 to 20 small biopsies (including, for example, a punch biopsy) of tumor
tissue from the donor.
10013131 In some embodiments, the invention provides the method
described in any of the
preceding paragraphs as applicable above modified such that the first
population of T cells is obtained
from 1 to 10 small biopsies (including, for example, a punch biopsy) of tumor
tissue from the donor.
10013141 In some embodiments, the invention provides the method
described in any of the
preceding paragraphs as applicable above modified such that the first
population of T cells is obtained
from 1, 2,3, 4, 5, 6, 7, 8,9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19 or 20
small biopsies (including, for
example, a punch biopsy) of tumor tissue from the donor.
10013151 In some embodiments, the invention provides the method
described in any of the
preceding paragraphs as applicable above modified such that the first
population of T cells is obtained
from 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 small biopsies (including, for example,
a punch biopsy) of tumor
tissue from the donor.
10013161 In some embodiments, the invention provides the method
described in any of the
preceding paragraphs as applicable above modified such that the first
population of T cells is obtained
from one or more core needle biopsies of tumor tissue from the donor.
10013171 In some embodiments, the invention provides the method
described in any of the
preceding paragraphs as applicable above modified such that the first
population of T cells is obtained
from 1 to 20 core needle biopsies of tumor tissue from the donor.
10013181 In some embodiments, the invention provides the method
described in any of the
preceding paragraphs as applicable above modified such that the first
population of T cells is obtained
from 1 to 10 core needle biopsies of tumor tissue from the donor.
10013191 In some embodiments, the invention provides the method
described in any of the
preceding paragraphs as applicable above modified such that the first
population of T cells is obtained
from 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19 or 20
core needle biopsies of tumor
tissue from the donor.
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10013201 In some embodiments, the invention provides the method
described in any of the
preceding paragraphs as applicable above modified such that the first
population of T cells is obtained
from 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 core needle biopsies of tumor tissue
from the donor.
10013211 In some embodiments, the invention provides a method for
expanding tumor
infiltrating lymphocytes (TILs) into a therapeutic population of TILs
comprising: i) obtaining and/or
receiving a first population of TILs from a tumor sample obtained from one or
more small biopsies,
core biopsies, or needle biopsies of a tumor in a subject by culturing the
tumor sample in a first cell
culture medium comprising IL-2 for about 3 days; (ii) performing a priming
first expansion by
culturing the first population of TILs in a second cell culture medium
comprising IL-2, OKT-3, and
antigen presenting cells (APCs) to produce a second population of TILs,
wherein the priming first
expansion is performed in a container comprising a first gas-permeable surface
area, wherein the
priming first expansion is performed for first period of about 7 or 8 days to
obtain the second
population of TILs, wherein the second population of TILs is greater in number
than the first
population of TILs; (iii) performing a rapid second expansion by supplementing
the second cell
culture medium of the second population of TILs with additional 1L-2, OKT-3,
and APCs, to produce
a third population of TILs, wherein the number of APCs added in the rapid
second expansion is at
least twice the number of APCs added in step (ii), wherein the rapid second
expansion is performed
for a second period of about 11 days to obtain the third population of TILs,
wherein the third
population of TILs is a therapeutic population of TILs, wherein the rapid
second expansion is
performed in a container comprising a second gas-permeable surface area; (iv)
harvesting the
therapeutic population of TILs obtained from step (iii); and (v) transferring
the harvested TIL
population from step (iv) to an infusion bag.
10013221 In some embodiments, the invention provides a method for
expanding tumor
infiltrating lymphocytes (TILs) into a therapeutic population of TILs
comprising: (i) obtaining and/or
receiving a first population of TILs from a tumor sample obtained from one or
more small biopsies,
core biopsies, or needle biopsies of a tumor in a subject by culturing the
tumor sample in a first cell
culture medium comprising IL-2 for about 3 days; (ii) performing a priming
first expansion by
culturing the first population of TILs in a second cell culture medium
comprising IL-2, OKT-3, and
antigen presenting cells (APCs) to produce a second population of TILs,
wherein the priming first
expansion is performed for first period of about 7 or 8 days to obtain the
second population of TILs,
wherein the second population of TILs is greater in number than the first
population of TILs; (iii)
performing a rapid second expansion by contacting the second population of
TILs with a third cell
culture medium comprising IL-2, OKT-3, and APCs, to produce a third population
of TILs, wherein
the rapid second expansion is performed for a second period of about 11 days
to obtain the third
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population of TILs, wherein the third population of TILs is a therapeutic
population of TILs; and (iv)
harvesting the therapeutic population of TILs obtained from step (iii).
10013231 In some embodiments, the invention provides the method
described in any of the
preceding paragraphs as applicable above modified such that after day 5 of the
second period the
culture is split into 2 or more subcultures, and each subculture is
supplemented with an additional
quantity of the third culture medium and cultured for about 6 days.
10013241 In some embodiments, the invention provides the method
described in any of the
preceding paragraphs as applicable above modified such that after day 5 of the
second period the
culture is split into 2 or more subcultures, and each subculture is
supplemented with a fourth culture
medium comprising IL-2 and cultured for about 6 days.
10013251 In some embodiments, the invention provides the method
described in any of the
preceding paragraphs as applicable above modified such that after day 5 of the
second period the
culture is split into up to 5 subcultures.
10013261 in some embodiments, the invention provides the method
described in any of the
preceding paragraphs as applicable above modified such that all steps in the
method are completed in
about 24 days. In some embodiments, the invention provides the method
described in any of the
preceding paragraphs as applicable above modified such that all steps in the
method are completed in
about 22 days.
10013271 In some embodiments, the invention provides a method of
expanding T cells
comprising: (i) performing a priming first expansion of a first population of
T cells from a tumor
sample obtained from one or more small biopsies, core biopsies, or needle
biopsies of a tumor in a
donor by culturing the first population of T cells to effect growth and to
prime an activation of the
first population of T cells; (ii) after the activation of the first population
of T cells primed in step (a)
begins to decay, performing a rapid second expansion of the first population
of T cells by culturing
the first population of T cells to effect growth and to boost the activation
of the first population of T
cells to obtain a second population of T cells; and (iv) harvesting the second
population of T cells. In
some embodiments, the tumor sample is obtained from a plurality of core
biopsies. In some
embodiments, the plurality of core biopsies is selected from the group
consisting of 2, 3, 4, 5, 6, 7, 8,
9 and 10 core biopsies.
10013281 In some embodiments, the invention the method described
in any of the preceding
paragraphs as applicable above modified such that T cells or TILs are obtained
from tumor digests. In
some embodiments, tumor digests are generated by incubating the tumor in
enzyme media, for
example but not limited to RPMI 1640, 2mM GlutaMAX, 10 mg/mL gentamicin, 30
U/mL DNase,
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and 1.0 mg/mL collagenase, followed by mechanical dissociation (GentleMACS,
Miltenyi Biotec,
Auburn, CA). In some embodiments, the tumor is placed in a tumor dissociating
enzyme mixture
including one or more dissociating (digesting) enzymes such as, but not
limited to, collagenase
(including any blend or type of collagenase), AccutaseTM, AccumaxTM,
hyaluronidase, neutral
protease (dispase), chymotrypsin, chymopapain, trypsin, caseinase, elastase,
papain, protease type
XIV (pronasc), dcoxyribonucicase I (DNasc), trypsin inhibitor, any other
dissociating or proteolytic
enzyme, and any combination thereof In other embodiments, the tumor is placed
in a tumor
dissociating enzyme mixture including collagenase (including any blend or type
of collagenase),
neutral protease (dispase) and deoxyribonuclease I (DNase).
1001329]
VI. Pharmaceutical Compositions, Dosages, and Dosing Regimens
10013301 In some embodiments, TILs, MILs, or PBLs expanded and/or genetically
modified
(including TILs, MILs, or PBLs genetically-modified to express a CCR) using
the methods of the
present disclosure are administered to a patient as a pharmaceutical
composition. In some
embodiments, the pharmaceutical composition is a suspension of TILs in a
sterile buffer. TILs
expanded using PBMCs of the present disclosure may be administered by any
suitable route as known
in the art. In some embodiments, the T-cells are administered as a single
intra-arterial or intravenous
infusion, which preferably lasts approximately 30 to 60 minutes. Other
suitable routes of
administration include intraperitoneal, intrathecal, and intralvmphatic
administration.
10013311Any suitable dose of TILs can be administered. In some embodiments,
from about 2.3 x10"
to about 13.7 x 10" TILs are administered, with an average of around 7.8>< 101
TILs, particularly if the
cancer is NSCLC or melanoma. In some embodiments, about 1.2 xleto about 4.3
x10" of TILs are
administered. In some embodiments, about 3 x 10' to about 12x 1010 TiLs are
administered. In some
embodiments, about 4 x 101 to about 10x 1010 TILs are administered. In some
embodiments, about
x 1010 to about 8x 1010 TILs are administered. In some embodiments, about 6
x1010to about 8 x 1010
TILs are administered. In some embodiments, about 7x 1010 to about 8 x 10"
TILs are administered. In
some embodiments, the therapeutically effective dosage is about 2.3 x101 to
about 13.7 x 10'. In some
embodiments, the therapeutically effective dosage is about 7.8x 10th TILs,
particularly of the cancer is
melanoma. In some embodiments, the therapeutically effective dosage is about
1.2< 10' to about
4.3 x1010 of TILs. In some embodiments, the therapeutically effective dosage
is about 3 x101 to about
12> 1010 TILs. In some embodiments, the therapeutically effective dosage is
about 4>< 10" to about
10x 1010 TILs. In some embodiments, the therapeutically effective dosage is
about 5 x101 to about
8x 1010 TILs. In some embodiments, the therapeutically effective dosage is
about 6x 1010 to about
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8 x10" TILs. In some embodiments, the therapeutically effective dosage is
about 7x 1010 to about
8 x 10" TILs.
10013321 In some embodiments, the number of the TILs provided in the
pharmaceutical compositions
of the invention is about 1 x 106, 2x 106, 3x106, 4x106, 5x106, 6x106, 7x 106,
8x 106, 9x 106, 1x10,
2x107, 3x107, 4x107,5x107, 6x107, 7x107, 8x107, 9x107, lx108, 2x108,3x108,
4x108, 5x108, 6x108,
7x108, 8x10s, 9x10s, lx 109, 2x109, 3x109, 4x109, 5x109, 6x109, 7x109, 8x109,
9x109, 1>11010,2>1010,
3x1010, 4x1e, 5><1010, 6x10", TAO", 8x10' , 9x1010, 1x1011, 2>10, 3x1011,
4x1011, 5><1011,
6><1011, 7><1011, 8x1011, 9><10", 1x1012, 2x1012, 3><1012, 4><1012, 5><1012,
6>11012,7>1012 8x1012,
9>11012, 1>1013, 2x1013, 3x1013, 41<1013, 5x1013, 6x1013, 71<1013, 8x 1013,
and 91<1013. In some
embodiments, the number of the TILs provided in the pharmaceutical
compositions of the invention is
inthe range of lx106to 5x106, 5x106to lx107, lx107to 5x107, 5x107to lx10x,
lx10x to 5x10x,
5x108to 1x109, lx109 to 5x109, 5x109to 1x1010, 1x101 to 5x1010, 5x101 to
1x10", 5x1011to
1x1012, 1x10"to 5x10", and 5x10"to lx1013.
10013331 In some embodiments, the concentration of the TILs provided in the
pharmaceutical
compositions of the invention is less than, for example, 100%, 90%, 80%, 70%,
60%, 50%, 40%,
30%, 20%, 19%, 18%, 17%, 16%, 15%, 14%, 13%, 12%, 11%, 10%, 9%, 8%, 7%, 6%,
5%, 4%, 3%,
2%, 1%, 0.5%, 0.4%, 0.3%, 0.2%, 0.1%, 0.09%, 0.08%, 0.07%, 0.06%, 0.05%,
0.04%, 0.03%, 0.02%,
0.01%, 0.009%, 0.008%, 0.007%, 0.006%, 0.005%, 0.004%, 0.003%, 0.002%, 0.001%,
0.0009%,
0.0008%, 0.0007%, 0.0006%, 0.0005%, 0.0004%, 0.0003%, 0.0002% or 0.0001% w/w,
w/v or v/v of
the pharmaceutical composition.
10013341In some embodiments, the concentration of the TILs provided in the
pharmaceutical
compositions of the invention is greater than 90%, 80%, 70%, 60%, 50%, 40%,
30%, 20%, 19.75%,
19.50%, 19.25% 19%, 18.75%, 18.50%, 18.25% 18%, 17.75%, 17.50%, 17.25% 17%,
16.75%,
16.50%, 16.25% 16%, 15.75%, 15.50%, 15.25% 15%, 14.75%, 14.50%, 14.25% 14%,
13.75%,
13.50%, 13.25% 13%, 12.75%, 12.50%, 12.25% 12%, 11.75%, 11.50%, 11.25% 11%,
10.75%,
10.50%, 10.25% 10%, 9.75%, 9.50%, 9.25% 9%, 8.75%, 8.50%, 8.25% 8%, 7.75%,
7.50%, 7.25%
7%, 6.75%, 6.50%, 6.25% 6%, 5.75%, 5.50%, 5.25% 5%, 4.75%, 4.50%, 4.25%, 4%,
3.75%, 3.50%,
3.25%, 3%, 2.75%, 2.50%, 2.25%, 2%, 1.75%, 1.50%, 125%, 1%, 0.5%, 0.4%, 0.3%,
0.2%, 0.1%,
0.09%, 0.08%, 0.07%, 0.06%, 0.05%, 0.04%, 0.03%, 0.02%, 0.01%, 0.009%, 0.008%,
0.007%,
0.006%, 0.005%, 0.004%, 0.003%, 0.002%, 0.001%, 0.0009%, 0.0008%, 0.0007%,
0.0006%,
0.0005%, 0.0004%, 0.0003%, 0.0002% or 0.0001% w/w, w/v, or v/v of the
pharmaceutical
composition.
10013351In some embodiments, the concentration of the TILs provided in the
pharmaceutical
compositions of the invention is in the range from about 0.0001% to about 50%,
about 0.001% to
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about 40%, about 0.01% to about 30%, about 0.02% to about 29%, about 0.03% to
about 28%, about
0.04% to about 27%, about 0.05% to about 26%, about 0.06% to about 25%, about
0.07% to about
24%, about 0.08% to about 23%, about 0.09% to about 22%, about 0.1% to about
21%, about 0.2% to
about 20%, about 0.3% to about 19%, about 0.4% to about 18%, about 0.5% to
about 17%, about
0.6% to about 16%, about 0.7% to about 15%, about 0.8% to about 14%, about
0.9% to about 12% or
about 1% to about 10% w/w, w/v or v/v of the pharmaceutical composition.
10013361 In some embodiments, the concentration of the TILs provided in the
pharmaceutical
compositions of the invention is in the range from about 0.001% to about 10%,
about 0.01% to about
5%, about 0.02% to about 4.5%, about 0.03% to about 4%, about 0.04% to about
3.5%, about 0.05%
to about 3%, about 0.06% to about 2.5%, about 0.07% to about 2%, about 0.08%
to about 1.5%, about
0.09% to about 1%, about 0.1% to about 0.9% w/w, w/v or v/v of the
pharmaceutical composition.
100133711n some embodiments, the amount of the TILs provided in the
pharmaceutical compositions
of the invention is equal to or less than 10 g, 9.5 g, 9.0 g, 8.5 g, 8.0 g,
7.5 g, 7.0 g, 6.5 g, 6.0 g, 5.5 g,
5.0 g, 4.5 g, 4.0 g, 3.5 g, 3.0 g, 2.5 g, 2.0 g, 1.5 g, 1.0 g, 0.95 g, 0.9 g,
0.85 g, 0.8 g, 0.75 g, 0.7 g, 0.65
g, 0.6 g, 0.55 g, 0.5 g, 0.45 g, 0.4 g, 0.35 g, 0.3 g, 0.25 g, 0.2 g, 0.15 g,
0.1 g, 0.09 g, 0.08 g, 0.07 g,
0.06 g, 0.05 g, 0.04 g, 0.03 g, 0.02 g, 0.01 g, 0.009 g, 0.008 g, 0.007 g,
0.006 g, 0.005 g, 0.004 g,
0.003 g, 0.002 g, 0.001 g, 0.0009 g, 0.0008 g, 0.0007 g, 0.0006 g, 0.0005 g,
0.0004 g, 0.0003 g,
0.0002 g, or 0.0001 g.
10013381 In some embodiments, the amount of the TILs provided in the
pharmaceutical compositions
of the invention is more than 0.0001 g, 0.0002 g, 0.0003 g, 0.0004 g, 0.0005
g, 0.0006 g, 0.0007 g,
0.0008 g, 0.0009g. 0.001 g, 0.0015 g, 0.002 g, 0.0025 g, 0.003 g, 0.0035 g,
0.004 g, 0.0045 g, 0.005
g, 0.0055 g, 0.006 g, 0.0065 g, 0.007 g, 0.0075 g, 0.008 g, 0.0085 g, 0.009 g,
0.0095 g, 0.01 g, 0.015
g, 0.02 g, 0.025 g, 0.03 g, 0.035 g, 0.04 g, 0.045 g, 0.05 g, 0.055 g, 0.06 g,
0.065 g, 0.07 g, 0.075 g,
0.08 g, 0.085 g, 0.09 g, 0.095 g, 0.1 g, 0.15 g, 0.2 g, 0.25 g, 0.3 g, 0.35 g,
0.4 g, 0.45 g, 0.5 g, 0.55 g,
0.6 g, 0.65 g, 0.7 g, 0.75 g, 0.8 g, 0.85 g, 0.9 g, 0.95 g, 1 g, 1.5 g, 2 g,
2.5,3 g, 3.5,4 g, 4.5 g, 5 g, 5.5
g, 6 g, 6.5 g, 7 g, 7.5 g, 8g. 8.5 g, 9 g, 9.5 g, or 10 g.
10013391 The TILs provided in the pharmaceutical compositions of the invention
are effective over a
wide dosage range. The exact dosage will depend upon the route of
administration, the form in which
the compound is administered, the gender and age of the subject to be treated,
the body weight of the
subject to be treated, and the preference and experience of the attending
physician. The clinically-
established dosages of the TILs may also be used if appropriate. The amounts
of the pharmaceutical
compositions administered using the methods herein, such as the dosages of
TILs, will be dependent
on the human or mammal being treated, the severity of the disorder or
condition, the rate of
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administration, the disposition of the active pharmaceutical ingredients and
the discretion of the
prescribing physician.
10013401111 some embodiments, TILs may be administered in a single dose. Such
administration may
be by injection, e.g., intravenous injection. In some embodiments, TILs may be
administered in
multiple doses. Dosing may be once, twice, three times, four times, five
times, six times, or more than
six times per year. Dosing may be once a month, once every two weeks, once a
week, or once every
other day. Administration of TILs may continue as long as necessary.
10013411In some embodiments, an effective dosage of TILs is about 1 x 106, 2 x
106, 3x106, 4 x 106,
5x106, 6x106, 7x106, 8x106, 9x106, lx107, 2x107, 3x107, 4x107, 5x107, 6x107,
7x107, 8x107, 9x107,
lx108, 2x108, 3x108, 4x108,5x108, 6x108, 7x108, 8x108, 9x108, lx109, 2x109,
3x109, 4x109, 5x109,
6x109, 7x109, 8x109, 9x109, lx10', 2x10', 3x10", 4x10',5x10', 6x101", 7x1019,
8x10', 9x10th,
lx 1011, 2x1011, 3x1011, 4x1011, 51<1011, 6x1011, 7x1011, 8x1011, 9x1011, lx
1012, 2x1012, 3x1012,
4x1012, 5x1012, 6x1012, 7x1012, 8x1012, 9x 1012, lx 1013, 2x1013, 3x 1013,
4x1013, 5x1013, 6x1013,
7x10'3, 8x10'3, and 9x10'3. In some embodiments, an effective dosage of TILs
is in the range of
lx106to 5x106,5x106to 1x107, lx107to 5x107, 5x107t0 1x108, lx108to
5x108,5x108to 1x109,
lx109to 5x109, 5x109 to lx101 , lx1016to 5x101 , 5x101 to 1x1011, 5x1011to
1x1012, lx1012to
5x1012, and 5x1012 to lx1012.
100134211n some embodiments, an effective dosage of TILs is in the range of
about 0.01 mg/kg to
about 4.3 mg/kg, about 0.15 mg/kg to about 3.6 mg/kg, about 0.3 mg/kg to about
3.2 mg/kg, about
0.35 mg/kg to about 2.85 mg/kg, about 0.15 mg/kg to about 2.85 mg/kg, about
0.3 mg to about 2.15
mg/kg, about 0.45 mg/kg to about 1.7 mg/kg, about 0.15 mg/kg to about 1.3
mg/kg, about 0.3 mg/kg
to about 1.15 mg/kg, about 0.45 mg/kg to about I mg/kg, about 0.55 mg/kg to
about 0.85 mg/kg,
about 0.65 mg/kg to about 0.8 mg/kg, about 0.7 mg/kg to about 0.75 mg/kg,
about 0.7 mg/kg to about
2.15 mg/kg, about 0.85 mg/kg to about 2 mg/kg, about I mg/kg to about 1.85
mg/kg, about 1.15
mg/kg to about 1.7 mg/kg, about 1.3 mg/kg mg to about 1.6 mg/kg, about 1.35
mg/kg to about 1.5
mg/kg, about 2.15 mg/kg to about 3.6 mg/kg, about 2.3 mg/kg to about 3.4
mg/kg, about 2.4 mg/kg to
about 3.3 mg/kg, about 2.6 mg/kg to about 3.15 mg/kg, about 2.7 mg/kg to about
3 mg/kg, about 2.8
mg/kg to about 3 mg/kg, or about 2.85 mg/kg to about 2.95 mg/kg.
[001343111i some embodiments, an effective dosage of TILs is in the range of
about 1 mg to about
500 mg, about 10 mg to about 300 mg, about 20 mg to about 250 mg, about 25 mg
to about 200 mg,
about 1 mg to about 50 mg, about 5 mg to about 45 mg, about 10 mg to about 40
mg, about 15 mg to
about 35 mg, about 20 mg to about 30 mg, about 23 mg to about 28 mg, about 50
mg to about 150
mg, about 60 mg to about 140 mg, about 70 mg to about 130 mg, about 80 mg to
about 120 mg, about
90 mg to about 110 mg, or about 95 mg to about 105 mg, about 98 mg to about
102 mg, about 150 mg
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to about 250 mg, about 160 mg to about 240 mg, about 170 mg to about 230 mg,
about 180 mg to
about 220 mg, about 190 mg to about 210 mg, about 195 mg to about 205 mg, or
about 198 to about
207 mg.
10013441 An effective amount of the TILs may be administered in either single
or multiple doses by
any of the accepted modes of administration of agents having similar
utilities, including intranasal and
transdermal routes, by intra-arterial injection, intravenously,
intraperitoneally, parenterally,
intramuscularly, subcutaneously, topically, by transplantation, or by
inhalation.
10013451 In some embodiments, the invention provides an infusion bag
comprising the therapeutic
population of TILs described in any of the preceding paragraphs above.
10013461 In some embodiments, the invention provides a tumor infiltrating
lymphocyte (TIL)
composition comprising the therapeutic population of TILs described in any of
the preceding
paragraphs above and a pharmaceutically acceptable carrier.
10013471 In some embodiments, the invention provides an infusion bag
comprising the TIL
composition described in any of the preceding paragraphs above.
10013481 In some embodiments, the invention provides a cryoprescrved
preparation of the therapeutic
population of TILs described in any of the preceding paragraphs above.
10013491 In some embodiments, the invention provides a tumor infiltrating
lymphocyte (TIL)
composition comprising the therapeutic population of TILs described in any of
the preceding
paragraphs above and a cryoprcscrvation media.
10013501 In some embodiments, the invention provides the TIL composition
described in any of the
preceding paragraphs above modified such that the cryopreservation media
contains DMSO.
100135111n some embodiments, the invention provides the TIL composition
described in any of the
preceding paragraphs above modified such that the cryopreservation media
contains 7-10% DMSO.
10013521 In some embodiments, the invention provides a cryopreserved
preparation of the TIL
composition described in any of the preceding paragraphs above.
10013531 In some embodiments, TILs expanded using the methods of the present
disclosure are
administered to a patient as a pharmaceutical composition. In some
embodiments, the pharmaceutical
composition is a suspension of TILs in a sterile buffer. TILs expanded using
PBMCs of the present
disclosure may be administered by any suitable route as known in the art. In
some embodiments, the
T-cells are administered as a single intra-arterial or intravenous infusion,
which preferably lasts
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approximately 30 to 60 minutes. Other suitable routes of administration
include intraperitoneal,
intrathecal, and intralymphatic administration.
10013541Any suitable dose of TILs can be administered. In some embodiments,
from about 2.3 x1010
to about 13.7 x 1010 TILs are administered, with an average of around 7.8x1010
TILs, particularly if the
cancer is NSCLC. In some embodiments, about 1.2x101 to about 4=3x 1010 of TILs
are administered.
In some embodiments, about 3 x1010to about 12 x1010TILs are administered. In
some embodiments,
about 4< 10' to about 10 x 101" TILs are administered. In some embodiments,
about 5 xlVto about
8x 1010 TILs are administered. In some embodiments, about 6 x10"to about 8x
101 TILs are
administered. In some embodiments, about 7x 101"to about 8x101 TILs are
administered. In some
embodiments, therapeutically effective dosage is about 2.3 x101"to about 13.7
x101 . In some
embodiments, therapeutically effective dosage is about 7.8x1010TILs,
particularly of the cancer is
NSCLC. In some embodiments, therapeutically effective dosage is about 1.2x
1010 to about 4=3x 101
of TILs. In some embodiments, therapeutically effective dosage is about 3
x101" to about 12x 1010
TILs. In some embodiments, therapeutically effective dosage is about 4x 1010
to about 10x 1010 TILs.
In some embodiments, therapeutically effective dosage is about 5 x 1010 to
about 8x1010 TILs. In some
embodiments, therapeutically effective dosage is about 6x1010to about 8x 101
TILs. In some
embodiments, therapeutically effective dosage is about 7x101"to about 8 x 1010
TILs.
10013551in some embodiments, the number of the TILs provided in the
pharmaceutical compositions
of the invention is about 1 x 106, 2x 106, 3x106, 4x106, 5x10", 6x 106, 7x
106, 8x 106, 9x 106, lx 107,
2x107, 3x107, 4x107,5x107, 6x107, 7x107, 8x107, 9x107, lx108,2x108,3x10s,
4x10', 5x10g, 6x10g,
7x10s, 8x108, 9x108, lx109, 2x109, 3x109, 4x109, 5x109, 6x109, 7x109, 8x109,
9x109, lx101", 2x101 ,
3x101 , 4x 1010, 5x 101 , 6x1010, 7x101 , 8x lOb, 9x101 , 11<1011, 2x1011,
3x10", 4x1011, 51<1011,
6x10", 7x10", 8x10", 9x10", lx1012, 2x1012,31<1012, 4x1012, 5x1012, 6x1012,
7x1012, 8x1012,
91<1012, 1x10', 2x1013, 3x1013, 4x1013, 5x 10',6x10', 7x1013, 8x 101s, and
9x1013. In some
embodiments, the number of the TILs provided in the pharmaceutical
compositions of the invention is
inthe range of lx106to 5x106, 5x106to lx107, lx107to 5x 107, 5x 10' to lx 108,
lx108to 5x108,
5x108to lx109, lx109 to 510, 5x109to lx1010, lx10mto 51<1010, 5x101 to
lx1011, 5x1011to
11<1012, lx1012 to 5x1012, and 5x1012 to lx10'.
10013561In some embodiments, the concentration of the TILs provided in the
pharmaceutical
compositions of the invention is less than, for example, 100%, 90%, 80%, 70%,
60%, 50%, 40%,
30%, 20%, 19%, 18%, 17%, 16%, 15%, 14%, 13%, 12%, 11%, 10%, 9%. 8%, 7%, 6%,
5%, 4%, 3%,
2%, 1%, 0.5%, 0.4%, 0.3%, 0.2%, 0.1%, 0.09%, 0.08%, 0.07%, 0.06%, 0.05%,
0.04%, 0.03%, 0.02%,
0.01%, 0.009%, 0.008%, 0.007%, 0.006%, 0.005%, 0.004%, 0.003%, 0.002%, 0.001%,
0.0009%,
0.0008%, 0.0007%, 0.0006%, 0.0005%, 0.0004%, 0.0003%, 0.0002% or 0.0001% w/w,
w/v or v/v of
the pharmaceutical composition.
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10013571In some embodiments, the concentration of the TILs provided in the
pharmaceutical
compositions of the invention is greater than 90%, 80%, 70%, 60%, 50%, 40%,
30%, 20%, 19.75%,
19.50%, 19.25% 19%, 18.75%, 18.50%, 18.25% 18%, 17.75%, 17.50%, 17.25% 17%,
16.75%,
16.50%, 16.25% 16%, 15.75%, 15.50%, 15.25% 15%, 14.75%, 14.50%, 14.25% 14%,
13.75%,
13.50%, 13.25% 13%, 12.75%, 12.50%, 12.25% 12%, 11.75%, 11.50%, 11.25% 11%,
10.75%,
10.50%, 10.25% 10%, 9.75%, 9.50%, 9.25% 9%, 8.75%, 8.50%, 8.25% 8%, 7.75%,
7.50%, 7.25%
7%, 6.75%, 6.50%, 6.25% 6%, 5.75%, 5.50%, 5.25% 5%, 4.75%, 4.50%, 4.25%, 4%,
3.75%, 3.50%,
3.25%, 3%, 2.75%, 2.50%, 2.25%, 2%, 1.75%, 1.50%, 125%, 1%, 0.5%, 0.4%, 0.3%,
0.2%, 0.1%,
0.09%, 0.08%, 0.07%, 0.06%, 0.05%, 0.04%, 0.03%, 0.02%, 0.01%, 0.009%, 0.008%,
0.007%,
0.006%, 0.005%, 0.004%, 0.003%, 0.002%, 0.001%, 0.0009%, 0.0008%, 0.0007%,
0.0006%,
0.0005%, 0.0004%, 0.0003%, 0.0002% or 0.0001% w/w, w/v, or v/v of the
pharmaceutical
composition.
10013581 In some embodiments, the concentration of the TILs provided in the
pharmaceutical
compositions of the invention is in the range from about 0.0001% to about 50%,
about 0.001% to
about 40%, about 0.01% to about 30%, about 0.02% to about 29%, about 0.03% to
about 28%, about
0.04% to about 27%, about 0.05% to about 26%, about 0.06% to about 25%, about
0.07% to about
24%, about 0.08% to about 23%, about 0.09% to about 22%, about 0.1% to about
21%, about 0.2% to
about 20%, about 0.3% to about 19%, about 0.4% to about 18%, about 0.5% to
about 17%, about
0.6% to about 16%, about 0.7% to about 15%, about 0.8% to about 14%, about
0.9% to about 12% or
about 1% to about 10% w/w, w/v or v/v of the pharmaceutical composition.
10013591111 some embodiments, the concentration of the TILs provided in the
pharmaceutical
compositions of the invention is in the range from about 0.001% to about 10%,
about 0.01% to about
5%, about 0.02% to about 4.5%, about 0.03% to about 4%, about 0.04% to about
3.5%, about 0.05%
to about 3%, about 0.06% to about 2.5%, about 0.07% to about 2%, about 0.08%
to about 1.5%, about
0.09% to about 1%, about 0.1% to about 0.9% w/w, w/v or v-/v- of the
pharmaceutical composition.
10013601In some embodiments, the amount of the TILs provided in the
pharmaceutical compositions
of the invention is equal to or less than 10 g, 9.5 g, 9.0g. 8.5 g, 8.0 g, 7.5
g, 7.0 g, 6.5 g, 6.0 g, 5.5 g,
5.0 g, 4.5 g, 4.0 g, 3.5 g, 3.0 g, 2.5 g, 2.0 g, 1.5 g, 1.0 g, 0.95 g, 0.9 g,
0.85 g, 0.8 g, 0.75 g, 0.7 g, 0.65
g, 0.6 g, 0.55 g, 0.5 g, 0.45 g, 0.4 g, 0.35 g, 0.3 g, 0.25 g, 0.2 g, 0.15 g,
0.1 g, 0.09 g, 0.08 g, 0.07 g,
0.06 g, 0.05 g, 0.04 g, 0.03 g, 0.02 g, 0.01 g, 0.009 g, 0.008 g, 0.007 g,
0.006 g, 0.005 g, 0.004 g,
0.003 g, 0.002 g, 0.001 g, 0.0009 g, 0.0008 g, 0.0007g. 0.0006 g, 0.0005 g,
0.0004 g, 0.0003 g,
0.0002g. or 0.0001 g.
10013611ln some embodiments, the amount of the TILs provided in the
pharmaceutical compositions
of the invention is more than 0.0001 g, 0.0002 g, 0.0003 g, 0.0004 g, 0.0005
g, 0.0006 g, 0.0007 g,
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0.0008 g, 0.0009g. 0.001 g, 0.0015 g, 0.002 g, 0.0025 g, 0.003 g, 0.0035 g,
0.004 g, 0.0045 g, 0.005
g, 0.0055 g, 0.006 g, 0.0065 g, 0.007 g, 0.0075 g, 0.008 g, 0.0085 g, 0.009 g,
0.0095 g, 0.01 g, 0.015
g, 0.02 g, 0.025 g, 0.03 g, 0.035 g, 0.04 g, 0.045 g, 0.05 g, 0.055 g, 0.06 g,
0.065 g, 0.07 g, 0.075 g,
0.08g, 0.085 g, 0.09g, 0.095 g, 0.1 g, 0.15 g, 0.2g, 0.25 g, 0.3 g, 0.35 g,
0.4g, 0.45 g, 0.5 g, 0.55 g,
0.6 g, 0.65 g, 0.7 g, 0.75 g, 0.8 g, 0.85 g, 0.9 g, 0.95 g, 1 g, 1.5 g, 2 g,
2.5,3 g, 3.5,4 g, 4.5 g, 5 g, 5.5
g, 6g. 6.5 g, 7 g, 7.5 g, 8 g, 8.5 g, 9 g, 9.5 g, or 10 g.
10013621 The TILs provided in the pharmaceutical compositions of the invention
are effective over a
wide dosage range. The exact dosage will depend upon the route of
administration, the form in which
the compound is administered, the gender and age of the subject to be treated,
the body weight of the
subject to be treated, and the preference and experience of the attending
physician. The clinically-
established dosages of the TILs may also be used if appropriate. The amounts
of the pharmaceutical
compositions administered using the methods herein, such as the dosages of
TILs, will be dependent
on the human or mammal being treated, the severity of the disorder or
condition, the rate of
administration, the disposition of the active pharmaceutical ingredients and
the discretion of the
prescribing physician.
10013631 In some embodiments, TILs may be administered in a single dose. Such
administration may
be by injection, e.g., intravenous injection. In some embodiments, TILs may be
administered in
multiple doses. Dosing may be once, twice, three times, four times, five
times, six times, or more than
six times per year. Dosing may be once a month, once every two weeks, once a
week, or once every
other day. Administration of TILs may continue as long as necessary.
100136411n some embodiments, an effective dosage of TILs is about 1 x 106, 2 x
106, 3 x 106, 4 x 106,
5x106, 6x106, 73< 106, 83< 106, 9x106, 1x10, 2x107, 310, 4x107, 5x107, 610,
7x10, 8x107, 9x107,
lx108, 2x108, 3x108, 4x108,5x10s, 6x108, 7x108, 8x108, 9x108, lx109, 2x109,
3x109, 4x109, 5x109,
6x109, 7x109, 8x109, 9x109, x low, 2x low, 3xioio, 4x low, 5x
iu
6x101 , 7x101 , 8x 101 , 9x10",
lx1011, 2x1011, 3x1011, 4x1011, 5x1011, 6x1011, 7x1011, 8x1011, 9x1011, lx
1012, 2x1012, 3x1012,
4x1012, 5x 1012, 6x 1012, 7x1012, 8x1012, 9x 1012, lx10", 2x1013, 3x 1013,
4x1013, 5x1013, 6x10",
7x10'3 8 x 10", and 9x10'3. In some embodiments, an effective dosage of TILs
is in the range of
lx10f¨to 5x10',5x10'tolx107,1x107to 5x107,5x107to lx10', lx10'to 5x10',5x10to
lx109,
lx109to 5x109. 5x109 to lx101 , lx10"to 5x101 , 5x101 to lx10'1, 5x1011to
lx1012, lx1012to
5x1012, and 5x1012to lx1012.
10013651111 some embodiments, an effective dosage of TILs is in the range of
about 0.01 mg/kg to
about 4.3 mg/kg, about 0.15 mg/kg to about 3.6 mg/kg, about 0.3 mg/kg to about
3.2 mg/kg, about
0.35 mg/kg to about 2.85 mg/kg, about 0.15 mg/kg to about 2.85 mg/kg, about
0.3 mg to about 2.15
mg/kg, about 0.45 mg/kg to about 1.7 mg/kg, about 0.15 mg/kg to about 1.3
mg/kg, about 0.3 mg/kg
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to about 1.15 mg/kg, about 0.45 mg/kg to about 1 mg/kg, about 0.55 mg/kg to
about 0.85 mg/kg,
about 0.65 mg/kg to about 0.8 mg/kg, about 0.7 mg/kg to about 0.75 mg/kg,
about 0.7 mg/kg to about
2.15 mg/kg, about 0.85 mg/kg to about 2 mg/kg, about 1 mg/kg to about 1.85
mg/kg, about 1.15
mg/kg to about 1.7 mg/kg, about 1.3 mg/kg mg to about 1.6 mg/kg, about 1.35
mg/kg to about 1.5
mg/kg, about 2.15 mg/kg to about 3.6 mg/kg, about 2.3 mg/kg to about 3.4
mg/kg, about 2.4 mg/kg to
about 3.3 mg/kg, about 2.6 mg/kg to about 3.15 mg/kg, about 2.7 mg/kg to about
3 mg/kg, about 2.8
mg/kg to about 3 mg/kg, or about 2.85 mg/kg to about 2.95 mg/kg.
10013661ln some embodiments, an effective dosage of TILs is in the range of
about 1 mg to about
500 mg, about 10 mg to about 300 mg, about 20 mg to about 250 mg, about 25 mg
to about 200 mg,
about 1 mg to about 50 mg, about 5 mg to about 45 mg, about 10 mg to about 40
mg, about 15 mg to
about 35 mg, about 20 mg to about 30 mg, about 23 mg to about 28 mg, about 50
mg to about 150
mg, about 60 mg to about 140 mg, about 70 mg to about 130 mg, about 80 mg to
about 120 mg, about
90 mg to about 110 mg, or about 95 mg to about 105 mg, about 98 mg to about
102 mg, about 150 mg
to about 250 mg, about 160 mg to about 240 mg, about 170 mg to about 230 mg,
about 180 mg to
about 220 mg, about 190 mg to about 210 mg, about 195 mg to about 205 mg, or
about 198 to about
207 mg.
10013671 An effective amount of the TILs may be administered in either single
or multiple doses by
any of the accepted modes of administration of agents having similar
utilities, including intranasal and
transdermal routes, by intra-arterial injection, intravenously,
intraperitoneally, parenterally,
intramuscularly, subcutaneously, topically, by transplantation, or by
inhalation.
VII. Methods of Treating Patients
10013681 Methods of treatment begin with the initial TIL collection and
culture of TILs. Such
methods have been both described in the art by, for example, Jin et al., I
Inimunotherapy, 2012,
35(3):283-292, incorporated by reference herein in its entirety. Embodiments
of methods of treatment
are described throughout the sections below, including the Examples.
10013691 The expanded TILs produced according the methods described herein,
including for
example as described in Steps A through F above or according to Steps A
through F above (also as
shown, for example, in Figure 1 and/or Figure 8) find particular use in the
treatment of patients with
cancer (for example, as described in Goff, et al., I Clinical Oncology, 2016,
34(20):2389-239, as well
as the supplemental content; incorporated by reference herein in its entirety.
In some embodiments,
TIL were grown from resected deposits of metastatic melanoma as previously
described (see, Dudley,
et al., õI Immunother., 2003, 26:332-342; incorporated by reference herein in
its entirety). Fresh tumor
can be dissected under sterile conditions. A representative sample can be
collected for formal
pathologic analysis. Single fragments of 2 mm3to 3 min3 may be used. In some
embodiments, 5, 10,
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15, 20, 25 or 30 samples per patient are obtained. In some embodiments, 20,
25, or 30 samples per
patient are obtained. In some embodiments, 20, 22, 24, 26, or 28 samples per
patient are obtained. In
some embodiments, 24 samples per patient are obtained. Samples can be placed
in individual wells of
a 24-well plate, maintained in growth media with high-dose IL-2 (6,000 IU/mL),
and monitored for
destruction of tumor and/or proliferation of TIL. Any tumor with viable cells
remaining after
processing can be enzymatically digested into a single cell suspension and
cryopreserved, as
described herein.
[0013701M some embodiments, successfully grown TIT, can be sampled for
phenotype analysis
(CD3, CD4, CD8, and CD56) and tested against autologous tumor when available.
TIL can be
considered reactive if overnight coculture yielded interferon-gamma (IFN-7)
levels > 200 pg/mL and
twice background. (Goff, et al., J Immunother. , 2010, 33:840-847;
incorporated by reference herein in
its entirety). In some embodiments, cultures with evidence of autologous
reactivity or sufficient
growth patterns can be selected for a second expansion (for example, a second
expansion as provided
in according to Step D of Figure 1 and/or Figure 8), including second
expansions that are sometimes
referred to as rapid expansion (REP). In some embodiments, expanded TILs with
high autologous
reactivity (for example, high proliferation during a second expansion), are
selected for an additional
second expansion. In some embodiments, TILs with high autologous reactivity
(for example, high
proliferation during second expansion as provided in Step D of Figure 1 and/or
Figure 8), are selected
for an additional second expansion according to Step D of Figure 1 and/or
Figure 8.
10013711 Cell phenotypes of cryopreseryed samples of infusion bag TIL can be
analyzed by flow
cytometry (e.g. , Floyd()) for surface markers CD3, CD4, CD8, CCR7, and CD45RA
(BD
BioSciences), as well as by any of the methods described herein. Serum
cytokines were measured by
using standard enzyme-linked immunosorbent assay techniques. A rise in serum
IFN-g was defined as
>100 pg/mL and greater than 4 3 baseline levels.
10013721ln some embodiments, the TILs produced by the methods provided herein,
for example
those exemplified in Figure 1 and/or Figure 8, provide for a surprising
improvement in clinical
efficacy of the TILs. In some embodiments, the TILs produced by the methods
provided herein, for
example those exemplified in Figure 1 and/or Figure 8, exhibit increased
clinical efficacy as
compared to TILs produced by methods other than those described herein,
including for example,
methods other than those exemplified in Figure 1 and/or Figure 8. In some
embodiments, the methods
other than those described herein include methods referred to as process 1C
and/or Generation 1 (Gen
1). In some embodiments, the increased efficacy is measured by DCR, ORR,
and/or other clinical
responses. In some embodiments, the TILs produced by the methods provided
herein, for example
those exemplified in Figure 1, exhibit a similar time to response and safety
profile compared to TILs
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produced by methods other than those described herein, including for example,
methods other than
those exemplified in Figure 1 and/or Figure 8.
10013731ln some embodiments, IFN-gamma (IFN-y) is indicative of treatment
efficacy and/or
increased clinical efficacy. In some embodiments, 1FN-y in the blood of
subjects treated with TILs is
indicative of active TILs. In some embodiments, a potency assay for IFN-y
production is employed.
IFN-y production is another measure of cytotoxic potential. IFN-y production
can be measured by
determining the levels of the cytokine IFN-y in the blood, serum, or TILs ex
vivo of a subject treated
with TILs prepared by the methods of the present invention, including those as
described for example
in Figure 1 and/or Figure 8. In some embodiments, an increase in IFN-y is
indicative of treatment
efficacy in a patient treated with the TILs produced by the methods of the
present invention. In some
embodiments, IFN-y is increased one-fold, two-fold, three-fold, four-fold, or
five-fold or more as
compared to an untreated patient and/or as compared to a patient treated with
TILs prepared using
other methods than those provide herein including for example, methods other
than those embodied in
Figure 1 and/or Figure 8. In some embodiments, IFN-y secretion is increased
one-fold as compared to
an untreated patient and/or as compared to a patient treated with TILs
prepared using other methods
than those provide herein including for example, methods other than those
embodied in Figure 1
and/or Figure 8. In some embodiments, IFN-y secretion is increased two-fold as
compared to an
untreated patient and/or as compared to a patient treated with TILs prepared
using other methods than
those provide herein including for example, methods other than those embodied
in Figure 1 and/or
Figure 8. in some embodiments, IFN-y secretion is increased three-fold as
compared to an untreated
patient and/or as compared to a patient treated with TILs prepared using other
methods than those
provide herein including for example, methods other than those embodied in
Figure 1 and/or Figure 8.
In some embodiments, 1FN-y secretion is increased four-fold as compared to an
untreated patient
and/or as compared to a patient treated with TILs prepared using other methods
than those provide
herein including for example, methods other than those embodied in Figure 1
and/or Figure 8. In
some embodiments, IFN-y secretion is increased five-fold as compared to an
untreated patient and/or
as compared to a patient treated with TILs prepared using other methods than
those provide herein
including for example, methods other than those embodied in Figure 1 and/or
Figure 8. In some
embodiments, IFN-y is measured using a Quantikine ELISA kit. In some
embodiments, IFN-y is
measured in TILs ex vivo of a subject treated with TILs prepared by the
methods of the present
invention, including those as described for example in Figure 1 and/or Figure
8. In some
embodiments, IFN-y is measured in blood of a subject treated with TILs
prepared by the methods of
the present invention, including those as described for example in Figure 1
and/or Figure 8. In some
embodiments, IFN-y is measured in TILs serum of a subject treated with TILs
prepared by the
methods of the present invention, including those as described for example in
Figure 1 and/or Figure
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8. In some embodiments, IFN-gamma (IFN-y) is indicative of treatment efficacy
and/or increased
clinical efficacy in the treatment of cancer
10013741In some embodiments, in the TILs prepared by the methods of the
present invention,
including those as described for example in Figure 1 and/or Figure 8 1FN -
gamma (1FN-y) is indicative
of treatment efficacy and/or increased clinical efficacy. In some embodiments,
IFN-y in the blood of
subjects treated with TILs is indicative of active TILs. In some embodiments,
a potency assay for
IFN-y production is employed. IFN-y production is another measure of cytotoxic
potential. IFN-y
production can be measured by determining the levels of the cytokine IFN-y in
the blood, senim, or
TILs ex vivo of a subject treated with TILs prepared by the methods of the
present invention,
including those as described for example in Figure 1 and/or Figure 8. In some
embodiments, an
increase in IFN-y is indicative of treatment efficacy in a patient treated
with the TILs produced by the
methods of the present invention. In some embodiments, IFN-y is increased one-
fold, two-fold, three-
fold, four-fold, or five-fold or more IFN-y as compared to an untreated
patient and/or as compared to
a patient treated with TILs prepared using other methods than those provide
herein including for
example, methods other than those embodied in Figure 1 and/or Figure 8.
10013751in some embodiments, the TILs prepared by the methods of the present
invention, including
those as described for example in Figure 1 and/or Figure 8, exhibit increased
polyclonality as
compared to TILs produced by other methods, including those not exemplified in
Figure 1 and/or
Figure 8, including for example, methods referred to as process 1C methods. In
some embodiments,
significantly improved polyclonality and/or increased polyclonality is
indicative of treatment efficacy
and/or increased clinical efficacy. In some embodiments, polyclonality refers
to the T-cell repertoire
diversity. In some embodiments, an increase in polyclonality can be indicative
of treatment efficacy
with regard to administration of the TILs produced by the methods of the
present invention. In some
embodiments, polyclonality is increased one-fold, two-fold, ten-fold, 100-
fold, 500-fold, or 1000-fold
as compared to TILs prepared using methods than those provide herein including
for example,
methods other than those embodied in Figure 1 and/or Figure 8. In some
embodiments, polyclonality
is increased one-fold as compared to an untreated patient and/or as compared
to a patient treated with
TILs prepared using other methods than those provide herein including for
example, methods other
than those embodied in Figure 1 and/or Figure 8. In some embodiments,
polyclonality is increased
two-fold as compared to an untreated patient and/or as compared to a patient
treated with TILs
prepared using other methods than those provide herein including for example,
methods other than
those embodied in Figure 1 and/or Figure 8. In some embodiments, polyclonality
is increased ten-fold
as compared to an untreated patient and/or as compared to a patient treated
with TILs prepared using
other methods than those provide herein including for example, methods other
than those embodied in
Figure 1 and/or Figure 8. In some embodiments, polyclonality is increased 100-
fold as compared to an
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untreated patient and/or as compared to a patient treated with TILs prepared
using other methods than
those provide herein including for example, methods other than those embodied
in Figure 1 and/or
Figure 8. in some embodiments, polyclonality is increased 500-fold as compared
to an untreated
patient and/or as compared to a patient treated with TILs prepared using other
methods than those
provide herein including for example, methods other than those embodied in
Figure 1 and/or Figure 8.
In some embodiments, polyclonalrty is increased 1000-fold as compared to an
untreated patient and/or
as compared to a patient treated with TILs prepared using other methods than
those provide herein
including for example, methods other than those embodied in Figure 1 and/or
Figure 8.
10013761 Measures of efficacy can include the disease control rate (DCR) as
well as overall response
rate (ORR), as known in the art as well as described herein.
10013771 In some embodiments, the invention includes a method of treating
NSCLC with a
population of TILs, wherein a patient is pre-treated with non-myeloablative
chemotherapy prior to an
infusion of TILs according to the present disclosure. In some embodiments, the
non-mveloablative
chemotherapy is cyclophosphamide 60 mg/kg/d for 2 days (days 27 and 26 prior
to TIL infusion) and
fludarabine 25 mg/m2/d for 5 days (days 27 to 23 prior to TIL infusion). In
some embodiments, the
non-myeloablative chemotherapy is cyclophosphamide 60 mg/kg/d for 2 days (days
27 and 26 prior to
TIL infusion) and fludarabine 25 mg/m2/d for 3 days (days 27 to 25 prior to
TIL infusion). In some
embodiments, the non-myeloablative chemotherapy is cyclophosphamide 60 mg/kg/d
for 2 days (days
27 and 26 prior to TIL infusion) followed by fludarabine 25 mg/m2/d for 3 days
(days 25 to 23 prior
to TIL infusion). In some embodiments, after non-myeloablative chemotherapy
and TIL infusion (at
day 0) according to the present disclosure, the patient receives an
intravenous infusion of IL-2
intravenously at 720,000 IU/kg every 8 hours to physiologic tolerance.
10013781 Efficacy of the compounds and combinations of compounds described
herein in treating,
preventing and/or managing the indicated diseases or disorders can be tested
using various models
known in the art, which provide guidance for treatment of human disease. For
example, models for
determining efficacy of treatments for lung cancer are described, e.g., in
Meuwissen, et al., Genes &
Development, 2005, 19, 643-664. Models for detemaining efficacy of treatments
for lung cancer are
described, e.g., in Kim, Clin. Exp. Otorhinolaryngol. 2009, 2, 55-60; and
Sano, Head Neck Oncol.
2009, 1, 32.
100137911n some embodiments, 1FN-gamma (1FN-y) is indicative of treatment
efficacy for NSCLC
treatment. In some embodiments, IFN-y in the blood of subjects treated with
TILs is indicative of
active TILs. In some embodiments, a potency assay for IFN-y production is
employed. IFN-y
production is another measure of cytotoxic potential. IFN-y production can be
measured by
determining the levels of the cytokine IFN-y in the blood of a subject treated
with TILs prepared by
the methods of the present invention, including those as described for example
in Figure 8 (in
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particular, e.g., Figure 8A and/or Figure 8B and/or Figure 8C and/or Figure
8D). In some
embodiments, the TILs obtained by the present method provide for increased IFN-
y in the blood of
subjects treated with the TTLs of the present method as compared subjects
treated with TILs prepared
using methods referred to as the Gen 3 process, as exemplified Figure 8 (in
particular, e.g., Figure 8A
and/or Figure 8B and/or Figure 8C and/or Figure 8D) and throughout this
application. In some
embodiments, an increase in _ITN -y is indicative of treatment efficacy in a
patient treated with the
TILs produced by the methods of the present invention. In some embodiments,
IFN-y is increased
one-fold, two-fold, three-fold, four-fold, or five-fold or more as compared to
an untreated patient
and/or as compared to a patient treated with TILs prepared using other methods
than those provide
herein including, for example, methods other than those embodied in Figure 8
(in particular, e.g.,
Figure 8A and/or Figure 8B and/or Figure 8C and/or Figure 8D). In some
embodiments, IFN-y
secretion is increased one-fold as compared to an untreated patient and/or as
compared to a patient
treated with TILs prepared using other methods than those provide herein
including, for example,
methods other than those embodied in Figure 8 (in particular, e.g., Figure 8A
and/or Figure 8B and/or
Figure 8C and/or Figure 8D). In some embodiments, IFN-y secretion is increased
two-fold as
compared to an untreated patient and/or as compared to a patient treated with
TILs prepared using
other methods than those provide herein including, for example, methods other
than those embodied
in Figure 8 (in particular, e.g., Figure 8A and/or Figure 8B and/or Figure 8C
and/or Figure 8D). In
some embodiments, IFN-y secretion is increased three-fold as compared to an
untreated patient and/or
as compared to a patient treated with TILs prepared using other methods than
those provide herein
including, for example, methods other than those embodied in Figure 8 (in
particular, e.g., Figure 8A
and/or Figure 8B and/or Figure 8C and/or Figure 8D). In some embodiments, IFN-
y secretion is
increased four-fold as compared to an untreated patient and/or as compared to
a patient treated with
TILs prepared using other methods than those provide herein including, for
example, methods other
than those embodied in Figure 8 (in particular, e.g., Figure 8A and/or Figure
8B and/or Figure 8C
and/or Figure 8D). In some embodiments, IFN-y secretion is increased five-fold
as compared to an
untreated patient and/or as compared to a patient treated with TILs prepared
using other methods than
those provide herein including, for example, methods other than those embodied
in Figure 8 (in
particular, e.g., Figure 8A and/or Figure 8B and/or Figure 8C and/or Figure
8D). In some
embodiments, IFN-y is measured using a Quantikine ELISA kit. In some
embodiments, IFN-y is
measured using a Quantikine EL1SA kit. In some embodiments, 1FN-y is measured
in TILs ex vivo
from a patient treated with the TILs produced by the methods of the present
invention. In some
embodiments, IFN-y is measured in blood in a patient treated with the TILs
produced by the methods
of the present invention. In some embodiments, IFN-y is measured in serum in a
patient treated with
the TILs produced by the methods of the present invention.
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10013801111 some embodiments, the TILs prepared by the methods of the present
invention, including
those as described for example in Figure 8 (in particular, e.g., Figure 8A
and/or Figure 8B and/or
Figure 8C and/or Figure 8D and/or Figure 8E and/or Figure 8F and/or Figure
8G), exhibit increased
polyclonality as compared to TILs produced by other methods, including those
not exemplified in
Figure 8 (in particular, e.g., Figure 8A and/or Figure 8B and/or Figure 8C
and/or Figure 8D), such as
for example, methods referred to as process IC methods. In some embodiments,
significantly
improved polyclonality and/or increased polyclonality is indicative of
treatment efficacy and/or
increased clinical efficacy for cancer treatment. In some embodiments,
polyclonality refers to the T-
cell repertoire diversity. In some embodiments, an increase in polyclonality
can be indicative of
treatment efficacy with regard to administration of the TILs produced by the
methods of the present
invention. In some embodiments, polyclonality is increased one-fold, two-fold,
ten-fold, 100-fold,
500-fold, or 1000-fold as compared to TILs prepared using methods than those
provide herein
including, for example, methods other than those embodied in Figure 5 (in
particular, e.g., Figure SA
and/or Figure 8B and/or Figure 8C and/or Figure 8D). In some embodiments,
polyclonality is
increased one-fold as compared to an untreated patient and/or as compared to a
patient treated with
TILs prepared using other methods than those provide herein including, for
example, methods other
than those embodied in Figure 8 (in particular, e.g., Figure 8A and/or Figure
8B and/or Figure 8C
and/or Figure 8D). In some embodiments, polyclonality is increased two-fold as
compared to an
untreated patient and/or as compared to a patient treated with TILs prepared
using other methods than
those provide herein including, for example, methods other than those embodied
in Figure 8 (in
particular, e.g., Figure 8A and/or Figure 8B and/or Figure 8C and/or Figure
8D). In some
embodiments, polyclonality is increased ten-fold as compared to an untreated
patient and/or as
compared to a patient treated with TILs prepared using other methods than
those provide herein
including, for example, methods other than those embodied in Figure 8 (in
particular, e.g., Figure 8A
and/or Figure 8B and/or Figure SC and/or Figure SD). in some embodiments,
polyclonality is
increased 100-fold as compared to an untreated patient and/or as compared to a
patient treated with
TILs prepared using other methods than those provide herein including, for
example, methods other
than those embodied in Figure 8 (in particular, e.g., Figure 8A and/or Figure
8B and/or Figure 8C
and/or Figure 8D). In some embodiments, polyclonality is increased 500-fold as
compared to an
untreated patient and/or as compared to a patient treated with TILs prepared
using other methods than
those provide herein including, for example, methods other than those embodied
in Figure 8 (in
particular, e g , Figure RA and/or Figure RB and/or Figure RC and/or Figure
RD). In some
embodiments, polyclonality is increased 1000-fold as compared to an untreated
patient and/or as
compared to a patient treated with TILs prepared using other methods than
those provide herein
including, for example, methods other than those embodied in Figure 8 (in
particular, e.g., Figure 8A
and/or Figure 8B and/or Figure 8C and/or Figure 8D).
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1. Methods of Treating NSCLC
10013811 The compositions and methods described herein can be used in a method
for treating non-
small-cell lung cancer (NSCLC), wherein the NSCLC is refractory to treatment
with an anti-PD-1 or
anti-PD-L1 antibody. In some embodiments the NSCLC is metatstatic NSCLC. In
some embodiments
the anit-PD-1 antibody includes, e.g., but is not limited to nivolumab (BMS-
936558, Bristol-Myers
Squibb; Opdivon pembrolizumab (lambrolizumab, MK03475 or MK-3475, Merck;
Keytruda ),
humanized anti-PD-1 antibody JS001 (ShangHai JunShi), monoclonal anti-PD-1
antibody TSR-042
(Tesaro, Inc.), Pidilizumab (anti-PD-1 niAb CT-011, Medivation), anti -PD-1
monoclonal Antibody
BGB-A317 (BeiGene), and/or anti-PD-1 antibody SHR-1210 (ShangHai HengRui),
human
monoclonal antibody REGN2810 (Regeneron), human monoclonal antibody MDX-1106
(Bristol-
Myers Squibb), and/or humanized anti-PD-1 IgG4 antibody PDR001 (Novartis). In
some
embodiments, the PD-1 antibody is from clone: RMP1-14 (rat A) - BioXcell cat#
BP0146. Other
suitable antibodies suitable for use in co-administration methods with TILs
produced according to
Steps A through F as described herein are anti-PD-1 antibodies disclosed in
U.S. Patent No.
8,008,449, herein incorporated by reference. In sonic embodiments, the
antibody or antigen-binding
portion thereof binds specifically to PD-L1 and inhibits its interaction with
PD-1, thereby increasing
immune activity. Any antibodies known in the art which bind to PD-Li and
disrupt the interaction
between the PD-1 and PD-L1, and stimulates an anti-tumor immu2740274ne
response, are include.
For example, antibodies that target PD-Li and are in clinical trials, include
BMS-936559 (Bristol-
Myers Squibb) and MPDL3280A (Genentech). Other suitable antibodies that target
PD-Li are
disclosed in U.S. Patent No. 7,943,743, herein incorporated by reference. It
will be understood by one
of ordinary skill that any antibody which binds to PD-1 or PD-L1, disrupts the
PD-1/PD-L1
interaction, and stimulates an anti-tumor immune response, are included.
10013821In some embodiments, the NSCLC is refractory to the anti-CTLA-4 and/or
anti-PD-1 and a
chemotherapeutic agent. In some embodiments, the NSCLC is refractory to the
anti-CTLA-4 and/or
anti-PD-1 and chemotherapy, wherein the chemotherapeutic agent is carboplatin,
paclitaxel,
pemetrexed, cisplatin. In some embodiments, the NSCLS is refractory to an anti-
CTLA-4 antibody,
such as ipilimumab (Yervoyk).
10013831 In some embodiments, the NSCLC is refractory to treatment with
combined PD-1
(including for example pembrolizumab) and a chemotherapeutic agent. In some
embodiments, the
chemotherapeutic agent(s) is a platinum doublet chemotherapeutic agent. In
some embodiments, the
platinum doublet therapy comprises a first chemotherapeutic agent selected
from the group consisting
of cisplatin and carboplatin and a second chemotherapeutic agent selected from
the group consisting
of vinorelbine, gemcitabine and a taxane (including for example, paclitaxel,
docetaxel or nab-
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paclitaxel). In some embodiments, the platinum doublet chemotherapeutic agent
is in combination
with pemetrexed.
10013841In some embodiments, the NSCLC is refractory to a combination
treatment or combination
therapy comprising an anti-PD-1 and a chemotherapeutic agent. In some
embodiments, the anti-PD-1
or the anti-PD-Li antibody is selected from the group consisting of nivolumab,
pembrolizumab,
JS001, TSR-042, pidilizumab, (BGB-A317, SHR-1210, REGN2810, MDX-1106, PDR001,
anti-PD-1
from clone: RMP1-14, an anti-PD-1 antibodies disclosed in U.S. Patent No.
8,008,449, durvalumab,
atezolizumab, avelumab, and fragments, derivatives, variants, as well as
biosimila.rs thereof. In some
embodiments, the anti-PD-1 is pembrolizumab. In some embodiments, the
chemotherapeutic agent is
a platinum doublet chemotherapeutic agent. In some embodiments, the
chemotherapeutic agent is in
combination with pemetrexed. In some embodiments, the NSCLC is refractory to a
combination
therapy comprising carboplatin, paclitaxel, pemetrexed, and cisplatin. In some
embodiments, the
NSCLC is refractory to a combination therapy comprising carboplatin,
paclitaxel, pemetrexed,
cisplatin, nivolumab, and ipilimumab.
[0013851ln some embodiments, the NSCLS has undergone no prior therapy.
10013861ln some embodiments, the NSCI,C is PD-1 negative and/or is from a
subject that is PD-1.
10013871In some embodiments, the NSCLC is refractory to a combination therapy
comprising the
anti-PD-1 or the anti-PD-Li and a platinum doublet therapy, wherein the
platinum doublet therapy
comprises:
i) a first chemotherapeutic agent selected from the group consisting of
cisplatin and
carboplatin,
ii) and a second chemotherapeutic agent selected from the group consisting of
vinorelbine,
gemcitabine and a taxane (including for example, paclitaxel, docetaxel or nab-
paclitaxel).
10013881 In some embodiments, the NSCLC is refractory to a combination therapy
comprising the
anti-PD-1 or the anti-PD-L1, pemetrexed, and a platinum doublet therapy,
wherein the platinum
doublet therapy comprises:
i) a first chemotherapeutic agent selected from the group consisting of
cisplatin and
carboplatin,
ii) and a second chemotherapeutic agent selected from the group consisting of
vinorelbine,
gcmcitabinc and a taxanc (including for example, paclitaxcl, docctaxcl or nab-
paclitaxcl).
[0013891In some embodiments, the NSCLC has been treated with an anti-PD-1
antibody. In some
embodiments, the NSCLC has been treated with an anti-PD-Li antibody. In some
embodiments. the
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NSCLC subject is treatment naive. In some embodiments, the NSCLC has not been
treated with an
anti-PD-1 antibody. In some embodiments, the NSCLC has not been treated with
an anti-PD-Li
antibody. In some embodiments, the NSCLC has been previously treated with a
chemotherapeutic
agent. In some embodiments, the NSCLC has been previously treated with a
chemotherapeutic agent
but is not longer being treated with the chemotherapeutic agent. In some
embodiments, the NSCLC
patient is anti-PD-1/PD-L1 naive. In some embodiments, the NSCLC subject has
low expression of
PD-Li. In some embodiments, the NSCLC subject has treatment naïve NSCLC or is
post-
chemotherapeutic treatment but anti-PD-1/PD-L1 naive. In some embodiments, the
NSCLC subject
ist reatment naive NSCLC or post-chemotherapuetic treament but anti-PD-1/PD-L1
naive and has low
expression of PD-Li. In some embodiments, the NSCLC subject has bulky disease
at baseline. In
some embodiments, the subject has bulky disease at baseline and has low
expression of PD-Li. In
some embodiments, the NSCLC subject has no detectable expression of PD-Li. In
some
embodiments, the NSCLC subject ist rcatment naive NSCLC or post-
chemotherapuetic treament but
anti-PD-1/PD-L1 naive and has no detectable expression of PD-Li. In some
embodiments, the subject
has bulky disease at baseline and has no detectable expression of PD-Li. in
some embodiments, the
NSCLC subject has treatment naive NSCLC or post chemotherapy (e.g., post
chemotherapeutic agent)
but anti-PD-1/PD-Li naïve who have low expression of PD-Li and/or have bulky
disease at baseline.
In some embodiments, bulky disease is indicated where the maximal tumor
diameter is greater than 7
cm measured in either the transverse or coronal plane. In some embodiments,
bulky disease is
indicated when there are swollen lymph nodes with a short-axis diameter of 20
mm or greater. In
some embodiments, the chemotherapeutic includes a standard of care therapeutic
for NSCLC.
10013901 In some embodiments, PD-L1 expression is determined by the tumor
proportion score. In
some embodiments, the subject with a refractory NSCLC tumor has a < 1% tumor
proportion score
(TPS). In some embodiments, the subject with a refractory NSCLC tumor has a >
1')/0 TPS. In some
embodiments, subject with the refractory NSCLC has been previously treated
with an anti-PD-1
and/or anti-PD-Li antibody and the tumor proportion score was determined prior
to said anti-PD-1
and/or anti-PD-Li antibody treatment. In some embodiments, subject with the
refractory NSCLC has
been previously treated with an anti-PD-L1 antibody and the tumor proportion
score was determined
prior to said anti-PD-Li antibody treatment.
[001391] in some embodiments, the TILs prepared by the methods of the present
invention, including
those as described for example in Figure 1 and/or Figure 8, exhibit increased
polyclonality as
compared to TILs produced by other methods, including those not exemplified in
Figure 1 and/or
Figure 8, such as for example, methods referred to as process 1C methods. In
some embodiments,
significantly improved polyclonality and/or increased polyclonality is
indicative of treatment efficacy
and/or increased clinical efficacy for cancer treatment. In some embodiments,
polyclonality refers to
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the T-cell repertoire diversity. In some embodiments, an increase in
polyclonality can be indicative of
treatment efficacy with regard to administration of the TILs produced by the
methods of the present
invention. In some embodiments, polyclonality is increased one-fold, two-fold,
ten-fold, 100-fold,
500-fold, or 1000-fold as compared to TILs prepared using methods than those
provide herein
including for example, methods other than those embodied in Figure 1. In some
embodiments,
polyclonality is increased one-fold as compared to an untreated patient and/or
as compared to a
patient treated with TILs prepared using other methods than those provide
herein including for
example, methods other than those embodied in Figure 1. In some embodiments,
polyclonality is
increased two-fold as compared to an untreated patient and/or as compared to a
patient treated with
TILs prepared using other methods than those provide herein including for
example, methods other
than those embodied in Figure 1. In some embodiments, polyclonality is
increased ten-fold as
compared to an untreated patient and/or as compared to a patient treated with
TILs prepared using
other methods than those provide herein including for example, methods other
than those embodied in
Figure 1. In some embodiments, polyclonality is increased 100-fold as compared
to an untreated
patient and/or as compared to a patient treated with TILs prepared using other
methods than those
provide herein including for example, methods other than those embodied in
Figure 1. In some
embodiments, polyclonality is increased 500-fold as compared to an untreated
patient and/or as
compared to a patient treated with TILs prepared using other methods than
those provide herein
including for example, methods other than those embodied in Figure 1. In some
embodiments,
polyclonality is increased 1000-fold as compared to an untreated patient
and/or as compared to a
patient treated with TILs prepared using other methods than those provide
herein including for
example, methods other than those embodied in Figure 1.
a. Exemplary PD-L1 Testing Methods
1001392] In some embodiments, PD-Li expression is determined by the tumor
proportion score using
one more testing methods as described herein. In some embodiments, the subject
or patient with a
NSCLC tumor has a < 1% tumor proportion score (TPS). In some embodiments, the
NSCLC tumor
has a > 1% TPS. In some embodiments, the subject or patient with the NSCLC has
been previously
treated with an anti-PD-1 and/or anti-PD-L1 antibody and the tumor proportion
score was determined
prior to the anti-PD-1 and/or anti-PD-Li antibody treatment. In some
embodiments, the subject or
patient with the NSCLC has been previously treated with an anti-PD-Ll antibody
and the tumor
proportion score was determined prior to the anti-PD-Li antibody treatment. In
some embodiments,
the subject or patient with a refractory or resistant NSCLC tumor has a < 1%
tumor proportion score
(TPS). In some embodiments, the subject or patient with a refractory or
resistant NSCLC tumor has a
> 1% TPS. In some embodiments, the subject or patient with the refractory or
resistant NSCLC has
been previously treated with an anti-PD-1 and/or anti-PD-Li antibody and the
tumor proportion score
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was determined prior to the anti-PD-1 and/or anti-PD-Li antibody treatment. In
some embodiments,
the subject or patient with the refractory or resistant NSCLC has been
previously treated with an anti-
PD-Li antibody and the tumor proportion score was determined prior to the anti-
PD-Li antibody
treatment.
10013931 In some embodiments, the NSCLC is an NSCLC that exhibits a tumor
proportion score
(TPS), or the percentage of viable tumor cells from a patient taken prior to
anti-PD-1 or anti-PD-Li
therapy, showing partial or complete membrane staining at any intensity, for
the PD-Li protein that is
less than 1% (TPS <1%). in some embodiments, the NSCLC is an NSCT,C that
exhibits a TPS
selected from the group consisting of <50%, <45%, <40%, <35%, <30%, <25%,
<20%, <15%, <10%,
<9%, <8%, <7%, <6%, <5%, <4%, <3%, <2%, <1%, <0.9%, <0.8%, <0.7%, <0.6%,
<0.5%, <0.4%,
<0.3%, <0.2%, <0.1%, <0.09%, <0.08%, <0.07%, <0.06%, <0.05%, <0.04%, <0.03%,
<0.02%, and
<0.01%. In some embodiments, the NSCLC is an NSCLC that exhibits a TPS
selected from the group
consisting of about 50%, about 45%, about 40%, about 35%, about 30%, about
25%, about 20%,
about 15%, about 10%, about 9%, about 8%, about 7%, about 6%, about 5%, about
4%, about 3%,
about 2%, about 1%, about 0.9%, about 0.8%, about 0.7%, about 0.6%, about
0.5%, about 0.4%,
about 0.3%, about 0.2%, about 0.1%, about 0.09%, about 0.08%, about 0.07%,
about 0.06%, about
0.05%, about 0.04%, about 0.03%, about 0.02%, and about 0.01%. In some
embodiments, the NSCLC
is an NSCLC that exhibits a TPS between 0% and 1%. In some embodiments, the
NSCLC is an
NSCLC that exhibits a TPS between 0% and 0.9%. In some embodiments, the NSCLC
is an NSCLC
that exhibits a TPS between 0% and 0.8%. in some embodiments, the NSCLC is an
NSCLC that
exhibits a TPS between 0% and 0.7%. In some embodiments, the NSCLC is an NSCLC
that exhibits a
TPS between 0% and 0.6%. In some embodiments, the NSCLC is an NSCLC that
exhibits a TPS
between 0% and 0.5%. In some embodiments, the NSCLC is an NSCLC that exhibits
a TPS between
0% and 0.4%. In some embodiments, the NSCLC is an NSCLC that exhibits a TPS
between 0% and
0.3%. In some embodiments, the NSCLC is an NSCLC that exhibits a TPS between
0% and 0.2%. In
some embodiments, the NSCLC is an NSCLC that exhibits a TPS between 0% and
0.1%. TPS may be
measured by methods known in the art, such as those described in Hirsch, et
al. J. Thorac. Oncol.
2017:12, 208-222 or those used for the determination of TPS prior to treatment
with pembrolizumab
or other anti-PD-1 or anti-PD-Li therapies. Methods for meansurement of TPS
that have been
approved by the U.S. Food and Drug Administration may also be used. In some
embodiments, the
PD-Li is exosomal PD-Li. In some embodiments, the PD-Li is found on
circulating tumor cells.
10013941 in some embodiments, the partial membrane staining includes 1%, 5%,
10%, 15%, 20%,
25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%,
97%, 99%, or
more. In some embodiments, the completed membrane staining includes
approzimatley 100%
membrane staining.
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[0013951M some embodiments, testing for PD-Li can incoclve measuring levels of
PD-Li in patient
serum. In these embodiments, measurement of PD-L1 in patient serum removes the
uncertainty of
tumor heterogeneity and the patient discomfort of serial biopsies.
10013961 hi some embodiments, elevated soluble PD-Li as compared to a baseline
or standard level
correlates with worsened prognosis in NSCLC. See, for example, Okuma, Y. et
al., 2018, Clinical
Lung Cancer, 19(5): 410-417, Vecchiarelli, S., et al., 2018, Oncotarget,
9(25):17554-17563. In some
embodiments, the PD-Li is exosomal PD-Li. In some embodiments, the PD-Li is
expressed on
circulating tumor cells.
2. Driver Mutations
10013971 As used herein, the phrases "driver mutation" and/or "actionable
mutation" and/or
"oncogenic driver mutation- refer to mutations that are typically considered
oncogenic drivers (i.e.,
cancer drivers or cancer inducers). The presence of one or more of these
mutations has traditionally
been the utilized as the target for a targeted therapy. Often driver mutations
are examined and/or
analyzed for treatment with targeted therapeutic moieties, including for
example tyrosine kinase
inhibitors (TKIs). Such driver mutations can, in some embodiments, impact or
affect response to a
first line therapeutic treatment. TIL therapy methods and compositions
described herein are effective
for treatment whether such driver mutations are present or absent in the
patient or subject. Such driver
mutations can be tested and determined by any method known in the art,
including whole exome
sequencing or methods targeted to the detection of a specific driver mutation.
100139811n some embodiments, the NSCLC is an NSCLC that exhibits the presence
or absence of
one or more driver mutations. In some embodiments, the NSCLC is an NSCLC that
exhibits the
presence of one or more driver mutations. In some embodiments, the NSCLC is an
NSCLC that
exhibits the absence of one or more driver mutations. In some embodiments, the
NSCLC has been
analyzed for the absence or presence of one or more driver mutations. In some
embodiments, the one
or more driver mutations are not present. In some embodiments, the the NSCLC
treatment is
independent of the presence or absence of one or more driver mutations. In
some embodiments, the
one or more driver mutations is selected from the group consisting of an EGFR
mutation, an EGFR
insertion, EGFR exon20, a KRAS mutation, a BRAF-mutation, a BRAF V600E
mutation, a BRAF
V600K mutation, a BRAF V600 mutation, an ALK mutation, a c-ROS mutation (ROS1-
mutation), a
ROS I fusion, a RET mutation, a RET fusion, an ERBB2 mutation, an ERBB2
amplification, a BRCA
mutation, a MAP2K1 mutation, PIK3CA, CDKN2A, a PTEN mutation, an UMD mutation,
an NRAS
mutation, a KRAS mutation, an NF1 mutation,a MET mutation, a MET splice and/or
altered MET
signaling, a TP53 mutation, a CREBBP mutation, a KMT2C mutation, a KMT2D
mutation, an
ARID1A mutation, a RB1 mutation, an ATM mutation, a SETD2 mutation, a FLT3
mutation, a
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PTPN11 mutation, a FGFR1 mutation, an EP300 mutation, a MYC mutation, an EZH2
mutation, a
JAK2 mutation, a FBXIA/7 mutation, a CCND3 mutation, and a GNAll mutation. In
some
embodiments, the NSCLC exhibits a TPS of < 1% and has a predetermined absence
of one or more
driver mutations.
10013991In some embodiments, the NSCLC is an NSCLC that is not indicated for
treatment by an
EGFR inhibitor, a BRAF inhibitor, an ALK inhibitor, a c-Ros inhibitor, a RET
inhibitor, an ERBB2
inhibitor, BRCA inhibitor, a MAP2K1 inhibitor, PIK3CA inhibitor, CDKN2A
inhibitor, a PTEN
inhibitor, an UMD inhibitor, an NRAS inhibitor, a KRAS inhibitor, an NF1
inhibitor, MET inhibitor a
TP53 inhibitor, a CREBBP inhibitor, a KMT2C inhibitor, a KMT2D mutation, an
ARID IA mutation,
a RB1 inhibitor, an ATM inhibitor, a SETD2 inhibitor, a FLT3 inhibitor, a
PTPN11 inhibitor, a
FGFR1 inhibitor, an EP300 inhibitor, a MYC inhibitor, an EZH2 inhibitor, a
JAK2 inhibitor, a
FBXW7 inhibitor, a CCND3 inhibitor, and a GNAll inhibitor.
10014001In some embodiments, the NSCLC exhibits a TPS of < 1% and is a NSCLC
that is not
indicated for treatment by an EGFR inhibitor, a BRAF inhibitor, an ALK
inhibitor, a c-Ros inhibitor,
a RET inhibitor, an ERBB2 inhibitor, BRCA inhibitor, a MAP2K1 inhibitor,
PIK3CA inhibitor,
CDKN2A inhibitor, a PTEN inhibitor, an UMD inhibitor, an NRAS inhibitor, a
KRAS inhibitor, an
NF1 inhibitor, MET inhibitor a TPS 3 inhibitor, a CREBBP inhibitor, a KMT2C
inhibitor, a KMT2D
mutation, an ARID1A mutation, a RB1 inhibitor, an ATM inhibitor, a SETD2
inhibitor, a FLT3
inhibitor, a PTPN11 inhibitor, a FGFR1 inhibitor, an EP300 inhibitor, a MYC
inhibitor, an EZH2
inhibitor, a JAK2 inhibitor, a FBX7W7 inhibitor, a CCND3 inhibitor, and a
GNAll inhibitor
10014011 In some embodiments, the EGFR mutation results in tumor
transformation from NSCLC to
small cell lung cancer (SCLC).
10014021In some embodiments, the NSCLC (or a biopsy thereof) exhibits high-
tumor mutational
burden (high-TMB; > 10 mut/kb) and/or microsatellite instability high (MSI-
high). In some
embodiments, the NSCLC (or a biopsy thereof) exhibits high-tumor mutational
burden (high-TMB; >
mut/kb). In some embodiments, the NSCLC (or a biopsy thereof) exhibits
microsatellite instability
high (MSI-high). Methods and systems for evaluating tumor mutational burden
are known in the art.
Exemplary disclosures of such methods and systems can be found in US Patent
No. 9,792,403, US
Application Publication No. US20180363066A1, International Application
publication Nos.
W02013070634 and W02018106884, as well as Metzker, M. (2010) Nature
Biotechnology Reviews
11:31-46, incorporated herein by reference, all of which are incorporated by
reference in their
entireties.
100140311n some embodiments, the NSCLC (or a biopsy thereof) exhibits high-
tumor mutational
burden (high-TMB; > 10 mut/kb) and/or microsatellite instability high (MSI-
high). In some
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embodiments, the NSCLC (or a biopsy thereof) exhibits high-tumor mutational
burden (high-TMB; >
mut/kb). In some embodiments, the NSCLC (or a biopsy thereof) exhibits
microsatellite instability
high (MST-high). Methods and systems for evaluating tumor mutational burden
are known in the art.
Exemplary disclosures of such methods and systems can be found in US Patent
No. 9,792,403, US
Application Publication No. US20180363066A1, International Application
publication Nos.
W02013070634 and W02018106884, as well as Metzker, M. (2010) Nature
Biotechnology Reviews
11:31-46, incorporated herein by reference, all of which are incorporated by
reference in their
entireties.
10014041ln some embodiments, the EGFR mutation includes, for example, but is
not limited to
T790M, Ex19Del, L858R, Exon 20 insertion, delE709-T710insD,
1744_K745insKIPVAI,
K745_E746insTPVAIK, E709X, E709K, E709A, Exon 18 deletion, 6719X, G719A,
G719S, L861Q,
S768I, L747P, A763 764insFQEA, D770 N771insNPG, A763 764insFQEA, P772
H773insDNP
exon 20 insertion, H773_V774insNPH exon 20 insertion, S7681, D770_N771insSVD,
V769 D770InsASV, p.K745 E746insIPVAIK, p.K745 E746insTPVAIK, p.I744
K745insKIPVAI,
D770_N771insNPG, P772_H773insPNP, A763 Y764insFQEA, and/or EGFR kinase domain
duplication (EGFR-KDD). In some embodiments, the EGFR mutation is selected
from the group
consisting of T790M, Ex19Del, L858R, Exon 20 insertion, delE709-T710insD,
1744 K745insKIPVAI, K745 E746insTPVAIK, E709X, E709K, E709A, Exon 18 deletion,
G719X,
G719A, G719S, L861Q, S768I, L747P, A763 764insFQEA, D770 N771insNPG,
A763 764insFQEA, P772_H773insDNP exon 20 insertion, H773_V774insNPH exon 20
insertion,
S768I, D770_N771insSVD, V769_D770InsASV, p.K745_E746insIPVAIK,
p.K745 E746insTPVAIK, p.I744 K745insKIPVAI, D770 N771insNPG, P772 H773insPNP,
A763_Y764insFQEA, and EGFR kinasc domain duplication (EGFR-KDD).
10014051ln some embodiments, the EGFR mutation is a double mutation including
for example, but
not limited to, L858R/T790M, Exl9Del/T790M, G719X/L861Q, G719X/S7681 (or
S768I/G719X),
S768I/L858R, L858R/E709A, and/or E746_T751delinsA+T790M. In some embodiments,
the EGFR
mutation is a double mutation selected from group consisting of L858R/T790M,
Ex19Dcl/T790M,
G719X/L861Q, G719X/S7681 (or S7681/G719X), S768I/L858R, L858R/E709A, and
E746 T75 ldelinsA+T790M.
10014061Additional disclosures of the EGFR mutation are provided in
International Application
Publication No. W02010020618, which is incorporated by referenced herein in
its entirety.
10014071 In some embodiments, the ALK mutation includes, but not limited to,
EML4-ALK Variant
1 (AB274722.1; BAF73611.1), EML4-ALK Variant 2 (AB275889.1; BAF73612.1), EML4-
ALK
Variant 3a (AB374361.1; BAG55003.1), EML4-ALK Variant 3b (AB374362.1;
BAG55004.1),
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EML4-ALK Variant 4 (AB374363.1; BAG75147.1), EML4-ALK Variant 5a (AB374364.1;
BAG75148.1), EML4-ALK Variant 5b (AB374365.1; BAG75149.1), EML4-ALK Variant 6
(AB462411.1; BAH57335.1), EML4-ALK Variant 7 (AB462412.1; BAH57336.1), KIF5B-
ALK
(AB462413.1; BAH57337.1), NPM-ALK, TPM3-ALK, TFGXL-ALK, TEGL-ALK, TFGS-ALK,
A 11C-ALK, CLTC-ALK, MSN-ALK, TPM4-ALK, MYH9-ALK, RANBP2-ALK, AL017-ALK, and
CARS-ALK (sec, for example. Pulford ct al., (2004) J. Cell. Physiol. 199:330-
358). In addition, a
skilled artisan will understand that ALK kinase variants can arise depending
upon the particular
fusion event between an ALK kinase and its fusion partner (e.g., EML4 can fuse
at least exon 2, 6a,
6b, 13, 14, and/or 15, as described, for example, in Horn and Pao, (2009) J.
Clin. Oncol. 27:4247-
4253) (incorporated by reference in its entirety).
10014081Additional examples of ALK mutations are described in US Patent Nos.
9,018,230 and
9,458,508, the disclosures of which are incorporated by reference herein.
10014091111 some embodiments, the ROS1 mutation of the present invention is a
ROS1 fusion, where
a portion of the ROS1 polypeptide that includes the kinase domain of the ROS1
protein (or
polynucleotide encoding the same) fused to all or a portion of another
polypeptide (or polynucleotide
encoding the same) and where the name of that second polypeptide or
polynucleotide is named in the
fusion. In some embodiments, the ROS1 mutation is determined as ROS1-fusion
protein (e.g. by IHC)
and/or ROS -fusion gene (e.g. by FISH), and/or ROS1 mRNA (e.g. by qRT-PCR),
preferably
indicative of a ROS1 -fusion protein selected from the group consisting of
SLC34A2-ROS1
(SLC34A2 exons 13de12046 and 4 fused to ROS1 exons 32 and 34), CD74-ROS1 (CD74
exon 6
fused to ROS1 exons 32 and 34), EZR-ROS1 (EZR exon 10 fused to ROS1 exon 34),
TPM3-ROS1
(TPM3 exon 8 fused to ROS1 exon 35), LRIG3-ROS1 (LRIG3 exon 16 fused to ROS1
exon 35),
SDC4-ROS1 (SDC4 exon 2 and 4 fused to ROS1 exon 32 and SDC4 exon 4 fused to
ROS1 exon 34),
GOPC-ROS1 , also known as FIG-ROS1 , (GOPC exon 8 fused to ROS1 exon 35 and
GOPC exon 4
fused to ROS1 exon 36), and G2032R, i.e. ROS1 G2 32R.
10014101Additional disclosures of the ROS1 mutations and the ROS fusion have
been provided in
U.S. Patent Publication Nos. 20100221737, 20150056193, and 20100143918, and
PCT Publication
No, W02010/093928, all of which are hereby incorporated by reference in their
entirety.In some
embodiments, the RET mutation is a RET fusion or point mutation.
10014111In some embodiments, the RET point mutation includes but is not
limited to H6650,
K666E, K666M, S686N, G691S, R694Q, M700L, V706M, V706A, E713K, G736R, G748C,
A750P,
S765P, P766S, E768Q, E768D, L769L, R770Q, D771N, N777S, V7781, Q781R, L790F,
Y791F,
Y791N, V804L, Vg04M, V804E, E8O5K, E806C, Y806E, Y806F, Y806S, Y806GY806C E81
8K
S819I G823E Y826M R833C P841L P841P E843D R844W, R844Q, R844L, M848T, 1852M
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A866W R873W A876V L88 1V A883F A883S A883T E884K R886W, S891A, R8970, D898V,
E901K 5904F S904C2 K907E K907M R908K G911D R912P R912Q M918T, M918V, M918L6,
A919V, E921K, S922P S922Y T930M F961L R972G R982C M1009V D1017N V10416, and
M1064T.
[0014121M some embodiments, the RET fusion is a fusion between RET and a
fusion partner that is
selected from the group consisting of BCR, BCR, CLIP 1, KIFSB, CCDC6, PTClex9,
NCOA4,
TRIM33, ERC1, FGFRIOP, MBD1, RAB61P2, PRKARIA, TRIM24, KTN1, GOLGA5, HOOK3,
KIA A1468, TRIM27, AK AP13, FKBP15, SPECCIIõ TBL1XR1, CFP55, CUX1, ACBD5,
MYH13,
PIBF1, KIAA1217, and MPRIP.
10014131Additional disclosures of the RET mutations has been provided in U.S.
Patent No.
10035789, which is hereby incorporated by reference in their entirety.
100141411n some embodiments, the BRAF mutation is BRAF V600E/K mutation. In
other
embodiments, the BRAF mutation is a non-V600E/K mutation.
100141 511n some embodiments, the non-V600E/K BRAF mutation is a kinase-
activated mutation, a
kinase-impaired mutation, or a kinase-unknown mutation, and combinations
thereof. In some
embodiments, the kinase-activated mutation is selected from the group
consisting of R4621, 1463S,
G464E, G464R, G464V, G466A, G469A, N58 is, E586K, F595L, L597Q, L597R, L5975,
L597V,
A598V, T599E, V600R, K601E, 5602D, A728V, and combinations thereof. In some
embodiments,
the kinase-impaired mutation is selected from the group consisting of G466E,
G466R_, G466V,
Y472C, K483M, D594A, D594E, D594G, D594H, D594N, D594V, G596R, T599A, 5602A,
and
combinations thereof In some embodiments, the kinase-unknovvn mutation is
selected from the group
consisting of T4401, 5467L, G469E, G469R, G4695. G469V, L584F, L588F, V600
K6Olde1insE,
56051, Q609L, E611Q, and combinations thereof. In some embodiments, the non-
V600E/K BRAF
mutation is selected from the group consisting of D594, G469, K601E, L597,
T599 duplication,
L485W, F247L, G466V, BRAF fusion, BRAF-AGAP3 rearrangement, BRAF exon 15 slice
variant,
and combinations thereof.
In some embodiments, the Met mutation includes point mutation, deletion
mutation, insertion
mutation, inversion, aberrant splicing, missense mutation, or gene
magnification that causes the
increase of at least one bioactivity of c-Met protein, the tyrosine kinase
activity such as improved,
receptor homolog dimerization Ligand binding of formation, enhancing of body
and heterodimer etc.
The Met mutation can be located at any part of c-Met genes. In one embodiment,
the mutation is in
the kinase domain of c-Met protein encoded by the c-MET gene. In some
embodiments, the c-Met
mutations are point mutation at N375, V13, V923, R175, V136, L229, S323, R988,
S1058/T1010 and
E168.
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1001416] In some embodiments, the ERBB2 mutation is a point mutation in the
amino acid sequence
of ERBB2. In some embodiments, the point mutation of ERBB2 is one that causes
amino acid
substitutions, causes mRNA splicing, or is a point mutation in the upstream
region. Wherein the
mutation comprises a nucleotide mutation causing at least one amino acid
substitution selected from
the group consisting of Q568E, P601R, I628M, P885S, R143Q, R434Q, and E874K.
10014171 In some embodiments, the ERBB2 mutation is ERBB2 amplification. In
some
embodiments, the ERBB2 amplification includes point mutation selected from the
group consisting of
V659E, G309A, G309E, S310F, D769H, D769Y, V777L, P780ins, P780-Y781insGSP,
V842I,
R896C, K753E, and L755S and can be detected by Polymerase Chain Reaction or
any sequencing
technique (Bose et al. Cancer Discov. 2013, 3(2), 224-237; Zuo et al. Clin
Cancer Res 2016, 22(19),
4859-4869).
100141811n some embodiments, the BRCA mutation is a mutation in BRCA1 and/or
BRCA2,
preferably BRCA1, and/or in one or more other genes of which the protein
product associates with
BRCA1 and/or BRCA2 at DNA damage sites, including ATM, ATR, Chk2, H2AX, 53BP1,
NFBD1,
Mrell, Rad50, Nibrin, BRCAl-associated RING domain (BARD]), Abraxas, and MSH2.
A mutation
in one or more of these genes may result in a gene expression pattern that
mimics a mutation in
BRCA1 and/or BRCA2.
10014191 In certain cmbodimcnts the BRCA mutation comprises a non-synonymous
mutation. In
sonic embodiments, the BRCA mutation comprises a nonsense mutation. In some
embodiments, the
BRCA mutation comprises a frameshift mutation. In some embodiments, the BRCA
mutation
comprises a splicing mutation. In some embodiments, the BRCA mutation is
expressed as a mutant
mRNA and ultimately a mutant protein. In some embodiments, the BRCA1/2 protein
is functional. In
other embodiments, the BRCA1/2 protein has reduced activity. In other
embodiments, the BRCA1/2
protein is non-functional.
10014201 As used herein with regard to susbtitions, the "="- sign with regard
to mutaitons generally
refers to synonymous substitutions, silent codons, and/or silent
substitutions. In particular, a
synonymous substitution (also called a silent substitution or silent codon)
refers to the substitution of
one nucleotide base for another in an exon of a gene encoding a protein,
wherein the produced amino
acid sequence is not modified. This is due to the fact that the genetic code
is "degenerate", i.e. that
some amino acids are coded for by more than one three-base-pair codon. Because
some of the codons
for a given amino acid vary by just one base pair from others coding for the
same amino acid, a point
mutation that replaces the wild-type base by one of the alternatives will
result in incorporation of the
same amino acid into the elongating polypeptide chain during translation of
the gene. In some
embodiments, synonymous substitutions and mutations affecting noncoding DNA
are often
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considered silent mutations; however, it is not always the case that the
mutation is silent and without
any impact. For example, a synonymous mutation can affect transcription,
splicing, mRNA transport,
and translation, any of which could alter the resulting phenotype, rendering
the synonymous mutation
non-silent. The substrate specificity of the tRNA to the rare codon can affect
the timing of translation,
and in turn the co-translational folding of the protein. This is manifested in
the codon usage bias that
has been observed in many species. A nonsynonymous substitution/mutation
results in a change in
amino acid that may be arbitrarily classified as conservative (a change to an
amino acid with similar
physiochemical properties), semi-conservative (e.g. negatively to positively
charged amino acid), or
radical (vastly different amino acid) In some embodiments, the BRCA mutation
is a BRCA1 mutation
that includes, but is not limited to P871L, K1183R, D693N, S1634G, E1038G,
S1040N, S694-
silience codon), M16731, Q356R, S1436=, L771=, K654Sfs*47, S198N, R496H,
R841W, R1347G,
H619N, S15331, L30=, A622V, Y655Vfs*18, R496C, E597K, R1443*, E23Vfs*17, L30F,
El 11Gfs*3, K339Rfs*2, L512F, D693N, P871S, Si 140G. Q1240*, P1770S, R7=,
L52F, T176M,
A224S ,L347=, S561F, E597*, K820E, K893Rfs*107, E962K, M10141, R1028H, E1258D,
E1346K,
R1347T, L1439F,H1472R, Q1488*, S1572C, E1602K, R1610C, L1621=, Q1625*, Q1625=,
D1754N, R1772Q, R1856*, and any combination thereof.
[001421] In some embodiments, the BRCA mutation is a BRCA2 mutation that
includes, but is not
limited to V2466A, N289H, N991D, S455= (=: silience codon), N372H, H743=,
V1269=, S2414=,
V2171=, L1521=, T3033Nfs*11, K1132=, T3033Lfs*29, R2842C, N1784Tfs*7, K3326*,
K3326*,
D1420Y, 1605Y1s*9, 13412V, A2951T, T3085N1s*26, R2645N1s*3, S1013*, T1915M,
F3090-,
V3244I, A1393V, R2034C, L1356=, E2981Rfs*37, N1784Kfs*3, K3416Nfs*11,
K1691Nfs*15,
S1982Rfs*22, and any combination thereof.
10014221 In some embodiments, the NRAS mutation of the present invention
includes but is not
limited to E63K, Q61R, Q61K, G12D, G13D, Q61R, Q61L, Q61K, G12S, G12C, G13R,
Q61H,
G12V, G12A, Q61L, G13V, Q61H, Q61H, G12R, Gl3C, Q61P, Gl3S, G12D, G13A, G13D,
A18T,
Q61X, G60E, G12S, Q61= (=: silience codon), Q61E, Q61R, A146T, A59T, A59D,
Q61=, R681,
A146T, Gl2A, E62Q, G75=, A91V, and any combination thereof.
[001423] E132K1n some embodiments, the PIK3CA mutation includes substitution
mutations,
deletion mutations, and insertion mutations. In some embodiments, mutations
occur in PIK3CA's
helical domain and in its kinase. In other embodiments, in PIK3CA's P85BD
domain. In some
embodiments, the PIK3CA mutation is in exon 1. 2, 4, 5, 7, 9, 13, 18, and 20.
In some embodiments,
the PIK3CA mutation is in exons 9 and 20. In yet other embodiments, the PIK3CA
mutation is a
combination of the any mutations listed above. Any combination of these exons
can be tested,
optionally in conjunction with testing other exons. Testing for mutations can
be done along the whole
coding sequence or can be focused in the areas where mutations have been found
to cluster. Particular
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hotspots of mutations occur at nucleotide positions 1624, 1633, 1636, and 3140
of PIK3CA coding
sequence.
1001424] In some embodiments, the size of the PIK3CA mutation is small,
ranging from 1 to 3
nucleotides. In some embodiments, the PIK3CA mutations include, but are not
limited to G1624A,
G1633A, C1636A, A3140G, G113A, T1258C, G3129T, C3139T, E542K, E545K, Q546R,
H1047L,
H1047R and G2702T.
10014251 In some embodiments, the MAP2K1 mutation is a somatic MAP2K1
mutation, optionally a
MAP2K1 mutation that upregulates MEKI levels. In some embodiments, the MAP2K1
mutation is a
mutation in one or more genes associated with the RAS/MAPK pathway,
comprising: HRAS, KRAS,
NRAS, ARAF, BRAF, RAF1, MAP2K2, MAPK1, MAPK3, MAP3K3. In certain embodiments,
the
MAP2K1 mutation is in one or more genes selected from the group consisting of
RASA, PTEN,
ENG, ACVRL1, SMAD4, GDF2 or combinations thereof.
10014261 In some embodiments, the MAP2K1 mutation includes, but is not limited
to, P124S, Q56P,
K57N, E203K, G237*, P124L, G128D, D67N, K57E, E102 I103del, C121S, K57T, K57N,
Q56P,
P124L, K57N, G128V, Q58 E62del, F53L, I126=, 1103 K104del, and any combination
thereof
[001427] In certain embodiments the KRAS mutation comprises anon-synonymous
mutation. In
some embodiments, the KRAS mutation comprises a nonsense mutation. In some
embodiments, the
KRAS mutation comprises a frameshift mutation. In some embodiments, the KRAS
mutation
comprises a splicing mutation. In some embodiments, the KRAS mutation is
expressed as a mutant
mRNA and ultimately a mutant protein. In some embodiments, the mutated KRAS
protein is
functional. In other embodiments, the mutated KRAS protein has reduced
activity. In other
embodiments, the mutated KRAS protein is non-functional.
10014281In some embodiments, the KRAS mutation includes but is not limited to
G12D, G12V,
G13D, G12C, Gl2A, Gl2S, G12R, G13C, Q61H, A146T, Q61R, Q61H, Q61L, G13S,
A146V,
Q61K, G13R, Gl2F, K1 17N, G13A, G13V, A59T, V141, K1 17N, Q22K, Q61P, A146P,
G13D,
L19F, L19F, Q61K, G12V, G60=, G12=, G13=, A18D, T58I, Q61E, E63K, G12L, G13V,
A59G,
G60D, G1OR, GIOdup, D57N, A59Eõ V14G, D33E, G121, G13dup, and any combination
thereof,
wherein = is indiciative of silence coding.
10014291 In some embodiments, the NFI mutation includes substitution
mutations, deletion
mutations, missense mutations, aberrant splicing mutations, and insertion
mutations. In some
embodiments, the NF1 mutation is a loss of function (LOF) mutation. In some
embodiments, the NF1
mutation is selected from the group consisting of R1947X (C5839T), R304X, exon
37 mutation, exon
4b mutation, exon 7 mutation, exon 10b mutation, and exon 10c mutation (e.g.,
1570G4T, E524X).
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10014301ln some embodiments, the CDKN2A mutation includes but is not limited
to R2413, D108G,
DI08N, DIO8Y, GI25R, P114L, R80*, R58*, H83Y, W110*, P114L. E88*, WI10*,
E120*, DIO8Y,
D84Y, D84N, E69*, P81L, Q50*, L78Hfs*41, D108N, S12*, P48L, E61*, Y44*, E88K,
R80*,
D84G, L16Pfs*9, Y129*, D108H,A148T, A36G, A102V, W15*, H83R, A57V, E33*, D74Y,
A76V,
E153K, D74N, H83D, V82M, R58*, Y129*, E119*, Y44*, D74A, TI8_A I9dup,
Y44Lfs*76,
L32_L37del, V28 E33del, D14_L16del, A68T, or any combination thereof.
10014311 In certain embodiments the PTEN mutation comprises a non-synonymous
mutation. In
some embodiments, the PTEN mutation comprises a nonsense mutation. In some
embodiments, the
PTEN mutation comprises a frameshift mutation. In some embodiments, the PTEN
mutation
comprises a splicing mutation. In some embodiments, the mutated PTEN is
expressed as an mRNA
and ultimately a protein. In some embodiments, the mutated PTEN protein is
functional. In other
embodiments, the mutated PTEN protein has reduced activity. In other
embodiments, the mutated
PTEN protein is non-functional. In some embodiments, the PTEN mutation
includes, but is not
limited to, R130Q, R130G, T319*, R233*, R130*, K267Rfs*9, N323Mfs*21,
N323Kfs*2, R173C,
R173H, R335*, Q171*, Q245*, E7*, D268Gfs*30, R130Q, Q214*, R130L, C136R,
Q298*, Q17*,
H93R, P248Tfs*5, 133del, R233*, E299*, G132D, Y68H, T319Kfs*24, N329Kfs*14,
V166Sfs*14,
V290*, T319Nfs*6, R142W, P38S, A126T, H61R, F278L, S229*, R130P, G129R,
R130Qfs*4,
P246L, R130*, G165R, C136Y, R173C, I101T, Y155C, D92E, K164Rfs*3, N184Efs*6,
GI29E,R130G, G36R, F34IV, HI23Y, CI24S, M35VGI27E, GI65E and any combination
thereof.
10014321 In some embodiments, the TP53 mutation includes, but is not limited
to, R175H, G245S,
R248Q, R248W, R249S, R273C, R273H, R282W, C135Y, C141Y, P151S, V157F, R158L,
Y163C,
V173L, V173M, C176F, H179R, H179Y, H179Q, Y205C, Y220C, Y234C. M237I, C238Y,
S241F,
G245D, G245C, R248L, R249M, V272M, R273L, P278L, R280T, E285K, E286K, R158H,
C176Y,
II95T, G2I4R, G245V, G266R, G266E, P278S, R280K, or any combination thereof.
In some further
embodiments, the TP53 mutation is selected from the group consisting of:
G245S; R249S; R273C;
R273H; C141Y, V157F, R158L, Y163C, V173L, V173M, Y205C, Y220C, G245C, R249M,
V272M,
R273L, and E286K. In some embodiments, the TP53 mutation includes one or more
of the mutations
above.
10014331 In some embodiments, the CREBBP mutation includes, but is not limited
to, R1446C,
R1446H, S1680del, 11084Sfs*15, P1948L, 11084Nfs*3, ?R386*, S893L, R1341*,
P1423Lfs*36,
P1488L, Y1503H, R1664C, A1824T, R1173*, R1360*, Y1450C, H2228D, S71L, P928=,
D1435N,
W1502C, Y1503D, R483*, R601Q, S945L, R1103*, R1288W, R1392*, C1408Y, D1435G,
R1446L,
H1485Y, Q1491K, Q96*, L361M, L524Wfs*6, Q540*, Q1073*, Al 100V, R1169C,
C1237Y,
RI347W, G1411E, W1472C, I1483F, PI488T, RI498*, YI503F, Q1856*, R1985C,
R2I04C,
S2328L, V2349=, S2377L, and any combination thereof.
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10014341In some embodiments, the KMT2C mutation includes, but is not limited
to, D348N, P350-,
R380L, C391*, P309S. C988F, Y987H, S990G, K2797Rfs*26, V346=, R894Q, R284Q,
S806=,
R1690=, P986=, A1685S, G315S, Q755*, R909K, T316S, S772L, G838S, L291F, P335=,
C988F,
Q2680=, E765G, K339N, Y816*, R526P, N729D, G845E, 1817Nfs*11, G892R, C1103*,
S3660L,
F4496Lfs*21, G315C, R886C, D348N, S793=, V919L, R2481S, R2884*, R4549C,
M305Dfs*28,
T316S, P377=, I455M, T820I, S965=, S730Y, P860S, Q873Hfs*40, R904*, R2610Q,
R4478*, and
any combination thereof.
10014351 In some embodiments, the KMT2D mutation includes, but is not limited
to, L1419P,
E640D, E541D, E455D, T2131P, K1420R. P2354Lfs*30, G2493=, Q3612=, I942=,
T1195Hfs*17,
P4170=, P1194H, G1235Vfs*95, P4563=, P647Hfs*283, L449 P457del, P3557=,
Q3603=, R1702*,
P648Tfs*2, R5501*, R4198*, R4484*, R83Q, R1903*õR2685* , R4282*, L5326-,
R5432W,
R2734*, Q2800*, R2830*. Q3745dup, S4010P, R4904*, G5182Afs*61, R5214H, R1615*,
Q2380*,
R2687*, R2771*, V3089Wfs*30, Q3799Gfs*212, R4536*, R5030C, R5048C, R5432Q,
A221Lfs*40,
A476T, A2119Lfs*25,P2557L, R2801*, Q3913*, R4420W, G4641=, R5097*, and any
combination
thereof.
1001436] In some embodiments, the ARID lA mutation includes, but is not
limited to, For example,
subject has a mutation of ARID IA selected from the group consisting of a
C884* (*: nonsense
mutation), E966K, Q1411*, F1720fs (fs: frameshift), G1847fs, C1874fs, D1957E,
Q1430, R1721fs,
G1255E, G284fs, RI722*, M274fs, G1847fs, P559fs, R1276*, Q2176fs, H203fs, A59
ifs, Q1322*,
S2264*, Q586*, Q548fs, and N756fs.
10014371111 some embodiments, the RBI mutation includes, but is not limited
to, R320X, R467X,
R579X, R455X, R358X, R25IX, R787X, R552X, R255X, R556X, Y790X, Q575X, E323X,
R661W,
R579*, R455*, R556*. R787*, R661W, R445*, R467*, Q217* ,Q471*, W195*, Q395*,
1680T,
E137*, R255*, Q344*, Q62*, E440K, A488V, P777Lfs*33, E322K, R656W, G617Rfs*36,
C221*,
E440*,Q93*, Q504*, E125*, S834*, E323*, Q685*, S829*, W516*, G435*, Q257*,
E79*, S567L,
V654M, V654Sfs*14,G100Efs*11, K715*, and any combination thereof
10014381In some embodiments, the ATM mutation is a mutation in the ATM gene
sequence
including, but is not limited to, 10744A>G;10744A>G; 11482G>A; IVS3-558A>T;
146C>G;
381delA; IVS8-3delGT; 1028delAAAA; 1120C>T; 1930ins16; IVS16+21>C; 2572T>C;
1VS21+1G>A; 3085delA; 3381delTGAC; 3602delTT; 4052delT; 4396C>T; 5188C>T;
5290delC;
5546delT; 5791G>CCT; 6047A>G; IVS44-1G>T; 6672delGC/6677delTACG; 6736de11
1/6749de17;
7I59insAGCC; 7671delGTTT; 7705dell4; 7865C>T; 7979delTGT; 8I77C>T; 8545C>T;
8565T>A;
IVS64+1G>T; and 9010de128.
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100143911n some embodiments of the present invention, the SETD2 mutation is an
alteration in the
gene sequence encoding the SETD2 protein, when the transcription initiation
codon position of the
mRNA sequence of NCBI accession number NM 014159 is set to 1. In some
embodiments, the
7558th G (guanine) is substituted with T (thymine) , 4774 th C (cytosine) is
substituted by T, 1210 th
A (adenine) is substituted by T, 4883 th is substituted by G, 5290 th C is
replaced by T, 7072 th C is
replaced by T, 4144 th G Is substituted by T, 1297 is replaced by T, 755 is
replaced by G, 7261 is
substituted by G, 6700 is replaced by T, 2536 is substituted by T, 7438 is
replaced by T Substitution,
insertion of A at position 3866, insertion of T at position 6712, insertion of
T at position 7572,
deletion of 913th A, deletion of 5619th C, deletion of bases 4603-4604, 894-
897 Deletion of the 1st
base, deletion of the 1936th C, deletion of the 3094-3118 base, insertion of A
in the 5289th position,
and deletion of the 6323-6333 base One or more mutations selected from the
group true luer included.
[0014401M one embodiment of the present invention, the mutation of the SETD2
protein is stopped
after 2520 glutamic acid, stop after 1592 arginine, stop after 404 arginine,
stop after 1764 glutamine,
1032 of SETD2 amino acid sequence corresponding to NCBI accession number
NP_054878
Frameshift after the first serine, Frameshift after the 646 histidine,
Frameshift after the 2108th valine,
Frameshift after the 1764 glutamine, Frameshift after the 298th isoleucine,
Frameshift after the 1289th
asparagine, Frameshift since the 289th serine Frameshift after, stop after
2525 lysine, frame shift after
305 threonine, frame shift after 1873 prolinc, framc shift after 1535
asparagine, stop after 2234
glutamic acid, replace 2536 alanine with threonine, 7438 Glutamine stopped,
1628 Group consisting
of spargine substituted by serine, 2358 th proline by serine, 1382 alanine by
serine, 433 th arginine by
cytosine, 252nd alanine by glycine, and 2421 threonine by alanine One or more
mutations selected
from.
10014411In some embodiments, the FLT3 mutation includes, but is not limited
to, (Q569_E648)ins,
D835X, (Q569_E648)delins, (D835 1836), D835Y, D835V, D835Y, D835H, T227M,
1836de1,
N676K, D835E, Y597_E598insDYVDFREY,D835E, D835del, F594_D600dup, A680V, D839G,
D96=, D835H, V491L, D835E, Q989*, D835V, L561=, 1836del, P986Afs*27, D7G,
D324N, S451F,
D835N, L576P, Y597 E598insDVDFREY, V491L, N841T, D324N, Y572C, R595 L60 ldup,
K663R, N676K,F691L, D835A, I836H, N841K, S993L, L832F, I836M, A66V, and any
combination
thereof
100144211n some embodiments, the PTPN11 mutation includes, but is not limited
to, c E76K, A72V,
A72T, D61Y, D61VõG60V , E69K, E76G, 6507V, S506L, G507A, T73I, E76A, E76Q,
S506P,
D61N, F71L, E76V, F71L, A72D, V432M, T472M, P495L, N58Y, F285S, S506A, S189A,
A465T,
R502W, G507R, T511K, D61H, D61G, G507E, G6OR, G60A, Q514L, E139D, Y197*,
N308D,
Q514H, Q514H, N58S, E123D, L206=, A465G, P495S, G507R, and any combination
thereof
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10014431In some embodiments, the FGFR1 mutation includes, but is not limited
to, N577K, K687E,
N577K, D166del, T371M, R476W, T350=, E498K, N577D, D683G, R87C, A154D, N303=,
A374V,
D550=, S633=, V695L, G728=, R765W, P803S, W19C, P56=, R1 13C, V149I, 5158L,
D166dupR220C, N224Kfs*8, D249N, R281W, R281Q, A299S, S424L, 5461F, S467F,
R506Q, and
any combination thereof
1001444] In some embodiments, the EP300 mutation includes, but is not limited
to, D1399N,
Y1414C, M1470Cfs*26, Y1111*, H2324Pfs*55, RI627W, N2209 Q2213delinsK,
Q2268del, L415P,
M1470Nfs*3, E1514K, C1201Y, P1452L, S952*, C1164Y, D1399Y, S507G, Q824*,
D1507N,
H2324Tfs*29, P925T, P1440L, W1466C, P1502L, A1629V, R1645*, N1700Tfs*9,
P1869L, Q65*,
A171V, R202*, R580Q, A627V, Q1082*, N1236Kfs*2, N1286S, R1312*, R1356*,
C1385F,
H1451L, R1462*, Y1467N, Y1467H, R1478H, R1627Q, R86*, R370H, R397*, R754C,
P842S,
I997V, E1014*,and any combination thereof.
[0014451M some embodiments, the MYC mutation includes, but is limited to,
E61T, E681, R74Q,
R75N, W135E, W136E, V394D, L420P, W96E, V325D, L35 1P, a MYC protein with 41
amino acid
deleted at the N-terminus (dN2MYC), N26S, S161L, P74L, V7M, F153S, E54D, P246,
Li 64V, P74S,
A59V, T731, P72T,T73A, H374R, P17S, T73N, S264N, P72S, Q52del, 521T, P74A,
5107N, P75S,
S77P, P261S, P74Q, S190R, A59T, F153C, P75H, T73I, S77F, Ni IS, 521N, P78L,
P72L, N9K,
S190N, S267F, 173P, P78S, G105D, S187C, L71M, Q10H, L191x, Q50x, L191F, R25K,
F130L,
Y27S, D195N, D2G, V20A, V6G, V20I, D2H, P75A, G152D, P74T, C40Y, E8K, Q48x,
and any
combination thereof.
10014461 In some embodiments, the EZH2 mutation is associated with altered
histone methylation
patterns. In some embodiments, the EZH2 mutation leads to the conversion of
amino acid Y641
(equivalent to Y646, catalytic domain), to either F, N, H, S or C resulting in
hypertrimethylation of
H3K27 and drives lymphomagenesis. In some embodiments, the EZH2 mutation
includes EZH2 SET-
domain mutations, overexpression of EZH2, overexpression of other PRC2
subunits, loss of function
mutations of histone acetyl transferases (HATs), and loss of function of MLL2.
Cells that are
heterozygous for EZH2 Y646 mutations result in hypeitrimethylation of H3K27
relative to cells that
are homozygous wild-type (WT) for the EZH2 protein, or to cells that are
homozygous for the Y646
mutation.
10014471In some embodiments, the EZH2 mutation includes, but is not limited
to, Y646F, Y646N,
D185H, Y646F, Y646S, Y646H, R690H, Y646X, E745K, Y646C, V626M, V679M, R690H,
R684H,
A682G, E249K, G159R, R288Q, N3225, A692V, R690C, D730* (insertion frameshift),
5695L,
R684C, M667T, .R288*, 5644*, D192N, K550T,Q653E, D664G, R347Q,Y646C,G660R,
R213C,
A255T, 5538L, N693K, I55M, R561H, A692V, K515R,Y733*, R63*, Q570*, Q328*,
R25Q, T467P
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A656V, T573I, C571Y, E725K, R16W, P577L, F145S, V680M, G686D, G135R, K634E,
S652F,
R298C, G648E, R566H, L149Rõ R502Q, Y731D, R313W, N675K, S652C, T374Hfs*3,
N152Ifs*15,
E401Kfs*22, K406Mfs*17, E246*, S624C, 1146T, V626M, L674S, H694R, A581S, and
any
combination thereof.
10014481111 some embodiments, the JAK2 mutation is a mutation in the JAK2 gene
includes, but is
not limited to, T1923C mutation in combination with a G1920T mutation, a
G19201 / C1922T
mutation, or a G1920A mutation. In some embodiments, the JAK2 mutation is a
mutant JAK2 protein
comprising one or more substitutions include, but are not limited to, V617F,
V617I, R683G,
N542_E543del, E543 D544del, R683S, R683X, F537_K539delinsL (deletion in
frame), K539L,
N1108S, R1113H, R1063H, R487C, 1540Mfs*3 (deletion-frameshift), R867Q, K539L,
G571S,
R1113C, R938Q, R228Q, L830*, E1080*, K539L, C618R, R564Q, D1036H, L1088S,
H538Nfs*4,
D873N, V392M, I682F, L393V, M535I, C618R, T875N, L611V, D319N, L611S, G921S,
H538Y,
S1035L, and any combination thereof.
[0014491M some embodiments, the FBXW7 mutation is a point mutation selected
from the group
consisting of W244* (*:stop codon), R222*, R278*, E192A, S282*, E113D,
R465H/C, 726+1 G>A
splice, R505C, R479Q, R465C, R367*, R499Vfs*25 (fs*: frameshift), R658*,
D600Y, D520N,
D520Y, and any combination thereof. In further embodiments, the FBXW7 mutation
is double- or
triple-mutation includes, but is not limited to, R479Q and S582L, R465H and
S582L, D520N, D520Y
and R14Q, and R367* and S582L.
10014501In some embodiments, the CCND3 mutation includes, but is not limited
to, S259A,
R271Pfs*53 (insertion-caused frameshift), E51*, Q260*, P199S, T283A, T283P,
V287D,
D286 T288del, R271Gfs*33, Q276*, R241Q, D238G, R33P, 1290K, 1290T, 1290R,
P267fs, P284S,
P284L, PlOOS, E253D, S262I, R14W, R114L, D238N, A266E, R167W, and any
combination thereof.
[0014511M some embodiments, the GNAll mutation includes, but is not limited
to, Q209L, R183C,
T257=, R183C, G208Afs*16, Q209H, R183C, Q209P, Q209R, Q209H, ?T96=, R210W,
R256Q,
T334=, G48D, S53Gõ Q209P, R213Q , and any combination thereof. In some
embodiments, the
GNAll mutation has two mutations in cxon 4, cg, a mutation in V182 and a
mutation in T175, or one
or more mutation in exon 5.
3. Combinations with PD-1 and PD-Li Inhibitors
10014521Programmed death 1 (PD-1) is a 288-amino acid transmembrane
immunocheckpoint
receptor protein expressed by T cells, B cells, natural killer (NK) T cells,
activated monocytes, and
dendritic cells. PD-1, which is also known as CD279, belongs to the CD28
family, and in humans is
encoded by the Pdcal gene on chromosome 2. PD-1 consists of one immunoglobulin
(1g) superfamily
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domain, a transmembrane region, and an intracellular domain containing an
immunoreceptor tyrosine-
based inhibitory motif (ITIM) and an immunoreceptor tyrosine-based switch
motif (ITSM). PD-1 and
its ligands (PD-Li and PD-L2) are known to play a key role in immune
tolerance, as described in
Keir, et al., Annu. Rev. Immunol. 2008, 26, 677-704. PD-1 provides inhibitory
signals that negatively
regulate T cell immune responses. PD-Li (also known as B7-H1 or CD274) and PD-
L2 (also known
as B7-DC or CD273) arc expressed on tumor cells and stromal cells, which may
be encountered by
activated T cells expressing PD-1, leading to immunosuppression of the T
cells. PD-Li is a 290
amino acid transmembrane protein encoded by the Cd274 gene on human chromosome
9. Blocking
the interaction between PD-1 and its ligands PD-Li and PD-L2 by use of a PD-1
inhibitor, a PD-Li
inhibitor, and/or a PD-L2 inhibitor can overcome immune resistance, as
demonstrated in recent
clinical studies, such as that described in Topalian, etal., N. Eng. I Med.
2012, 366, 2443-54. PD-Li
is expressed on many tumor cell lines, while PD-L2 is expressed is expressed
mostly on dendritic
cells and a few tumor lines. In addition to T cells (which inducibly express
PD-1 after activation), PD-
1 is also expressed on B cells, natural killer cells, macrophages, activated
monocytes, and dendritic
cells.
100145311n some embodiments, TILs and a PD-1 inhibitor are administered as a
combination therapy
or co-therapy for the treatment of NSCLC.
[0014541in some embodiments, the NSCLC has undergone no prior therapy. In some
embodiments,
a PD-1 inhibitor is administered as a front-line therapy or initial therapy.
In some embodiments, a PD-
1 inhibitor is administered as a front-line therapy or initial therapy in
combination with the TILs as
described herein.
10014551ln some embodiments, the PD-1 inhibitor may be any PD-1 inhibitor or
PD-1 blocker
known in the art. In particular, it is one of the PD-1 inhibitors or blockers
described in more detail in
the following paragraphs. The terms -inhibitor," -antagonist," and -blocker"
are used interchangeably
herein in reference to PD-1 inhibitors. For avoidance of doubt, references
herein to a PD-1 inhibitor
that is an antibody may refer to a compound or antigen-binding fragments,
variants, conjugates, or
biosimilars thereof. For avoidance of doubt, references herein to a PD-1
inhibitor may also refer to a
small molecule compound or a pharmaceutically acceptable salt, ester, solvate,
hydrate, cocrystal, or
prodrug thereof.
10014561ln some embodiments, the PD-1 inhibitor is an antibody (i.e., an anti-
PD-1 antibody), a
fragment thereof, including Fab fragments, or a single-chain variable fragment
(scFv) thereof. In
some embodiments the PD-1 inhibitor is a polyclonal antibody. In some
embodiments, the PD-1
inhibitor is a monoclonal antibody. in some embodiments, the PD-1 inhibitor
competes for binding
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with PD-1, and/or binds to an epitope on PD-1. In some embodiments, the
antibody competes for
binding with PD-1, and/or binds to an epitope on PD-1.
10014571In some embodiments, the PD-1 inhibitor is one that binds human PD-1
with a KD of about
100 pM or lower, binds human PD-1 with a KD of about 90 pM or lower, binds
human PD-1 with a
KD of about 80 pM or lower, binds human PD-1 with a KD of about 70 pM or
lower, binds human PD-
1 with a KD of about 60 pM or lower, binds human PD-1 with a KD of about 50 pM
or lower, binds
human PD-1 with a KD of about 40 pM or lower, binds human PD-1 with a KD of
about 30 pM or
lower, binds human PD-1 with a KD of about 20 pM or lower, binds human PD-1
with a KD of about
pM or lower, or binds human PD-1 with a KD of about 1 pM or lower.
10014581ln some embodiments, the PD-1 inhibitor is one that binds to human PD-
1 with a kassoc of
about 7.5 x 105 1/M- s or faster, binds to human PD-1 with a kassoc of about
7.5 x 105 1/M- s or faster,
binds to human PD-1 with a k5s50, of about 8 x 105 1/M. s or faster, binds to
human PD-1 with a kassoc
of about 8.5 x 10' 1/M. s or faster, binds to human PD-1 with a kassoc of
about 9 x 105 1/M= s or faster,
binds to human PD-1 with a kassdc of about 9.5 x 105 1/Ms or faster, or binds
to human PD-1 with a
kassoc of about 1 x 106 1/Ms or faster.
[0014591in some embodiments, the PD-1 inhibitor is one that binds to human PD-
1 with a kdisso, of
about 2 x 10-5 1/s or slower, binds to human PD-1 with a kdissdc of about 2.1
x 10' 1/s or slower,
binds to human PD-1 with a kdissdc of about 2.2 x 10-5 1/s or slower, binds to
human PD-1 with a kdissde
of about 2.3 x 10-5 1/s or slower, binds to human PD-1 with a kdissoc of about
2.4 x 10-5 1/s or slower,
binds to human PD-1 with a kdissdc of about 2.5 x 10-5 1/s or slower, binds to
human PD-1 with a kdissdc
of about 2.6 x 10' 1/s or slower or binds to human PD-1 with a kdisso, of
about 2.7 x 10-5 1/s or
slower, binds to human PD-1 with a kdisso, of about 2.8 x 10-5 1/s or slower,
binds to human PD-1 with
a kahsoc of about 2.9 x 10-5 1/s or slower, or binds to human PD-1 with a
kdissoc of about 3 x 10-5 1/s or
slower.
10014601In some embodiments, the PD-1 inhibitor is one that blocks or inhibits
binding of human
PD-L1 or human PD-L2 to human PD-1 with an TC50 of about 10 nM or lower,
blocks or inhibits
binding of human PD-Li or human PD-L2 to human PD-1 with an IC of about 9 nM
or lower,
blocks or inhibits binding of human PD-Li or human PD-L2 to human PD-1 with an
IC50 of about 8
nM or lower, blocks or inhibits binding of human PD-Li or human PD-L2 to human
PD-1 with an
IC50 of about 7 nM or lower, blocks or inhibits binding of human PD-Li or
human PD-L2 to human
PD-1 with an 1050 of about 6 nM or lower, blocks or inhibits binding of human
PD-Li or human PD-
L2 to human PD-1 with an IC50 of about 5 nM or lower, blocks or inhibits
binding of human PD-Li or
human PD-L2 to human PD-1 with an 1050 of about 4 nM or lower, blocks or
inhibits binding of
human PD-L1 or human PD-L2 to human PD-1 with an IC50 of about 3 nM or lower,
blocks or
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inhibits binding of human PD-Li or human PD-L2 to human PD-1 with an IC50 of
about 2 nM or
lower, or blocks or inhibits binding of human PD-Li or human PD-L2 to human PD-
1 with an IC50 of
about 1 nM or lower.
100146111n some embodiments, the PD-1 inhibitor is nivolumab (commercially
available as
OPDIVO from Bristol-Myers Squibb Co.), or biosimilars, antigen-binding
fragments, conjugates, or
variants thereof. Nivolumab is a fully human IgG4 antibody blocking the PD-1
receptor. In some
embodiments, the anti-PD-1 antibody is an immunoglobulin G4 kappa, anti-(human
CD274)
antibody. Nivolumab is assigned Chemical Abstracts Service (CAS) registry
number 946414-94-4
and is also known as 5C4, BMS-936558, MDX-1106, and ONO-4538. The preparation
and properties
of nivolumab are described in U.S. Patent No. 8,008,449 and International
Patent Publication No. WO
2006/121168, the disclosures of which are incorporated by reference herein.
The clinical safety and
efficacy of nivolumab in various forms of cancer has been described in Wang,
et at., Cancer Immunol
Res. 2014, 2, 846-56; Page, et at., Ann. Rev. Med., 2014, 65, 185-202; and
Weber, et at., .1 Cl/n.
Oncology, 2013, 31, 4311-4318, the disclosures of which are incorporated by
reference herein. The
amino acid sequences of nivolumab are set forth in Table 18. Nivolumab has
intra-heavy chain
disulfide linkages at 22-96,140-196, 254-314, 360-418, 22-96", 140"-196", 254-
314", and 360"-
418"; intra-light chain disulfide linkages at 23-88', 134'-194', 231"-88"1,
and 134"-194"; inter-heavy-
light chain disulfide linkages at 127-214', 127"-214", inter-heavy-heavy chain
disulfide linkages at
219-219" and 222-222"; and N-glycosylation sites (H CH2 84.4) at 290, 290".
[0014621kt some embodiments, a PD-1 inhibitor comprises a heavy chain given by
SEQ ID NO: 158
and a light chain given by SEQ ID NO: i59. In some embodiments, a PD-1
inhibitor comprises heavy
and light chains having the sequences shown in SEQ ID NO: i58 and SEQ ID NO:
i59, respectively,
or antigen binding fragments, Fab fragments, single-chain variable fragments
(scFv), variants, or
conjugates thereof In some embodiments, a PD-1 inhibitor comprises heavy and
light chains that are
each at least 99% identical to the sequences shown in SEQ ID NO: i58 and SEQ
ID NO:159,
respectively. In some embodiments, a PD-1 inhibitor comprises heavy and light
chains that are each at
least 98% identical to the sequences shown in SEQ ID NO: i58 and SEQ ID
NO:159, respectively. In
some embodiments, a PD-1 inhibitor comprises heavy and light chains that are
each at least 97%
identical to the sequences shown in SEQ ID NO: i58 and SEQ ID NO: 159,
respectively. In some
embodiments, a PD-1 inhibitor comprises heavy and light chains that are each
at least 96% identical
to the sequences shown in SEQ ID NO: i58 and SEQ ID NO: 159, respectively. In
some embodiments.
a PD-1 inhibitor comprises heavy and light chains that are each at least 95%
identical to the sequences
shown in SEQ ID NO: i58 and SEQ ID NO: i59, respectively.
10014631ln some embodiments, the PD-1 inhibitor comprises the heavy and light
chain CDRs or
variable regions (VRs) of nivolumab. In some embodiments, the PD-1 inhibitor
heavy chain variable
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region (VH) comprises the sequence shown in SEQ ID NO:160, and the PD-1
inhibitor light chain
variable region (VL) comprises the sequence shown in SEQ ID NO: 161, and
conservative amino acid
substitutions thereof. In some embodiments, a PD-1 inhibitor comprises VII and
VL regions that are
each at least 99% identical to the sequences shown in SEQ ID NO: 160 and SEQ
ID NO:161,
respectively. In some embodiments, a PD-1 inhibitor comprises VH and VL
regions that are each at
least 98% identical to the sequences shown in SEQ Ill NO: i60 and SEQ ID
NO:161, respectively. In
some embodiments, a PD-1 inhibitor comprises Vu and VL regions that are each
at least 97% identical
to the sequences shown in SEQ ID NO:160 and SEQ ID NO: 161, respectively. In
some embodiments,
a PD-1 inhibitor comprises VH and VL regions that are each at least 96%
identical to the sequences
shown in SEQ ID NO: 160 and SEQ ID NO:161, respectively. In some embodiments,
a PD-1 inhibitor
comprises VH and VL regions that are each at least 95% identical to the
sequences shown in SEQ ID
NO:160 and SEQ ID NO:161, respectively.
1001464] In some embodiments, a PD-1 inhibitor comprises heavy chain CDR1,
CDR2 and CDR3
domains having the sequences set forth in SEQ ID NO:162, SEQ ID NO:163, and
SEQ ID NO: i64,
respectively, or conservative amino acid substitutions thereof, and light
chain CDR1, CDR2 and
CDR3 domains having the sequences set forth in SEQ ID NO:165, SEQ ID NO:166,
and SEQ ID
NO:167, respectively, or conservative amino acid substitutions thereof. In
some embodiments, the
antibody competes for binding with, and/or binds to the same epitope on PD-1
as any of the
aforementioned antibodies.
1001465] In some embodiments, the PD-1 inhibitor is an anti-PD-1 biosimilar
monoclonal antibody
approved by drug regulatory authorities with reference to nivolumab. In some
embodiments, the
biosimilar comprises an anti-PD-1 antibody comprising an amino acid sequence
which has at least
97% sequence identity, e.g., 97%, 98%, 99% or 100% sequence identity, to the
amino acid sequence
of a reference medicinal product or reference biological product and which
comprises one or more
post-translational modifications as compared to the reference medicinal
product or reference
biological product, wherein the reference medicinal product or reference
biological product is
nivolumab. In some embodiments, the one or more post-translational
modifications are selected from
one or more of: glycosylation, oxidation, deamidation, and truncation. In some
embodiments, the
biosimilar is an anti-PD-1 antibody authorized or submitted for authorization,
wherein the anti-PD-1
antibody is provided in a formulation which differs from the formulations of a
reference medicinal
product or reference biological product, wherein the reference medicinal
product or reference
biological product is nivolumab. The anti-PD-1 antibody may be authorized by a
drug regulatory
authority such as the U.S. FDA and/or the European Union's EMA. In some
embodiments, the
biosimilar is provided as a composition which further comprises one or more
excipients, wherein the
one or more excipients are the same or different to the excipients comprised
in a reference medicinal
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product or reference biological product, wherein the reference medicinal
product or reference
biological product is nivolumab. In some embodiments, the biosimilar is
provided as a composition
which further comprises one or more excipients, wherein the one or more
excipients are the same or
different to the excipients comprised in a reference medicinal product or
reference biological product,
wherein the reference medicinal product or reference biological product is
nivolumab.
TABLE 18. Amino acid sequences for PD-1 inhibitors related to nivolumab.
Identifier Sequence (One-Letter Amino Acid Symbols)
SEQ ID NO:158 QVQLVESGGG VVQPGRSLRL DCKASGITFS NSGMHWVRQA PGKGLEWVAV
IWYDGSKRYY 60
nivolumab ADSVKGRFTI SRDNSKNTLF LQMNSLRAED TAVYYCATND DYWGQGTLVT
VSSAS=GPS L20
heavy chain VFPLA2CSRS TSESTAALGC LVKDYFPEPV TVSWNSGALT SGVHTRPAVL
QSSGLYSLSS 180
VVTVPSSSLG THTYTCNVDH KPSNTHVDKR VESKYGFPCP PCPAPEELGG PSVFLFP2KP
240
KDTLMISETP EVTCVVVDVS QEDDEVQFNW YVDGVEVHNA KTKPREEQrN STYRVVSVLT
300
VLHQDWLNGK EYKCKVSNAG LPSS_LEKIS KAKGQPKEPQ VYTLPPSQEE MTKNQVSLTC
360
LVKGFYPSDI AVEWESNCQP ENNYKTTPPV LDSDCSFFLY SRLTVDKSRW QEGNVFSCSV
420
MHEALNNNYT QKSLSLSLGK
440
SEQ ID NO:159 EIVLTQSPAT LSLSPGERAT LSCRASQSVS SYLAWYQQKP GQAPRLLIYD
ASNRATGIPA 60
nivolumab RRSGSGSGTD FTLTISSLEF ED2AVYYCQQ SSNWPRTFGQ GTKVEIKRTV
AAPSVFIFFP 120
light chain SDEQLKSGTA SVVCLLNNFY PREAKVQWKV DNALQSGNSQ ESVTEQDSKD
STYSLSSTLT 180
LSKADYEKEK VYACEVTIIQG LSSPVTKSFN RGEC
214
SEQ ID NO:160 QVQLVESGGG VVQPGRSLRL DCKASGITES NSGMHWVRQA PGHGLEWVAV
IWYDGSKRYY 60
nivolumab ADSVKGRRTI SRDNSKNTLF LQMNSLRAED TAVYYCATND DYWGQGTLVT
VSS 113
variable heavy
chain
SEQ ID NO:161 EIVLTQSPAT LSLSPGERAT LSCRASQSVS SYLAWYQQKP GQAPRLLIYD
ASNRATGIPA 60
nivolumab RFSGSGSGTD FTLTISSLEP EDFAVYYCQQ SSNWPRTFGQ GTKVEIK
107
variable light
chain
SEQ ID NO:162 NSGMH
3
nivoLumab
heavy chain
CDR1
SEQ _D NO:163 VIWY2GSKRY YADSVKG
17
nivoLumab
heavy chain
CDR2
SEQ ID NO:164 NDDY
4
nivolumab
heavy chain
CDR3
SEQ ID NO:165 RASQSVSSYL A
11
nivolumab
light chain
CDR1
SEQ ID NO:166 DASNRAT
7
nivolumab
light chain
CDR2
SEQ ID NO:167 QQSSNWPRT
9
nivolumab
light chain
C2H3
10014661
In some embodiments, the PD-1 inhibitor is nivolumab or a biosimilar
thereof, and
the nivolumab is administered at a dose of about 0.5 mg/kg to about 10 mg/kg.
In some embodiments,
the PD-1 inhibitor is nivolumab or a biosimilar thereof, and the nivolumab is
administered at a dose of
about 0.5 mg/kg, about 1 mg/kg, about 1.5 mg/kg, about 2 mg/kg, about 2.5
mg/kg, about 3 mg/kg,
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about 3.5 mg/kg, about 4 mg/kg, about 4.5 mg/kg, about 5 mg/kg, about 5.5
mg/kg, about 6 mg/kg,
about 6.5 mg/kg, about 7 mg/kg, about 7.5 mg/kg, about 8 mg/kg, about 8.5
mg/kg, about 9 mg/kg,
about 9.5 mg/kg, or about 10 mg/kg.
10014671 In some embodiments, the PD-1 inhibitor is nivolumab or a
biosimilar thereof, and
the nivolumab is administered at a dose of about 200 mg to about 500 mg. In
some embodiments, the
PD-1 inhibitor is nivolumab or a biosimilar thereof, and the nivolumab is
administered at a dose of
about 200 mg, about 220 mg, about 240 mg, about 260 mg, about 280 mg, about
300 mg, about 320
mg, about 340 mg, about 360 mg, about 380 mg, about 400 mg, about 420 mg,
about 440 mg, about
460 mg, about 480 mg, or about 500 mg.
10014681 In some embodiments, the nivolumab is administered to
treat metastatic non-small
cell lung cancer. In some embodiments, the nivolumab is administered to treat
metastatic non-small
cell lung cancer at about 3 mg/kg every 2 weeks along with ipilimumab at about
1 mg/kg every 6
weeks. In some embodiments, the nivolumab is administered to treat metastatic
non-small cell lung
cancer at about 360 mg every 3 weeks with ipilimumab 1 mg/kg every 6 weeks and
2 cycles of
platinum-doublet chemotherapy. In some embodiments, the nivolumab is
administered to treat
metastatic non-small cell lung cancer at about 240 mg every 2 weeks or 480 mg
every 4 weeks.
10014691ln some embodiments, the PD-1 inhibitor comprises pembrolizumab
(commercially
available as KEYTRUDA from Merck & Co., Inc., Kenilworth, NJ, USA), or antigen-
binding
fragments, conjugates, or variants thereof. Pembrolizumab is assigned CAS
registry number 1374853-
91-4 and is also known as lambrolizumab, MK-3475, and SCH-900475.
Pembrolizumab has an
immunoglobulin 64, anti-(human protein PDCD1 (programmed cell death 1)) (human-
Mus musculus
monoclonal heavy chain), disulfide with human-Mus musculus monoclonal light
chain, dimer
structure. The structure of pembrolizumab may also be described as
immunoglobulin G4, anti-(human
programmed cell death 1); humanized mouse monoclonal [228-L-proline(H10-
S>P)Iy4 heavy chain
(134-218)-disulfide with humanized mouse monoclonal K light chain dimer (226-
226":229-229")-
bisdisulfide. The properties, uses, and preparation of pembrolizumab are
described in International
Patent Publication No. WO 2008/156712 Al, U.S. Patent No. 8,354,509 and U.S.
Patent Application
Publication Nos. US 2010/0266617 Al, US 2013/0108651 Al, and US 2013/0109843
A2, the
disclosures of which are incorporated herein by reference. The clinical safety
and efficacy of
pembrolizumab in various forms of cancer is described in Fuerst, Oncology
Times, 2014, 36, 35-36;
Robert, et al., Lancet, 2014, 384, 1109-17; and Thomas, et al., Exp. Op/n.
Biol. Ther., 2014,14, 1061-
1064. The amino acid sequences of pembrolizumab are set forth in Table 19.
Pembrolizumab includes
the following disulfide bridges: 22-96, 22-96", 23'-92', 23"-92", 134-218',
134"-218'", 138'498',
138'"-198", 147-203, 147"-203", 226-226", 229-229", 261-321, 261-321", 367-
425, and 367-425",
and the following glycosylation sites (N): Asn-297 and Asn-297". Pembrolizumab
is an IgG4/kappa
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isotype with a stabilizing S228P mutation in the Fc region; insertion of this
mutation in the IgG4
hinge region prevents the formation of half molecules typically observed for
IgG4 antibodies.
Pembrolizumab is heterogeneously glycosylated at Asn297 within the Fc domain
of each heavy chain,
yielding a molecular weight of approximately 149 kDa for the intact antibody.
The dominant
glycoform of pembrolizumab is the fucosylated agalacto diantennary glycan form
(GOF).
10014701In some embodiments, a PD-1 inhibitor comprises a heavy chain given by
SEQ ID NO:168
and a light chain given by SEQ ID NO: 169. In some embodiments, a PD-1
inhibitor comprises heavy
and light chains having the sequences shown in SEQ ID NO:168 and SF() Ti)
NO:169, respectively,
or antigen binding fragments, Fab fragments, single-chain variable fragments
(scFv), variants, or
conjugates thereof. In some embodiments, a PD-1 inhibitor comprises heavy and
light chains that are
each at least 99% identical to the sequences shown in SEQ ID NO: 168 and SEQ
ID NO:169,
respectively. In some embodiments, a PD-1 inhibitor comprises heavy and light
chains that are each at
least 98% identical to the sequences shown in SEQ ID NO: 168 and SEQ ID
NO:169, respectively. In
some embodiments, a PD-1 inhibitor comprises heavy and light chains that are
each at least 97%
identical to the sequences shown in SEQ ID NO: i68 and SEQ ID NO: 169,
respectively. In some
embodiments, a PD-1 inhibitor comprises heavy and light chains that are each
at least 96% identical
to the sequences shown in SEQ ID NO:168 and SEQ ID NO: 169, respectively. In
some embodiments,
a PD-1 inhibitor comprises heavy and light chains that are each at least 95%
identical to the sequences
shown in SEQ ID NO: 168 and SEQ ID NO: i69, respectively.
10014711hi some embodiments, the PD-1 inhibitor comprises the heavy and light
chain CDRs or
variable regions (VRs) of pcmbrolizumab. In some embodiments, the PD-1
inhibitor heavy chain
variable region (VH) comprises the sequence shown in SEQ ID NO:170, and the PD-
1 inhibitor light
chain variable region (VL) comprises the sequence shown in SEQ ID NO:171, or
conservative amino
acid substitutions thereof In some embodiments, a PD-1 inhibitor comprises VH
and VL regions that
are each at least 99% identical to the sequences shown in SEQ ID NO: 170 and
SEQ ID NO:171,
respectively. In some embodiments, a PD-1 inhibitor comprises VH and VL
regions that are each at
least 98% identical to the sequences shown in SEQ ID NO: 170 and SEQ ID
NO:171, respectively. In
some embodiments, a PD-1 inhibitor comprises Vu and VL regions that are each
at least 97% identical
to the sequences shown in SEQ ID NO:170 and SEQ ID NO: 171, respectively. In
some embodiments,
a PD-1 inhibitor comprises VH and VL regions that are each at least 96%
identical to the sequences
shown in SEQ ID NO: 170 and SEQ ID NO: i71, respectively. In some embodiments,
a PD-1 inhibitor
comprises VII and VL regions that are each at least 95% identical to the
sequences shown in SEQ ID
NO:170 and SEQ ID NO:171, respectively.
100147211n some embodiments, a PD-1 inhibitor comprises the heavy chain CDR1,
CDR2 and
CDR3 domains having the sequences set forth in SEQ ID NO: i72, SEQ ID NO: 173,
and SEQ ID
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NO:174, respectively, or conservative amino acid substitutions thereof, and
light chain CDR1, CDR2
and CDR3 domains having the sequences set forth in SEQ ID NO:175, SEQ ID
NO:176, and SEQ ID
NO:177, respectively, or conservative amino acid substitutions thereof. In
some embodiments, the
antibody competes for binding with, and/or binds to the same epitope on PD-1
as any of the
aforementioned antibodies.
10014731111 some embodiments, the PD-1 inhibitor is an anti-PD-1 biosimilar
monoclonal antibody
approved by drug regulatory authorities with reference to pembrolizumab. In
some embodiments, the
biosimilar comprises an anti-PD-1 antibody comprising an amino acid sequence
which has at least
97% sequence identity, e.g., 97%, 98%, 99% or 100% sequence identity, to the
amino acid sequence
of a reference medicinal product or reference biological product and which
comprises one or more
post-translational modifications as compared to the reference medicinal
product or reference
biological product, wherein the reference medicinal product or reference
biological product is
pembrolizumab. in some embodiments, the one or more post-translational
modifications are selected
from one or more of: glycosylation, oxidation, deamidation, and truncation. In
some embodiments,
the biosimilar is an anti-PD-1 antibody authorized or submitted for
authorization, wherein the anti-
PD-1 antibody is provided in a formulation which differs from the formulations
of a reference
medicinal product or reference biological product, wherein the reference
medicinal product or
reference biological product is pembrolizumab. The anti-PD-1 antibody may be
authorized by a drug
regulatory authority such as the U.S. FDA and/or the European Union's EMA. In
some embodiments,
the biosimilar is provided as a composition which further comprises one or
more excipients, wherein
the one or more excipients are the same or different to the excipients
comprised in a reference
medicinal product or reference biological product, wherein the reference
medicinal product or
reference biological product is pembrolizumab. In some embodiments, the
biosimilar is provided as a
composition which further comprises one or more excipients, wherein the one or
more excipients are
the same or different to the excipients comprised in a reference medicinal
product or reference
biological product, wherein the reference medicinal product or reference
biological product is
pembrolizumab.
TABLE 19. Amino acid sequences for PD-1 inhibitors related to pembrolizumab.
Identifier Sequence (One-Letter Amino Acid Symbols)
SEQ =D NO:168 QVQLVQSGVE VKKPGASVKV SCKASGY=T NYYMYWVRQA PGQGLEWMGG
INPSNGGTNF 60
pembrolizumab NEKEHNRVTL TTDSSTTTAY MELKSLQFDD TAVYYCARRD YRFDMGFEYW
GQGTTVTVSS 020
heavy chain ASTKGPSVFP LAPCSRSTSE STAALGCLVK DYFPEPVTVS WNSGALTSGV
HTEPAVLQSS 080
GLYSLSSVVT VPSSSLGTET YTCNVDHKPS NTKVDKRVES KYGPPCPPCP APEFLGGPSV
240
FLEPPKPKDT LMISRTPEVT CVVVDVSQED PEVQFNWYVD GVEVENAKTK PREEQFNSTY
300
RVVSVLTVLH QDWLNGKEYK CKVSNYGLPS SIEKTISKAK GQPREPQVYT LPPSQEEMTK
360
NQVSLTCLVK GFYPSDIAVE WESNGQPENN YKTTPPVLDS DGSFFLYSRL TVDXSRWQEG
420
NVESCSVMHE ALIINHYTQHS LSLSLGH
447
SEQ ED NO:169 EIVLTQSPAT LSLSPGERAT LSCRASHGVS TSGYSYLHWY QQKPGQAPRL
LIYLASYLES 60
GVPARFSGSG SGTDFTLTIS SLEPEDFAVY YCQHSRELPL TYGGGTKVEI KRTVAAPSVF
020
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Identifier Sequence (One-Letter Amino Acid Symbols)
pembrolizumab IFPFSDEQLK SGTASVVOLL NNEYPREAKV QWEN-DNALQS GNSQESV-
TEQ DSEDSFYSLS .. 180
light chain STLTLSKAllY EKHKVYACEV THQGLSSPVT ASENRGEC
218
SEQ =D NO:170 QVQLVQSGVE VKKPGASVEV SCKASGY=T NYYMYWVRQA PGQGLEWMGG
INPSNGGTNF 60
pembrolizumab NEKEKNRVTL TTOSSTMAY MELKSLQFDD TAVYYUARRD YRYDMGEDYW
GQGTTVTVSS 120
variable heavy
chain
SEQ ID NO:171 EIVLTQSPAT LSLSPGERAT LSCRASKGVS TSGYSYLHWY QQKPGQAPRL
LIYLASYLES .. 60
pembrolizumab GVPARFSGSG SGTDFTLTIS SLEPEDEAVY YCQHSRDLPL TYGGGTKVEI
K Ill
variable light
chain
SEQ ID NO:172 NYYMY
pembrolizumab
heavy chain
CDR1
SEQ =D NO:173 GINPSNGGTN FNEKFX
16
pembrolizumab
heavy chain
CDR2
SEQ ID NO:174 RDYRFDMGFD Y
11
pembrolizumab
heavy chain
CDR3
SEQ =D NO:175 RASKGVSTSG YSYLH
13
pembrolizumab
light chain
CDR1
SEQ ID NO:176 LASYLES
7
pembrolizumab
light chain
CDR2
SEQ =D NO:177 QHSRDLPLT
9
pembrolizumab
light chain
CDR3
10014741
In some embodiments, the PD-1 inhibitor is pembrolizumab or a biosimilar
thereof,
and the pembrolizumab is administered at a dose of about 0.5 mg/kg to about 10
mg/kg. In some
embodiments, the PD-1 inhibitor is pembrolizumab or a biosimilar thereof, and
the pembrolizumab is
administered at a dose of about 0.5 mg/kg, about 1 mg/kg, about 1.5 mg/kg,
about 2 mg/kg, about 2.5
mg/kg, about 3 mg/kg, about 3.5 mg/kg, about 4 mg/kg, about 4.5 mg/kg, about 5
mg/kg, about 5.5
mg/kg, about 6 mg/kg, about 6.5 mg/kg, about 7 mg/kg, about 7.5 mg/kg, about 8
mg/kg, about 8.5
mg/kg, about 9 mg/kg, about 9.5 mg/kg, or about 10 mg/kg.
10014751
In some embodiments, the PD-1 inhibitor is pembrolizumab or a biosimilar
thereof,
wherein the pembrolizumab is administered at a dose of about 200 mg to about
500 mg. In some
embodiments, the PD-1 inhibitor is pembrolizumab or a biosimilar thereof, and
the nivohunab is
administered at a dose of about 200 mg, about 220 mg, about 240 mg, about 260
mg, about 280 mg,
about 300 mg, about 320 mg, about 340 mg, about 360 mg, about 380 mg, about
400 mg, about 420
mg, about 440 mg, about 460 mg, about 480 mg, or about 500 mg.
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10014761 In some embodiments, the PD-1 inhibitor is pembrolizumab
or a biosimilar thereof,
wherein the pembrolizumab is administered every 2 weeks, every 3 weeks, every
4 weeks, every 5
weeks, or every 6 weeks.
10014771 In some embodiments, the pembrolizumab is administered to
treat NSCLC. In some
embodiments, the pembrolizumab is administered to treat NSCLC at about 200 mg
every 3 weeks. In
some embodiments, the pembrolizumab is administered to treat NSCLC at about
400 mg every 6
weeks.
10014781 In some embodiments, if the patient or subject is an
adult, i.e. , treatment of adult
indications, and additional dosing regimen of 400 mg every 6 weeks can be
employed.
100147911n some embodiments, the PD-1 inhibitor is a commercially-available
anti-PD-1
monoclonal antibody, such as anti-m-PD-1 clones J43 (Cat # BE0033-2) and RMP1-
14 (Cat #
BE0146) (Bio X Cell, Inc.; West Lebanon, NH, USA). A number of commercially-
available anti-PD-
1 antibodies are known to one of ordinary skill in the art.
10014801In some embodiments, the PD-1 inhibitor is an antibody disclosed in
U.S. Patent No.
8,354,509 or U.S. Patent Application Publication Nos. 2010/0266617 Al,
2013/0108651 Al,
2013/0109843 A2, the disclosures of which are incorporated by reference
herein. In some
embodiments, the PD-1 inhibitor is an anti-PD-1 antibody described in U.S.
Patent Nos. 8,287,856,
8,580,247, and 8,168,757 and U.S. Patent Application Publication Nos.
2009/0028857 Al,
2010/0285013 Al, 2013/0022600 Al, and 2011/0008369 Al, the teachings of which
are hereby
incorporated by reference. In some embodiments, the PD-1 inhibitor is an anti-
PD-1 antibody
disclosed in U.S. Patent No. 8,735,553 Bl, the disclosure of which is
incorporated herein by
reference. In some embodiments, the PD-1 inhibitor is pidilizumab, also known
as CT-011, which is
described in U.S. Patent No. 8,686,119, the disclosure of which is
incorporated by reference herein.
10014811In some embodiments, the PD-1 inhibitor may be a small molecule or a
peptide, or a
peptide derivative, such as those described in U.S. Patent Nos. 8,907,053;
9,096,642; and 9,044,442
and U.S. Patent Application Publication No. US 2015/0087581; 1,2,4-oxadiazole
compounds and
derivatives such as those described in U.S. Patent Application Publication No.
2015/0073024; cyclic
peptidomimetic compounds and derivatives such as those described in U.S.
Patent Application
Publication No. US 2015/0073042; cyclic compounds and derivatives such as
those described in U.S.
Patent Application Publication No. US 2015/0125491; 1.3,4-oxadiazole and 1,3,4-
thiadiazole
compounds and derivatives such as those described in International Patent
Application Publication
No. WO 2015/033301; peptide-based compounds and derivatives such as those
described in
International Patent Application Publication Nos. WO 2015/036927 and WO
2015/04490, or a
macrocyclic peptide-based compounds and derivatives such as those described in
U.S. Patent
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Application Publication No. US 2014/0294898; the disclosures of each of which
are hereby
incorporated by reference in their entireties.
100148211n some embodiments, TILs and a PD-Li inhibitor or a PD-L2 inhibitor
are administered as
a combination therapy or co-therapy for the treatment of NSCLC.
10014831 In some embodiments, the NSCLC has undergone no prior therapy. In
some embodiments,
a PD-Li inhibitor or a PD-L2 inhibitor is administered as a front-line therapy
or initial therapy. In
some embodiments, a PD-L1 inhibitor or a PD-L2 inhibitor is administered as a
front-line therapy or
initial therapy in combination with the TILs as described herein.
10014841 In some embodiments, the PD-Li or PD-L2 inhibitor may be any PD-Li or
PD-L2
inhibitor, antagonist, or blocker known in the art. In particular, it is one
of the PD-Li or PD-L2
inhibitors, antagonist, or blockers described in more detail in the following
paragraphs. The terms
-inhibitor," -antagonist,' and -blocker" are used interchangeably herein in
reference to PD-L1 and
PD-L2 inhibitors. For avoidance of doubt, references herein to a PD-Li or PD-
L2 inhibitor that is an
antibody may refer to a compound or antigen-binding fragments, variants,
conjugates, or biosimilars
thereof For avoidance of doubt, references herein to a PD-Li or PD-L2
inhibitor may refer to a
compound or a pharmaceutically acceptable salt, ester, solvate, hydrate,
cocrystal, or prodrug thereof.
10014851 In some embodiments, the compositions, processes and methods
described herein include a
PD-Li or PD-L2 inhibitor. In some embodiments, the PD-Li or PD-L2 inhibitor is
a small molecule.
In some embodiments, the PD-Li or PD-L2 inhibitor is an antibody (i.e., an
anti-PD-1 antibody), a
fragment thereof, including Fab fragments, or a single-chain variable fragment
(scFv) thereof In
some embodiments the PD-Ll or PD-L2 inhibitor is a polyclonal antibody. In
some embodiments, the
PD-L1 or PD-L2 inhibitor is a monoclonal antibody. In some embodiments, the PD-
L1 or PD-L2
inhibitor competes for binding with PD-Li or PD-L2, and/or binds to an epitope
on PD-Li or PD-L2.
In some embodiments, the antibody competes for binding with PD-Li or PD-L2,
and/or binds to an
epitope on PD-L I or PD-L2.
10014861 In some embodiments, the PD-Li inhibitors provided herein are
selective for PD-L1, in
that the compounds bind or interact with PD-L I at substantially lower
concentrations than they bind
or interact with other receptors, including the PD-L2 receptor. In certain
embodiments, the
compounds bind to the PD-Li receptor at a binding constant that is at least
about a 2-fold higher
concentration, about a 3-fold higher concentration, about a 5-fold higher
concentration, about a 10-
fold higher concentration, about a 20-fold higher concentration, about a 30-
fold higher concentration,
about a 50-fold higher concentration, about a 100-fold higher concentration,
about a 200-fold higher
concentration, about a 300-fold higher concentration, or about a 500-fold
higher concentration than to
the PD-L2 receptor.
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100148711n some embodiments, the PD-L2 inhibitors provided herein are
selective for PD-L2, in that
the compounds bind or interact with PD-L2 at substantially lower
concentrations than they bind or
interact with other receptors, including the PD-Li receptor. In certain
embodiments, the compounds
bind to the PD-L2 receptor at a binding constant that is at least about a 2-
fold higher concentration,
about a 3-fold higher concentration, about a 5-fold higher concentration,
about a 10-fold higher
concentration, about a 20-fold higher concentration, about a 30-fold higher
concentration, about a 50-
fold higher concentration, about a 100-fold higher concentration, about a 200-
fold higher
concentration, about a 300-fold higher concentration, or about a 500-fold
higher concentration than to
the PD-L1 receptor.
10014881Without being bound by any theory, it is believed that tumor cells
express PD-Li, and that
T cells express PD-1. However, PD-Li expression by tumor cells is not required
for efficacy of PD-1
or PD-Li inhibitors or blockers. In some embodiments, the tumor cells express
PD-Li. In some
embodiments, the tumor cells do not express PD-Ll. In some embodiments, the
methods can include
a combination of a PD-1 and a PD-Li antibody, such as those described herein,
in combination with a
TIL. The administration of a combination of a PD-1 and a PD-Li antibody and a
TIL may be
simultaneous or sequential.
100148911n some embodiments, the PD-Li and/or PD-L2 inhibitor is one that
binds human PD-Li
and/or PD-L2 with a KD of about 100 pM or lower, binds human PD-Li and/or PD-
L2 with a K0 of
about 90 pM or lower, binds human PD-Li and/or PD-L2 with a Ku of about 80 pM
or lower, binds
human PD-Li and/or PD-L2 with a KD of about 70 pM or lower, binds human PD-Li
and/or PD-L2
with a KD of about 60 pM or lower, a KD of about 50 pM or lower, binds human
PD-Li and/or PD-L2
with a KD of about 40 pM or lower, or binds human PD-Li and/or PD-L2 with a KD
of about 30 pM
or lower,
100149011n some embodiments, the PD-Li and/or PD-L2 inhibitor is one that
binds to human PD-Li
and/or PD-L2 with a kaõ,,, of about 7.5 105 1/1\4* s or faster, binds to human
PD-Li and/or PD-L2
with a kasso, of about 8 x 105 1/M= s or faster, binds to human PD-Li and/or
PD-L2 with a kasso, of
about 8.5 x 105 I/M=s or faster, binds to human PD-Li and/or PD-L2 with a
kas50, of about 9 x 105
1/Ms or faster, binds to human PD-Li and/or PD-L2 with a kassoc of about 9.5 x
105 1/M= s and/or
faster, or binds to human PD-Li and/or PD-L2 with a kassoc of about 1 x 106
1/Ms or faster.
100149111n some embodiments, the PD-Li and/or PD-L2 inhibitor is one that
binds to human PD-Li
or PD-L2 with a kdissoc of about 2 x 10-51/s or slower, binds to human PD-1
with a kaissoc of about 2.1
x 10-5 1/s or slower, binds to human PD-1 with a kaissoc of about 2.2 x 10'
1/s or slower, binds to
human PD-1 with a kdisso, of about 2.3 x 10' 1/s or slower, binds to human PD-
1 with a kdiõoc of about
2.4 x 10-5 1/s or slower, binds to human PD-1 with a kdissoc of about 2.5 ><
10 1/s or slower, binds to
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human PD-1 with a kaissoc of about 2.6>< 10-5 1/s or slower, binds to human PD-
Li or PD-L2 with a
kaisso, of about 2.7 x 10-5 1/s or slower, or binds to human PD-Li or PD-L2
with a kdiõoc of about 3
10-51/s or slower.
100149211n some embodiments, the PD-Li and/or PD-L2 inhibitor is one that
blocks or inhibits
binding of human PD-Li or human PD-L2 to human PD-1 with an ICso of about 10
nM or lower;
blocks or inhibits binding of human PD-L1 or human PD-L2 to human PD-1 with an
ICso of about 9
nM or lower; blocks or inhibits binding of human PD-Li or human PD-L2 to human
PD-1 with an
ICso of about ft nM or lower; blocks or inhibits binding of human PD-Li or
human PD-L2 to human
PD-1 with an ICso of about 7 nM or lower; blocks or inhibits binding of human
PD-L1 or human PD-
L2 to human PD-1 with an ICso of about 6 nM or lower; blocks or inhibits
binding of human PD-Li or
human PD-L2 to human PD-1 with an IC50 of about 5 nM or lower; blocks or
inhibits binding of
human PD-Li or human PD-L2 to human PD-1 with an ICso of about 4 nM or lower;
blocks or
inhibits binding of human PD-Ll or human PD-L2 to human PD-1 with an IC50 of
about 3 nM or
lower; blocks or inhibits binding of human PD-L1 or human PD-L2 to human PD-1
with an ICso of
about 2 nM or lower; or blocks human PD-1, or blocks binding of human PD-Li or
human PD-L2 to
human PD-1 with an 1050 of about 1 nM or lower.
10014931 In some embodiments, the PD-Li inhibitor is durvalumab, also known as
MEDI4736
(which is commercially available from Medimmune, LLC, Gaithersburg, Maryland,
a subsidiary of
AstraZeneca plc.), or antigen-binding fragments, conjugates, or variants
thereof. In some
embodiments, the PD-Li inhibitor is an antibody disclosed in U.S. Patent No.
8,779,108 or U.S.
Patent Application Publication No. 2013/0034559, the disclosures of which arc
incorporated by
reference herein. The clinical efficacy of durvalumab has been described in
Page, et at., Ann. Rev.
Med., 2014, 65, 185-202; Brahmer, et al., I C//n. Oncol. 2014, 32, 5s
(supplement, abstract 8021);
and McDermott, et at., Cancer Treatment Rev., 2014, 40, 1056-64. The
preparation and properties of
durvalumab are described in U.S. Patent No. 8,779,108, the disclosure of which
is incorporated by
reference herein. The amino acid sequences of durvalumab are set forth in
Table 20. The durvalumab
monoclonal antibody includes disulfide linkages at 22-96, 22"-96", 23-89',
23"1-89", 135'-195', 135"-
195", 148-204, 148-204", 215-224, 215-224", 230-230", 233-233", 265-325, 265-
325", 371-429,
and 371-429'; and N-glycosylation sites at Asn-301 and Asn-301".
100149411n some embodiments, a PD-Li inhibitor comprises a heavy chain given
by SEQ ID
NO: i78 and a light chain given by SEQ ID NO: i79. In some embodiments, a PD-
Li inhibitor
comprises heavy and light chains having the sequences shown in SEQ ID NO: i78
and SEQ ID
NO:179, respectively, or antigen binding fragments, Fab fragments, single-
chain variable fragments
(scFv), variants, or conjugates thereof. In some embodiments, a PD-Li
inhibitor comprises heavy and
light chains that are each at least 99% identical to the sequences shown in
SEQ ID NO: i78 and SEQ
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ID NO: 179, respectively. In some embodiments, a PD-Li inhibitor comprises
heavy and light chains
that are each at least 98% identical to the sequences shown in SEQ ID NO:178
and SEQ ID NO:179,
respectively. in some embodiments, a PD-Li inhibitor comprises heavy and light
chains that are each
at least 97% identical to the sequences shown in SEQ ID NO:178 and SEQ ID
NO:179, respectively.
In some embodiments, a PD-Li inhibitor comprises heavy and light chains that
are each at least 96%
identical to the sequences shown in SEQ Ill NO:178 and SEQ Ill NO: 179,
respectively. In some
embodiments, a PD-Li inhibitor comprises heavy and light chains that are each
at least 95% identical
to the sequences shown in SEQ ID NO:178 and SEQ ID NO: 179, respectively.
10014951 In some embodiments, the PD-L1 inhibitor comprises the heavy and
light chain CDRs or
variable regions (VRs) of durvalumab. In some embodiments, the PD-Li inhibitor
heavy chain
variable region (VL) comprises the sequence shown in SEQ ID NO:180, and the PD-
Li inhibitor light
chain variable region (VL) comprises the sequence shown in SEQ ID NO:181, or
conservative amino
acid substitutions thereof. in some embodiments, a PD-Li inhibitor comprises
VH and VL regions that
are each at least 99% identical to the sequences shown in SEQ ID NO: 180 and
SEQ ID NO:181,
respectively. In some embodiments, a PD-Li inhibitor comprises Vif and VL
regions that are each at
least 98% identical to the sequences shown in SEQ ID NO: 180 and SEQ ID
NO:181, respectively. In
some embodiments, a PD-Li inhibitor comprises Vii and VL regions that are each
at least 97%
identical to the sequences shown in SEQ ID NO:180 and SEQ ID NO: 181,
respectively. In some
embodiments, a PD-Li inhibitor comprises VH and VL regions that are each at
least 96% identical to
the sequences shown in SEQ ID NO; 180 and SEQ ID NO.181, respectively. In some
embodiments, a
PD-Li inhibitor comprises VH and VL regions that are each at least 95%
identical to the sequences
shown in SEQ ID NO: 180 and SEQ ID NO:181, respectively.
10014961 In some embodiments, a PD-Li inhibitor comprises heavy chain CDR1,
CDR2 and CDR3
domains having the sequences set forth in SEQ ID NO:182, SEQ ID NO: 183, and
SEQ ID NO: 184,
respectively, or conservative amino acid substitutions thereof, and light
chain CDR1, CDR2 and
CDR3 domains having the sequences set forth in SEQ ID NO:185, SEQ ID NO:186,
and SEQ ID
NO:187, respectively, or conservative amino acid substitutions thereof. In
some embodiments, the
antibody competes for binding with, and/or binds to the same epitope on PD-Li
as any of the
aforementioned antibodies.
1001497] In some embodiments, the PD-Li inhibitor is an anti-PD-Li biosimilar
monoclonal
antibody approved by drug regulatory authorities with reference to durvalumab.
In some
embodiments, the biosimilar comprises an anti-PD-Li antibody comprising an
amino acid sequence
which has at least 97% sequence identity, e.g., 97%, 98%, 99% or 100% sequence
identity, to the
amino acid sequence of a reference medicinal product or reference biological
product and which
comprises one or more post-translational modifications as compared to the
reference medicinal
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product or reference biological product, wherein the reference medicinal
product or reference
biological product is durvalumab. In some embodiments, the one or more post-
translational
modifications are selected from one or more of: gly-cosylation, oxidation,
deamidation, and truncation.
In some embodiments, the biosimilar is an anti-PD-L1 antibody authorized or
submitted for
authorization, wherein the anti-PD-L1 antibody is provided in a formulation
which differs from the
formulations of a reference medicinal product or reference biological product,
wherein the reference
medicinal product or reference biological product is durvalumab. The anti-PD-
Li antibody may be
authorized by a drug regulatory authority such as the U.S. FDA and/or the
European Union's EMA. In
some embodiments, the biosimilar is provided as a composition which further
comprises one or more
excipients, wherein the one or more excipients are the same or different to
the excipients comprised in
a reference medicinal product or reference biological product, wherein the
reference medicinal
product or reference biological product is durvalumab. In some embodiments,
the biosimilar is
provided as a composition which further comprises one or more excipients,
wherein the one or more
excipients are the same or different to the excipients comprised in a
reference medicinal product or
reference biological product, wherein the reference medicinal product or
reference biological product
is durvalumab.
TABLE 20. Amino acid sequences for PD-Ll inhibitors related to durvalumab.
Identifier Sequence (One-Letter Amino Acid Symbols)
SEQ 1D NO:170 EVQLVESGGG LVQPGGSLRL SCAASGFTFS RYWMSWVRQA PGKGLEWVAN
IKQDGSEKYY 60
durvalumab VDSVKGRFTI SRDNAKNSIY 1,QMNSLRAED TAVYYCAREG GWFGELAEDI
WGQGTIVI-VS 120
heavy chain SASTKGPSV.F PLAYSSKSTS GGAALGCLV Kl;YEPEPVTV SWNSGALTSG
VHTYPAVLQS 180
SGLYSLSSVV TVPSSSLGTQ TYICNVNHKP SNTKVDKRVE PKSCDKTHTC PPCPAPEFEG
240
GPSV8L822K PKOTLMISRT PEVTCVVVDV SHESPEVKFN WYVDGVEVNN AAVKPREEQY
300
NSTYRVVSVL TVLHQDWLNG KEYKCKVSNK ALPASIEKTI SKAKGQPRED QVYTLPPSRE
360
EMTKNWSLT CLVKGFYPSD IAVEWESNGQ PENNYKTTPP VLDSDGSFFL YSHLTVDKSR
420
WQQGNVFSCS VMHEALHNHY TQKSLSLSPG K
451
SEQ 1D NO:179 EVQLVESGGG LVQPGGSLRL SCAASGFTES RYWMSWVRQA PGKGLEWVAN
EIVLTQSPGT 60
durvalumab LSLSPGFRAT LSCRASQRVS SSYLAWYQQK PGQAPRLLIY DASSRATGIP
DRFSGSGSGT 120
light chain DFTLTISRLE PEDFAVYYCQ QYGSLPWTFG QGTKVEIKRT VAAPSVTIFP
PSDEQLKSGT 180
ASVVCLLNNF YPREAKVQWK VDNALQSGNS QESVTEQDEK DSTYSLSSTL TLSKADYEKH
240
KVYACDVTNQ GISSPVTASE NRGEC
265
SEQ ID NO:180 EVQLVESGGG LVQPGGSLRL SCAASGFTYS RYWMSWVRQA PGKGLEWVAN
IHQDGSEKYY 60
durvalumab VDSVKGRETI SRDNAKNSLY LQMNSLRAED TAVYYCAREG GWFGELAFEY
WGQGTL-VDVS 120
variable 5
12:
heavy chain
SEQ ID NO: 1811 EIVLTOSPGT LSLSPGERAT LSCRASQ)RVS SSYLANYOOK PGOAPRLLIY
DASSRATGIP 60
durvalumab DRFSGSGSGT DFTLTISRLE PEDFAVYYCQ QYGSL2WTEG QGTHVEIK
108
variable
light chain
SEQ _D NO:182 HYWMS
5
durvalumab
heavy chain
CDR1
SEQ ID NO:183 NIKCDGSEKY YVDSVKG
17
durvalumab
heavy chain
CDR2
SEQ 1D NO:184 EGGWFGELAF DY
12
durvalumab
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Identifier Sequence (One-Letter Amino Acid Symbols)
heavy chain
C2R3
SEQ =D NO:185 RASQRVSSSY LA
12
durvalumab
Light chain
CDR1
SEQ =D NO:186 DASSRAT
durvalumab
Light chain
CDR2
SEQ =D NO:187 QQYGSLPWT
9
durvalumab
Light chain
CDR3
100149811n some embodiments, the PD-Li inhibitor is avelumab, also known as
MSB0010718C
(commercially available from Merck KGaA/EMD Serono), or antigen-binding
fragments, conjugates,
or variants thereof The preparation and properties of avelumab are described
in U.S. Patent
Application Publication No. US 2014/0341917 Al, the disclosure of which is
specifically
incorporated by reference herein. The amino acid sequences of avelumab are set
forth in Table 21.
Avelumab has intra-heavy chain disulfide linkages (C23-C104) at 22-96, 147-
203, 264-324, 370-428,
22"-96", 147"-203", 264"-324", and 370%428"; intra-light chain disulfide
linkages (C23-C104) at 22'-
90', 138-197', 22-90'", and 138"-197"; intra-heavy-light chain disulfide
linkages (h 5-CL 126) at
223-215' and 223-215"; intra-heavy-heavy chain disulfide linkages (h 11, h 14)
at 229-229" and 232-
232'; N-glycosylation sites (H CH2 N84.4) at 300, 300"; fucosylated complex bi-
antennary CHO-type
glycans; and H CHS K2 C-terminal lysinc clipping at 450 and 450'.
[001499] In some embodiments, a PD-L1 inhibitor comprises a heavy chain given
by SEQ ID
NO:188 and a light chain given by SEQ ID NO: i89. In some embodiments, a PD-L1
inhibitor
comprises heavy and light chains having the sequences shown in SEQ ID NO:188
and SEQ ID
NO:189, respectively, or antigen binding fragments, Fab fragments, single-
chain variable fragments
(scFv), variants, or conjugates thereof. In some embodiments, a PD-Li
inhibitor comprises heavy and
light chains that are each at least 99% identical to the sequences shown in
SEQ ID NO:188 and SEQ
ID NO: 189, respectively. In some embodiments, a PD-Ll inhibitor comprises
heavy and light chains
that are each at least 98% identical to the sequences shown in SEQ ID NO:188
and SEQ ID NO: i89,
respectively. In some embodiments, a PD-Li inhibitor comprises heavy and light
chains that are each
at least 97% identical to the sequences shown in SEQ ID NO:188 and SEQ ID
NO:189, respectively.
In some embodiments, a PD-Li inhibitor comprises heavy and light chains that
are each at least 96%
identical to the sequences shown in SEQ ID NO:188 and SEQ ID NO: 189,
respectively. In some
embodiments, a PD-Li inhibitor comprises heavy and light chains that are each
at least 95% identical
to the sequences shown in SEQ ID NO:188 and SEQ ID NO: 189, respectively.
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10015001in some embodiments, the PD-Li inhibitor comprises the heavy and light
chain CDRs or
variable regions (VRs) of avelumab. In some embodiments, the PD-Li inhibitor
heavy chain variable
region (VH) comprises the sequence shown in SEQ ID NO: 190, and the PD-Li
inhibitor light chain
variable region (VL) comprises the sequence shown in SEQ ID NO:191, or
conservative amino acid
substitutions thereof In some embodiments, a PD-L1 inhibitor comprises VH and
VL regions that are
each at least 99% identical to the sequences shown in SEQ ID NO: i90 and SEQ
ID NO:191,
respectively. In some embodiments, a PD-Li inhibitor comprises VH and VL
regions that are each at
least 98% identical to the sequences shown in SEQ ID NO: 190 and SEQ ID NO:
191, respectively. In
some embodiments, a PD-L1 inhibitor comprises VH and VI, regions that arc each
at least 97%
identical to the sequences shown in SEQ ID NO: 190 and SEQ ID NO: 191,
respectively. In some
embodiments, a PD-Li inhibitor comprises VH and VL regions that are each at
least 96% identical to
the sequences shown in SEQ ID NO: 190 and SEQ ID NO:191, respectively. In some
embodiments, a
PD-Li inhibitor comprises VH and VL regions that are each at least 95%
identical to the sequences
shown in SEQ ID NO: 190 and SEQ ID NO:191, respectively.
[0015011in some embodiments, a PD-Li inhibitor comprises heavy chain CDR1,
CDR2 and CDR3
domains having the sequences set forth in SEQ ID NO:192, SEQ ID NO:193, and
SEQ ID NO:194,
respectively, or conservative amino acid substitutions thereof, and light
chain CDRI, CDR2 and
CDR3 domains having the sequences set forth in SEQ ID NO:195, SEQ ID NO: i96,
and SEQ ID
NO:197, respectively, or conservative amino acid substitutions thereof. In
some embodiments, the
antibody competes for binding with, and/or binds to the same epitope on PD-Li
as any of the
aforementioned antibodies.
[0015021M some embodiments, the PD-Li inhibitor is an anti-PD-Li biosimilar
monoclonal
antibody approved by drug regulatory authorities with reference to avelumab.
In some embodiments,
the biosimilar comprises an anti-PD-LI antibody comprising an amino acid
sequence which has at
least 97% sequence identity, e.g., 97%, 98%, 99% or 100% sequence identity, to
the amino acid
sequence of a reference medicinal product or reference biological product and
which comprises one or
more post-translational modifications as compared to the reference medicinal
product or reference
biological product, wherein the reference medicinal product or reference
biological product is
avelumab. In some embodiments, the one or more post-translational
modifications are selected from
one or more of: glycosylation, oxidation, deamidation, and truncation. In some
embodiments, the
biosimilar is an anti-PD-Li antibody authorized or submitted for
authorization, wherein the anti-PD-
Li antibody is provided in a formulation which differs from the formulations
of a reference medicinal
product or reference biological product, wherein the reference medicinal
product or reference
biological product is avelumab. The anti-PD-Li antibody may be authorized by a
drug regulatory
authority such as the U.S. FDA and/or the European Union's EMA. In some
embodiments, the
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biosimilar is provided as a composition which further comprises one or more
excipients, wherein the
one or more excipients are the same or different to the excipients comprised
in a reference medicinal
product or reference biological product, wherein the reference medicinal
product or reference
biological product is avelumab. In some embodiments, the biosimilar is
provided as a composition
which further comprises one or more excipients, wherein the one or more
excipients are the same or
different to the excipicnts comprised in a reference medicinal product or
reference biological product,
wherein the reference medicinal product or reference biological product is
avelumab.
TABLE 21. Amino acid sequences for PD-Li inhibitors related to avelumab.
Identifier Sequence (One-Letter Amino Acid Symbols)
SEQ ED NO:108 EVQLLESGGG LVQPGGSLRL SCAASGFTFS SYIMMWVRQA PGXGLEWVSS
IYPSGGITEY 60
avelumab ADTVKGRETI SRDNSKNTLY LQNNSLRAED TAVYYCARIK LGTVTTVDYW
GQGTLVTVSS 120
heavy chain ASTKGPSVFP LAPSSKSTSG GTAALGCLVK DYFPEPVTVS WNSGALTSGV
HTFPAVLQSS 180
GLYSLSSVVT V2SSSLG1QT YICNVNNKPS NTKVDKKVEP ASCDKTHTCP 2CPAPELLGG
240
PSVELFP2KP KDTLMISRTP EVTCVVVDVS HEDPEVKFNW YVDGVEVHNA KTHPREEQYN
300
STYRVVSVLT VLHQDWLNGK EYKCKVSNKA LPAPIEKTIS KAKGQPREPQ VYTLPPSRDE
360
LTNNQVSLTC LVHGFYPSDI AVEWESNGQP ENNYKTTPPV LDSDGSFFLY SKLTVDKSRW
420
QQGNVFSCSV MHEALHNHYT QHSLSLSPGK
450
SEQ ID NO:189 QSALTQPASV SGSPGQSITI SCTGTSSDVG GYNYVSWYQQ 14PGKAPKLMI
YDVSNRPSGV 60
avelumab SNRFSGSKSG NTASLTISGL QAEDEADYYC SSYTSSSTRV FGTGTKVTVL
GQPKANPTVT 120
light chain LFPPSSEELQ ANKATLVCLI SDFYPGAVTV AWRADCSPV7 AGVETTKPSR
QSNNRYAASS 180
YLSLTPEQWK SHRSYSCQVT HEGSTVERTV APTECS
216
SEQ ED NO:190 EVQLLESGGG LVQPGGSLRL SCAASGFTFS SYINMWVRQA PGKGLFWVSS
IYPSGGITFY 60
avelumab ADTVKGRYTI SRDNSKNTLY LQMNSLRAED TAVYYCARIK LGTVTTVDYW
GQGTLVTVSS 120
variable
heavy chain
SEQ ED NO:191 QSALTQPASV SGSPGQSITI SCTGTSSDVG GYNYVSWYQQ NPGKAPKLMI
YDVSNRPSGV 60
avelumab SNRFSGSKSG NTASLTISGL QAEDEADYYC SSYTSSSTRV FGTGTKVTVL
110
variable
light chain
SEQ ED NO:192 SYINM
avelumab
heavy chain
CDR1
SEQ ED NO:193 SIYPSGGITF YADTVKG
17
avelumab
heavy chain
CDR2
SEQ ED NO:194 IKLGTVTTVD Y
11
avelumab
heavy chain
CDR3
SEQ ED NO:195 TGTSSDVGGY NYVS
14
avelumab
light chain
CDR1
SEQ ID NO:196 DVSNRPS
7
avelumab
light chain
CDR2
SEQ ED NO:197 SSYTSSSTRV
10
avelumab
light chain
CDP3
10015031in some embodiments, the PD-Li inhibitor is atezolizumab, also known
as MPDL32g0A or
RG7446 (commercially available as TECENTRIQ from Genentech, Inc., a subsidiary
of Roche
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Holding AG, Basel, Switzerland), or antigen-binding fragments, conjugates, or
variants thereof. In
some embodiments, the PD-Li inhibitor is an antibody disclosed in U.S. Patent
No. 8,217,149, the
disclosure of which is specifically incorporated by reference herein. In some
embodiments, the PD-Li
inhibitor is an antibody disclosed in U.S. Patent Application Publication Nos.
2010/0203056 Al,
2013/0045200 Al, 2013/0045201 Al, 2013/0045202 Al, or 2014/0065135 Al, the
disclosures of
which are specifically incorporated by reference herein. The preparation and
properties of
atezolizumab are described in U.S. Patent No. 8,217,149, the disclosure of
which is incorporated by
reference herein. The amino acid sequences of atezolizumab are set forth in
Table 22. Atezolizumab
has intra-heavy chain disulfide linkages (C23-C104) at 22-96, 145-201, 262-
322, 368-426, 22-96",
145'-201", 262"-322", and 368-426"; intra-light chain disulfide linkages (C23-
C104) at 23'-88', 134'-
194', 23-88", and 134'194'; intra-heavy-light chain disulfide linkages (h 5-CL
126) at 221-214'
and 221-214'; intra-heavy-heavy chain disulfide linkages (h 11, h 14) at 227-
227" and 230-230"; and
N-glycosylation sites (H CH2N84.4>A) at 298 and 298'.
100150411n some embodiments, a PD-L1 inhibitor comprises a heavy chain given
by SEQ ID
NO:198 and a light chain given by SEQ ID NO: 199. In some embodiments, a PD-L1
inhibitor
comprises heavy and light chains having the sequences shown in SEQ ID NO:198
and SEQ ID
NO:199, respectively, or antigen binding fragments, Fab fragments, single-
chain variable fragments
(scFv), variants, or conjugates thereof. In some embodiments, a PD-L1
inhibitor comprises heavy and
light chains that are each at least 99% identical to the sequences shown in
SEQ ID NO:198 and SEQ
ID NO; 199, respectively. In some embodiments, a PD-Ll inhibitor comprises
heavy and light chains
that are each at least 98% identical to the sequences shown in SEQ ID NO:198
and SEQ ID NO:199,
respectively. In some embodiments, a PD-Li inhibitor comprises heavy and light
chains that are each
at least 97% identical to the sequences shown in SEQ ID NO:198 and SEQ ID
NO:199, respectively.
In some embodiments, a PD-L1 inhibitor comprises heavy and light chains that
are each at least 96%
identical to the sequences shown in SEQ ID NO:198 and SEQ ID NO: 199,
respectively. In some
embodiments, a PD-L1 inhibitor comprises heavy and light chains that are each
at least 95% identical
to the sequences shown in SEQ ID NO:198 and SEQ ID NO; 199, respectively.
10015051in some embodiments, the PD-L1 inhibitor comprises the heavy and light
chain CDRs or
variable regions (VRs) of atezolizumab. In some embodiments, the PD-L1
inhibitor heavy chain
variable region (Vu) comprises the sequence shown in SEQ ID NO:200, and the PD-
Li inhibitor light
chain variable region (VL) comprises the sequence shown in SEQ ID NO:201, or
conservative amino
acid substitutions thereof In some embodiments, a PD-Li inhibitor comprises
VII and VL regions that
arc each at least 99% identical to the sequences shown in SEQ ID NO:200 and
SEQ ID NO:201,
respectively. In some embodiments, a PD-Li inhibitor comprises VH and VL
regions that are each at
least 98% identical to the sequences shown in SEQ ID NO:200 and SEQ ID NO:201,
respectively. In
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some embodiments, a PD-Li inhibitor comprises VH and VL regions that are each
at least 97%
identical to the sequences shown in SEQ ID NO:200 and SEQ ID NO:201,
respectively. In some
embodiments, a PD-Li inhibitor comprises VH and VL regions that are each at
least 96% identical to
the sequences shown in SEQ ID NO:200 and SEQ ID NO:201, respectively. In some
embodiments, a
PD-Li inhibitor comprises VH and VL regions that are each at least 95%
identical to the sequences
shown in SEQ Ill NO:200 and SEQ Ill NO:201, respectively.
10015061In some embodiments, a PD-Li inhibitor comprises heavy chain CDR1,
CDR2 and CDR3
domains having the sequences set forth in SEQ ID NO:202, SEQ ID NO:203, and
SEQ ID NO:204,
respectively, or conservative amino acid substitutions thereof, and light
chain CDRI, CDR2 and
CDR3 domains having the sequences set forth in SEQ ID NO:205, SEQ ID NO:206,
and SEQ ID
NO:207, respectively, or conservative amino acid substitutions thereof. In
some embodiments, the
antibody competes for binding with, and/or binds to the same epitope on PD-Li
as any of the
aforementioned antibodies.
1001507] In some embodiments, the anti-PD-L1 antibody is an anti-PD-L I
biosimilar monoclonal
antibody approved by drug regulatory authorities with reference to
atezolizumab. In some
embodiments, the biosimilar comprises an anti-PD-Li antibody comprising an
amino acid sequence
which has at least 97% sequence identity, e.g., 97%, 98%, 99% or 100% sequence
identity, to the
amino acid sequence of a reference medicinal product or reference biological
product and which
comprises one or more post-translational modifications as compared to the
reference medicinal
product or reference biological product, wherein the reference medicinal
product or reference
biological product is atezolizumab. In some embodiments, the one or more post-
translational
modifications are selected from one or more of: glycosylation, oxidation,
deamidation, and truncation.
In some embodiments, the biosimilar is an anti-PD-Li antibody authorized or
submitted for
authorization, wherein the anti-PD-LI antibody is provided in a formulation
which differs from the
formulations of a reference medicinal product or reference biological product,
wherein the reference
medicinal product or reference biological product is atezolizumab. The anti-PD-
Li antibody may be
authorized by a drug regulatory authority such as the U.S. FDA and/or the
European Union's EMA. In
some embodiments, the biosimilar is provided as a composition which further
comprises one or more
excipients, wherein the one or more excipients are the same or different to
the excipients comprised in
a reference medicinal product or reference biological product, wherein the
reference medicinal
product or reference biological product is atezolizumab. In some embodiments,
the biosimilar is
provided as a composition which further comprises one or more excipients,
wherein the one or more
excipients arc the same or different to the cxcipicnts comprised in a
reference medicinal product or
reference biological product, wherein the reference medicinal product or
reference biological product
is atezolizumab.
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TABLE 22. Amino acid sequences for PD-Li inhibitors related to atezolizumab.
Identifier Sequence (One-Letter Amino Acid Symbols)
SEQ _D 50:198 EVQLVESGGG LVQ2GGSLRL SCAASGFS DSWIHWVRQA PGKGLEWVAW
ISPYGGSTYY 60
aLezolizumab ADSVKGRETI SADTSKNTAY LQNNSLRAED TAVYYCARRH WPGGFDYWGQ
GTLVTVSSAS 120
heavy chain TKGPSVFPLA PSSKSTSGGT AALGCLVXDY FPEPVTVSWN SGALTSGVHT
FPAVLQSSGL 180
YSLSSVVTVP SSSLGTQTYI CNVNHKPSNT KVDKKVEPKS CDKTHTCPPC PAPELLGGPS
240
VFLEPPK2KD TLMISRTPEV TCVVVDVSHE DPEVKFNWYV DGVEVIINAKT KPREEQYAST
300
YRVVSVLTVL HQDWLNGKEY KCKVSNKALP APIEKTISKA KGQPREPQVY TLPPSREEMT
360
KNQVSLTCLV KGFYPSDIAV EWESNGQPEN NYKTTPPVLD SDGSFFLYSK LTVDXSRWQQ
420
GNVFSCSVMH EALHNHYTQK SLSLSPGH
448
SEQ =D 50:199 DIQMTQSPSS LSASVGDRVT ITCRASQDVS TAVAWYQQKP GKAPHLLIYS
ASFLYSGVPS 60
aLezolizumab RFSGSGSGTD F=TISSLQP EDFATYYCQQ YLYHPATFGQ GTKVEIKRTV
AAPSVFIFFP 120
light chain SDEQLKSGTA SVVCLLNNFY PREAr{VQWKV DNALQSGNSQ ESVTEQDSKD
STYSLSSTLT 180
LSKADYEKHK VYACEVTHQG LSSPVTKSFN RGEC
214
SEQ ID 50:200 EVQLVESGGG LVQPGGSLRL SCAASGFTFS DSWIHWVRQA PGNGLEWVAW
ISPYGGSTYY 60
atezolizumab ADSVKGRYTI SADTSKNTAY LQNNSLRAED TAVYYCARRH WPGGFDYWGQ
GTLVTVSA 118
variable
heavy chain
SEQ =D 50:201 DIQMTQS2SS LSASVGDRVT ITCRASQDVS TAVAWYQQKP GKAPKLLIYS
ASFLYSGVPS 60
atezolizumab RFSGSGSGTD F=TISSLQP EDFATYYCQQ YLYHPATFGQ GTKVEIKR
108
variable
light chain
SEQ TD 50:202 GFTFSDSWIH
10
atezolizumab
heavy chain
0051
SEQ ID 50:203 AWISPYGGST YYADSVKG
10
atezolizumab
heavy chain
0652
SEQ =D 50:204 RHWPGGFDY
9
atezolizumab
heavy chain
0053
SEQ TD NO:205 RASQDVSTAV A
11
atezolizumab
light chain
CDR1
SEQ =D 50:206 aASFLYS
7
atezolizumab
light chain
0052
SEQ =D NO:207 QQYLYHPAT
9
atezolizumab
light chain
0053
[001508] In some embodiments, PD-Li inhibitors include those antibodies
described in U.S. Patent
Application Publication No. US 2014/0341917 Al, the disclosure of which is
incorporated by
reference herein. In some embodiments, antibodies that compete with any of
these antibodies for
binding to PD-Li arc also included. In some embodiments, the anti-PD-Li
antibody is MDX-1105,
also known as BMS-935559, which is disclosed in U.S. Patent No. US 7,943,743,
the disclosures of
which are incorporated by reference herein. In some embodiments, the anti-PD-
Li antibody is
selected from the anti-PD-Li antibodies disclosed in U.S. Patent No. US
7,943,743, which are
incorporated by reference herein.
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[0015091M some embodiments, the PD-Li inhibitor is a commercially-available
monoclonal
antibody, such as INVIVOMAB anti-m-PD-L1 clone 10F.9G2 (Catalog # BE0101, Bio
X Cell, Inc.,
West Lebanon, NH, USA). In some embodiments, the anti-PD-Li antibody is a
commercially-
available monoclonal antibody, such as AFFYMETRIX EBIOSCIENCE (MIH1). A number
of
commercially-available anti-PD-L1 antibodies are known to one of ordinary
skill in the art.
10015101ln some embodiments, the PD-L2 inhibitor is a commercially-available
monoclonal
antibody, such as BIOLEGEND 24F. 10C12 Mouse IgG2a, i isotype (catalog #
329602 Biolegend,
Inc., San Diego, CA), SIGMA anti-PD-L2 antibody (catalog # SAB3500395, Sigma-
Aldrich Co., St.
Louis, MO), or other commercially-available anti-PD-L2 antibodies known to one
of ordinary skill in
the art.
4. Combinations with CTLA-4 Inhibitors
10015111ln some embodiments, TILs and a CTLA-4 inhibitor are administered as a
combination
therapy or co-therapy for the treatment of NSCLC.
10015121In some embodiments, the NSCLC has undergone no prior therapy. In some
embodiments,
the CTLA-4 inhibitor is administered as a front-line therapy or initial
therapy. In some embodiments,
the CTLA-4 inhibitor is administered as a front-line therapy or initial
therapy in combination with the
TILs as described herein.
10015131 Cytotoxic T lymphocyte antigen 4 (CTLA-4) is a member of the
immunoglobulin
superfamily and is expressed on the surface of helper T cells. CTLA-4 is a
negative regulator of
CD28-dependent T cell activation and acts as a checkpoint for adaptive immune
responses. Similar to
the T cell costimulatory protein CD28, the CTLA-4 binding antigen presents
CD80 and CD86 on the
cells. CTLA-4 delivers a suppressor signal to T cells, while CD28 delivers a
stimulus signal. Human
antibodies against human CTLA-4 have been described as immunostimulatory
modulators in many
disease conditions, such as treating or preventing viral and bacterial
infections and for treating cancer
(WO 01/14424 and WO 00/37504). Various preclinical studies have shown that
CTLA-4 blockade by
CTLA-4 inhibitors such as monoclonal antibodies enhances host immune responses
against
immunogenic tumors and can even rule out established tumors. A number of fully
human anti-human
CTLA-4 monoclonal antibodies (mAbs) have been studied in clinical trials for
the treatment of
various types of solid tumors, including, but limited to, ipilimumab (MDX-010)
and tremelimumab
(CP-675,206).
10015141In some embodiments, a CTLA-4 inhibitor may be any CTLA-4 inhibitor or
CTLA-4
blocker known in the art. In particular, it is one of the CTLA-4 inhibitors or
blockers described in
more detail in the following paragraphs. The terms "inhibitor," "antagonist,"
and "blocker" are used
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interchangeably herein in reference to CTLA-4 inhibitors. For avoidance of
doubt, references herein
to a CTLA-4 inhibitor that is an antibody may refer to a compound or antigen-
binding fragments,
variants, conjugates, or biosimilars thereof. For avoidance of doubt,
references herein to a CTLA-4
inhibitor may also refer to a small molecule compound or a pharmaceutically
acceptable salt, ester,
solvate, hydrate, cocrystal, or prodrug thereof.
10015151 Suitable CTLA-4 inhibitors for use in the methods of the invention,
include, without
limitation, anti-CTLA-4 antibodies, human anti-CTLA-4 antibodies, mouse anti-
CTLA-4 antibodies,
mammalian anti-CTLA-4 antibodies, humanized anti-CT1A-4 antibodies, monoclonal
anti-CTLA-4
antibodies, polyclonal anti-CTLA-4 antibodies, chimeric anti-CTLA-4
antibodies, MDX-010
(ipilimumab), tremelimumab, anti-CD28 antibodies, anti-CTLA-4 adnectins, anti-
CTLA-4 domain
antibodies, single chain anti-CTLA-4 fragments, heavy chain anti-CTLA-4
fragments, light chain
anti-CTLA-4 fragments, inhibitors of CTLA-4 that agonize the co-stimulatory
pathway, the antibodies
disclosed in PCT Publication No. WO 2001/014424, the antibodies disclosed in
PCT Publication No.
WO 2004/035607, the antibodies disclosed in U.S. Publication No. 2005/0201994,
and the antibodies
disclosed in granted European Patent No. EP 1212422 Bl, the disclosures of
each of which are
incorporated herein by reference. Additional CTLA-4 antibodies are described
in U.S. Pat. Nos.
5,811,097, 5,855,887, 6,051,227, and 6,984,720; in PCT Publication Nos. WO
01/14424 and WO
00/37504; and in U.S. Publication Nos. 2002/0039581 and 2002/086014, the
disclosures of each of
which are incorporated herein by reference. Other anti-CTLA-4 antibodies that
can be used in a
method of the present invention include, for example, those disclosed in: WO
98/42752; U.S. Pat.
Nos. 6,682,736 and 6,207,156: Hurwitz et al., Proc. Natl. Acad. Sci. USA,
95(17):10067-10071
(1998); Camacho et al., J. Clin. Oncology, 22(145): Abstract No. 2505 (2004)
(antibody CP-675206);
Mokyr et al., Cancer Res., 58:5301-5304 (1998), and U.S. Pat. Nos. 5,977,318,
6,682,736, 7,109,003,
and 7,132,281, the disclosures of each of which are incorporated herein by
reference the non-
myeloablative lymphodepletion regimen.
10015161 Additional CTLA-4 inhibitors include, but are not limited to, the
following: any inhibitor
that is capable of disrupting the ability of CD28 antigen to bind to its
cognate ligand, to inhibit the
ability of CTLA-4 to bind to its cognate ligand, to augment T cell responses
via the co-stimulatory
pathway, to disrupt the ability of B7 to bind to CD28 and/or CTLA-4, to
disrupt the ability of B7 to
activate the co-stimulatory pathway, to disrupt the ability of CD80 to bind to
CD28 and/or CTLA-4,
to disrupt the ability of CD80 to activate the co-stimulatory pathway, to
disrupt the ability of CD86 to
bind to CD28 and/or CTLA-4, to disrupt the ability of CD86 to activate the co-
stimulatory pathway,
and to disrupt the co-stimulatory pathway, in general from being activated.
This necessarily includes
small molecule inhibitors of CD28, CD80, CD86, CTLA-4, among other members of
the co-
stimulatory pathway; antibodies directed to CD28, CD80, CD86, CTLA-4, among
other members of
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the co-stimulatory pathway; antisense molecules directed against CD28, CD80,
CD86, CTLA-4,
among other members of the co-stimulatory pathway; adnectins directed against
CD28, CD80, CD86,
CTLA-4, among other members of the co-stimulatory pathway, RNAi inhibitors
(both single and
double stranded) of CD28, CD80, CD86, CTLA-4, among other members of the co-
stimulatory
pathway, among other CTLA-4 inhibitors.
10015171In some embodiments a CTLA-4 inhibitor binds to CTLA-4 with a Kd of
about 10' M or
less, 10 7M or less, 10 M or less, 10 9 M or less, 10 M or less, 10 11M or
less, 10 12 M or less,
e.g., between 10'3 M and 10-16 M, or within any range having any two of the
afore-mentioned values
as endpoints. In some embodiments a CTLA-4 inhibitor binds to CTLA-4 with a Kd
of no more than
10-fold that of ipilimumab, when compared using the same assay. In some
embodiments a CTLA-4
inhibitor binds to CTLA-4 with a Kd of about the same as, or less (e.g., up to
10-fold lower, or up to
100-fold lower) than that of ipilimumab, when compared using the same assay.
In some embodiments,
the IC50 values for inhibition by a CTLA-4 inhibitor of CTLA-4 binding to CD80
or CD86 is no
more than 10-fold greater than that of ipilimumab-mediated inhibition of CTLA-
4 binding to CD80 or
CD86, respectively, when compared using the same assay. In some embodiments,
the IC50 values for
inhibition by a CTLA-4 inhibitor of CTLA-4 binding to CD80 or CD86 is about
the same or less (e.g.,
up to 10-fold lower, or up to 100-fold lower) than that of ipilimumab-mediated
inhibition of CTLA-4
binding to CD80 or CD86, respectively, when compared using the same assay.
10015181ln some embodiments a CTLA-4 inhibitor is used in an amount sufficient
to inhibit
expression and/or decrease biological activity of CTLA-4 by at least 20%, 30%,
40%, 50%, 60%,
70%, 80%, 90%, 95%, or 100% relative to a suitable control, e.g., between 50%
and 75%, 75% and
90%, or 90% and 100%. In some embodiments a CTLA-4 pathway inhibitor is used
in an amount
sufficient to decrease the biological activity of CTLA-4 by reducing binding
of CTLA-4 to CD80,
CD86, or both by at least 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 95%, or 100%
relative to a
suitable control, e.g., between 50% and 75%, 75% and 90%, or 90% and 100%
relative to a suitable
control. A suitable control in the context of assessing or quantifying the
effect of an agent of interest
is typically a comparable biological system (e.g., cells or a subject) that
has not been exposed to or
treated with the agent of interest, e.g., CTLA-4 pathway inhibitor (or has
been exposed to or treated
with a negligible amount). In some embodiments a biological system may serve
as its own control
(e.g., the biological system may be assessed before exposure to or treatment
with the agent and
compared with the state after exposure or treatment has started or finished.
In some embodiments a
historical control may be used.
10015191 In some embodiments, the CTLA-4 inhibitor is ipilimumab (commercially
available as
Yervoy from Bristol-Myers Squibb Co.), or biosimilars, antigen-binding
fragments, conjugates, or
variants thereof. As is known in the art, ipilimumab refers to an anti-CTLA-4
antibody, a fully human
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IgG 1 antibody derived from a transgenic mouse with human genes encoding heavy
and light chains
to generate a functional human repertoire. is there. Ipilimumab can also be
referred to by its CAS
Registry Number 477202-00-9, and in PCT Publication Number WO 01/14424, which
is incorporated
herein by reference in its entirety for all purposes. It is disclosed as
antibody 10DI. Specifically,
ipilimumab contains a light chain variable region and a heavy chain variable
region (having a light
chain variable region comprising SEQ Ill NO: 516 and having a heavy chain
variable region
comprising SEQ ID NO: 515). Represents a human monoclonal antibody or its
antigen binding site
that specifically binds to CTLA-4. A pharmaceutical composition of ipilimumab
includes all
pharmaceutically acceptable compositions containing ipilimumab and one or more
diluents, vehicles
and / or excipients. An example of a pharmaceutical composition containing
ipilimumab is described
in PCT Publication No. WO 2007/67959. Impilimumab can be administered
intravenously (IV).
10015201 In some embodiments, a CTLA-4 inhibitor comprises a heavy chain given
by SEQ ID
NO:208 and a light chain given by SEQ ID NO:209. In some embodiments, a CTLA-4
inhibitor
comprises heavy and light chains having the sequences shown in SEQ ID NO:208
and SEQ ID
NO:209, respectively, or antigen binding fragments, Fab fragments, single-
chain variable fragments
(scFv), variants, or conjugates thereof. In some embodiments, a CTLA-4
inhibitor comprises heavy
and light chains that are each at least 99% identical to the sequences shown
in SEQ ID NO:208 and
SEQ ID NO:209, respectively. In some embodiments, a CTLA-4 inhibitor comprises
heavy and light
chains that are each at least 98% identical to the sequences shown in SEQ ID
NO:208 and SEQ ID
NO:209, respectively. To some embodiments, a CTLA-4 inhibitor comprises heavy
and light chains
that are each at least 97% identical to the sequences shown in SEQ ID NO:208
and SEQ ID NO:209,
respectively. In some embodiments, a CTLA-4 inhibitor comprises heavy and
light chains that are
each at least 96% identical to the sequences shown in SEQ ID NO:208 and SEQ ID
NO:209.
respectively. In some embodiments, a CTLA-4 inhibitor comprises heavy and
light chains that are
each at least 95% identical to the sequences shown in SEQ ID NO:208 and SEQ ID
NO:209,
respectively.
10015211 In some embodiments, the CTLA-4 inhibitor comprises the heavy and
light chain CDRs or
variable regions (VRs) of ipilimumab. In some embodiments, the CTLA-4
inhibitor heavy chain
variable region (VH) comprises the sequence shown in SEQ ID NO:210, and the
CTLA-4 inhibitor
light chain variable region (VI) comprises the sequence shown in SEQ ID
NO:211, or conservative
amino acid substitutions thereof. In some embodiments, a CTLA-4 inhibitor
comprises VH and VL
regions that are each at least 99% identical to the sequences shown in SEQ ID
NO:210 and SEQ ID
NO:211, respectively. In some embodiments, a CTLA-4 inhibitor comprises VH and
VL regions that
are each at least 98% identical to the sequences shown in SEQ ID NO:210 and
SEQ ID NO:211,
respectively. In some embodiments, a CTLA-4 inhibitor comprises VH and VL
regions that are each at
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least 97% identical to the sequences shown in SEQ ID NO:210 and SEQ ID NO:211,
respectively. In
some embodiments, a CTLA-4 inhibitor comprises VH and VL regions that are each
at least 96%
identical to the sequences shown in SEQ ID NO:210 and SEQ ID NO:211,
respectively. In some
embodiments, a CTLA-4 inhibitor comprises VH and VL regions that are each at
least 95% identical to
the sequences shown in SEQ ID NO:210 and SEQ ID NO:211, respectively.
10015221IIn some embodiments, a CTLA-4 inhibitor comprises the heavy chain
CDR1, CDR2 and
CDR3 domains having the sequences set forth in SEQ ID NO:212, SEQ ID NO:213,
and SEQ ID
NO:214, respectively, or conservative amino acid substitutions thereof, and
light chain CDR1, CDR2
and CDR3 domains having the sequences set forth in SEQ ID NO:215, SEQ ID
NO:216, and SEQ ID
NO:217, respectively, or conservative amino acid substitutions thereof. In
some embodiments, the
antibody competes for binding with, and/or binds to the same epitope on CTLA-4
as any of the
aforementioned antibodies.
10015231 In some embodiments, the CTLA-4 inhibitor is a CTLA-4 biosimilar
monoclonal antibody
approved by drug regulatory authorities with reference to ipilimumab. In some
embodiments, the
biosimilar comprises an anti-CTLA-4 antibody comprising an amino acid sequence
which has at least
97% sequence identity, e.g., 97%, 98%, 99% or 100% sequence identity, to the
amino acid sequence
of a reference medicinal product or reference biological product and which
comprises one or more
post-translational modifications as compared to the reference in product or
reference
biological product, wherein the reference medicinal product or reference
biological product is
ipilimumab. In some embodiments, the one or more post-translational
modifications are selected from
one or more of: glycosylation, oxidation, dcamidation, and truncation. The
amino acid sequences of
ipilimumab are set forth in Table 23. In some embodiments, the biosimilar is
an anti-CTLA-4
antibody authorized or submitted for authorization, wherein the anti-CTLA-4
antibody is provided in
a formulation which differs from the formulations of a reference medicinal
product or reference
biological product, wherein the reference medicinal product or reference
biological product is
ipilimumab. The anti-CTLA-4 antibody may be authorized by a drug regulatory
authority such as the
U.S. FDA and/or the European Union's EMA. In some embodiments, the biosimilar
is provided as a
composition which further comprises one or more excipients, wherein the one or
more excipients are
the same or different to the excipients comprised in a reference medicinal
product or reference
biological product, wherein the reference medicinal product or reference
biological product is
ipilimumab. In some embodiments, the biosimilar is provided as a composition
which further
comprises one or more excipients, wherein the one or more excipients are the
same or different to the
excipients comprised in a reference medicinal product or reference biological
product, wherein the
reference medicinal product or reference biological product is ipilimumab.
TABLE 23. Amino acid sequences for ipilimumab.
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Identifier Sequence (One-Letter Amino Acid Symbols)
SEQ =D NO:208 1 QVQLVESGGG VVQPGRSLRI SCAASGFTFS SYTMHWVRQA
PGHGLEWVTF ISYDGNNKYY
ipilLmumab 61 AllSV.KGPFTI SKJMSKNTLY LQMNSLRAE2 TAIYYCARTG
WLGPFDYWGQ G_'LVTVSSAS
heavy chain 121 TKGPSVTPLA PSSKSTSGGT AALGCLVHDY FPEPVTVSWN
SGALTSGVHT FPAVLQSSGL
181 YSLSEVVTVP SSSLGTQTYI CNVNHKPSNT KVDKRVEPHS CDKTH
SEQ ED NO:209 1 EIVLTQSPGT LSLSPGERAT LSCRASQSVG SSYLAWYQQK
PGQAPRLLIY GAFSRATGIP
ipilLmumab 61 DRFSGSGSGT DFTLTISRLE PEDFAVYYCQ OYGSSPWTFG
QGTKVEIKRT VAAPSVFIFP
light chain 121 PSDEQLKSGT ASVVCLLNNF YPREAKVQWK VONALQSGNS
QESVTEQDSK DSTYSLSSTL
181 TISKADYEHN KVYACEVTNQ GLSSPVTKSF NRGEC
SEQ ID NO:810 1 QVQLVESGGG VVQPGRSLRL SCAASGFTES SYTMHWVRQA
PGKGLEWVTF ISYDGNNKYY
ipilimumab 61 ADSVKGRFTI SRDNSKNTLY LQMNSLRAED TAIYYCARTG
WLGPFDYWGQ GTLVTVSS
variable heavy
chain
SEQ ED NO:211 1 EIVLTQSPGT LSLSPGERAT LSCRASQSVG SSYLANYQQH
PGQAPRLLIY GAFSRATGIP
ipilLmumab 61 DRFSGEGSGT DFTLTISRLE PEDFAVYYCQ QYGSSPWTFG
QGTKVEIK
variable light
chain
SEQ =D NO:212 gFTFSSYT
8
1pillmumab
heavy chain
CDR1
SEQ ED NO:213 TFISYDGNEK
10
ipilimumab
heavy chain
CDR2
SEQ ED NO:214 ARTGWLGPFD Y
11
1pillmumab
heavy chain
CDR3
SEQ =D NO:215 QSVGSSY
7
ipilLmumab
light chain
CDR1
SEQ ED NO:216 GAF
3
ipilLmumab
light chain
CDR2
SEQ ID NO:817 QQYGSSPWT
9
ipilimumab
light chain
CDR3
10015241In some embodiments, the CTLA-4 inhibitor is ipilimumab or a
biosimilar thereof, and the
ipilimumab is administered at a dose of about 0.5 mg/kg to about 10 mg/kg. In
some embodiments,
the CTLA-4 inhibitor is ipilimumab or a biosimilar thereof, and the ipilimumab
is administered at a
dose of about 0.5 mg/kg, about 1 mg/kg, about 1.5 mg/kg, about 2 mg/kg, about
2.5 mg/kg, about 3
mg/kg, about 3.5 mg/kg, about 4 mg/kg, about 4.5 mg/kg, about 5 mg/kg, about
5.5 mg/kg, about 6
mg/kg, about 6.5 mg/kg, about 7 mg/kg, about 7.5 mg/kg, about 8 mg/kg, about
8.5 mg/kg, about 9
mg/kg, about 9.5 mg/kg, or about 10 mg/kg.
10015251ln some embodiments, the CTLA-4 inhibitor is ipilimumab or a
biosimilar thereof, and the
ipilimumab is administered at a dose of about 200 mg to about 500 mg. In some
embodiments, the
CTLA-4 inhibitor is ipilimumab or a biosimilar thereof, and the ipilimumab is
administered at a dose
of about 200 mg, about 220 mg, about 240 mg, about 260 mg, about 280 mg, about
300 mg, about
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320 mg, about 340 mg, about 360 mg, about 380 mg, about 400 mg, about 420 mg,
about 440 mg,
about 460 mg, about 480 mg, or about 500 mg.
10015261 In some embodiments, the CTLA-4 inhibitor is ipilimumab or a
biosimilar thereof, and the
ipilimumab is administered every 2 weeks, every 3 weeks, every 4 weeks, every
5 weeks, or every 6
weeks.
10015271 In some embodiments, the ipilimumab is administered to treat
metastatic non-small cell
lung cancer. In some embodiments, the ipilimumab is administered to treat
metastatic non-small cell
lung cancer at about 1 mg/kg every 6 weeks with nivolumab 3 mg/kg every 2
weeks. In some
embodiments, the ipilimumab is administered to treat metastatic non-small cell
lung cancer at about 1
mg/kg every 6 weeks with nivolumab 360 mg every 3 weeks and 2 cycles of
platinum-doublet
chemotherapy.
10015281 Tremelimumab (also known as CP-675,206) is a fully human IgG2
monoclonal antibody
and has the CAS number 745013-59-6. Tremelimumab is disclosed as antibody
11.2.1 in U.S. Patent
No: 6,682,736 (incorporated herein by reference). The amino acid sequences of
the heavy chain and
light chain of tremelimumab are set forth in Table 24, respectively.
Tremelimumab has been
investigated in clinical trials for the treatment of various tumors, including
melanoma and breast
cancer; in which Tremelimumab was administered intravenously either as single
dose or multiple
doses every 4 or 12 weeks at the dose range of 0.01 and 15 mg/kg. In the
regimens provided by the
present invention, tremelimumab is administered locally, particularly
intradermally or
subcutaneously. The effective amount of tremelimumab administered
intradenually or subcutaneously
is typically in the range of 5 - 200 mg/dose per person. In some embodiments,
the effective amount of
tremelimumab is in the range of 10 -150 mg/dose per person per dose. In some
particular
embodiments, the effective amount of tremelimumab is about 10, 25, 37.5, 40,
50, 75, 100, 125, 150,
175, or 200 mg/dose per person.
10015291 In some embodiments, a CTLA-4 inhibitor comprises a heavy chain given
by SEQ ID
NO:218 and a light chain given by SEQ ID NO:219. In some embodiments, a CTLA-4
inhibitor
comprises heavy and light chains having the sequences shown in SEQ ID NO:218
and SEQ ID
NO:219, respectively, or antigen binding fragments, Fab fragments, single-
chain variable fragments
(scFv), variants, or conjugates thereof. In some embodiments, a CTLA-4
inhibitor comprises heavy
and light chains that are each at least 99% identical to the sequences shown
in SEQ ID NO:218 and
SEQ ID NO:219, respectively. In some embodiments, a CTLA-4 inhibitor comprises
heavy and light
chains that are each at least 98% identical to the sequences shown in SEQ ID
NO:218 and SEQ ID
NO:219, respectively in some embodiments, a CTLA-4 inhibitor comprises heavy
and light chains
that are each at least 97% identical to the sequences shown in SEQ ID NO:218
and SEQ ID NO:219,
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respectively. In some embodiments, a CTLA-4 inhibitor comprises heavy and
light chains that are
each at least 96% identical to the sequences shown in SEQ ID NO:218 and SEQ ID
NO:219,
respectively. in some embodiments, a CTLA-4 inhibitor comprises heavy and
light chains that are
each at least 95% identical to the sequences shown in SEQ ID NO:218 and SEQ ID
NO:219,
respectively.
10015301In some embodiments, the CTLA-4 inhibitor comprises the heavy and
light chain CDRs or
variable regions (VRs) of tremelimumab. In some embodiments, the CTLA-4
inhibitor heavy chain
variable region (VII) comprises the sequence shown in SEQ ID NO:220, and the
CTLA-4 inhibitor
light chain variable region (VL) comprises the sequence shown in SEQ ID
NO:221, or conservative
amino acid substitutions thereof. In some embodiments, a CTLA-4 inhibitor
comprises VH and VL
regions that are each at least 99% identical to the sequences shown in SEQ ID
NO:220 and SEQ ID
NO:221, respectively. In some embodiments, a CTLA-4 inhibitor comprises VH and
VL regions that
are each at least 98% identical to the sequences shown in SEQ ID NO:220 and
SEQ ID NO:221,
respectively. In some embodiments, a CTLA-4 inhibitor comprises VH and VL
regions that are each at
least 97% identical to the sequences shown in SEQ ID NO:220 and SEQ ID NO:221,
respectively. In
some embodiments, a CTLA-4 inhibitor comprises VH and VL regions that are each
at least 96%
identical to the sequences shown in SEQ ID NO:220 and SEQ ID NO:221,
respectively. In some
embodiments, a CTLA-4 inhibitor comprises VII and VL regions that are each at
least 95% identical to
the sequences shown in SEQ ID NO:220 and SEQ ID NO:221, respectively.
[0015311kt some embodiments, a CTLA-4 inhibitor comprises the heavy chain
CDR1, CDR2 and
CDR3 domains having the sequences set forth in SEQ ID NO:222, SEQ ID NO:223,
and SEQ ID
NO:224, respectively, or conservative amino acid substitutions thereof, and
light chain CDR1, CDR2
and CDR3 domains having the sequences set forth in SEQ ID NO:225, SEQ ID
NO:226, and SEQ ID
NO:227, respectively, or conservative amino acid substitutions thereof. In
some embodiments, the
antibody competes for binding with, and/or binds to the same epitope on CTLA-4
as any of the
aforementioned antibodies.
10015321 In some embodiments, the CTLA-4 inhibitor is an anti-CTLA-4
biosimilar monoclonal
antibody approved by drug regulatory authorities with reference to
tremelimumab. In some
embodiments, the biosimilar comprises an anti-CTLA-4 antibody comprising an
amino acid sequence
which has at least 97% sequence identity, e.g., 97%, 98%, 99% or 100% sequence
identity, to the
amino acid sequence of a reference medicinal product or reference biological
product and which
comprises one or more post-translational modifications as compared to the
reference medicinal
product or reference biological product, wherein the reference medicinal
product or reference
biological product is tremelimumab. In some embodiments, the one or more post-
translational
modifications are selected from one or more of: glycosylation, oxidation,
deamidation, and truncation.
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The amino acid sequences of tremelimumab are set forth in Table 24. In some
embodiments, the
biosimilar is an anti-CTLA-4 antibody authorized or submitted for
authorization, wherein the anti-
CTLA-4 antibody is provided in a formulation which differs from the
formulations of a reference
medicinal product or reference biological product, wherein the reference
medicinal product or
reference biological product is tremelimumab. The anti-CTLA-4 antibody may be
authorized by a
drug regulatory authority such as the U.S. FDA and/or the European Union's
EVIA. In some
embodiments, the biosimilar is provided as a composition which further
comprises one or more
excipients, wherein the one or more excipients are the same or different to
the excipients comprised in
a reference medicinal product or reference biological product, wherein the
reference medicinal
product or reference biological product is tremelimumab. In some embodiments,
the biosimilar is
provided as a composition which further comprises one or more excipients,
wherein the one or more
excipients are the same or different to the excipients comprised in a
reference medicinal product or
reference biological product, wherein the reference medicinal product or
reference biological product
is tremelimumab.
TABLE 24. Amino acid sequences for tremelimumab.
Identifier Sequence (One-Letter Amino Acid Symbols)
SEQ =D NO:218
1 QVQLVESGGG VVQPGRSLRL SCAASGFTFS SYGMHWVRQA PGHGLEWVAV IWYDGSNHYY
tremelimumab
61 ADSVKGRFTI SRDNSKNTLY LQMNSLRAED TAVYYCARDP RGATLYYYYY GMDVWGQGTT
heavy chain
121 VTVSSASTHG PSVFPLAPCS RSTSESTAAL GCLVYDYFPE PVTVSWNSGA LTSGVHTFPA
181 VIQSSGLYSL SSVVTVPSSN FGTQTYTCNV DHKPSNTKVD KTVERKCCVE CPPCPAPPVA
241 GPSVFLEPPK PKDTLMISRT PEVTCVVVDV SHEDPEVQFN WYVDGVEVHN AKTKPREEQF
301 NSTFRVVSVL TVVHQDWLNG KEYKCKVSNK GLPAPIEKTI SKTHGQPREP QVYTLPPSRE
361 EMTKNOVSLT CLVKGFYPSD IAVEWESNGQ PENNYKTTPP MLDSDGSFFL YSKLTVDKSR
421 WQQGNVFSCS VMHEALHNHY TQKSLSLSPG K
SEQ =D NO:219
1 DIQMTQSPSS LSASVGDRVT ITCRASQSIN SYLDWYQQKP GKAPKLLIYA ASSLQSGVPS
tremelimumab
61 RFSGSGSGTD FTLTISSLQP EDFATYYCQQ YYSTPFTFGP GTKVEIKRTV AAPSVFIFPP
11011_ chain
121 SDEQLKSGTA SVVOLLNNFY PREAKVQWKV DNALQSGNSQ ESVTEQDSHD STYSLSSTLT
181 LSKADYEKHK VYACEVTHQG LSSPVTKSFN RGEC
SEQ =D NO:220
1 GVVQPGRSLR LSCAASGETY SSYGMHWVRQ APGKGLEWVA VIWYDGSNKY YADSVKGRFT
tremelimumab
61 ISRDNSKNTL YLQMNSLRAE DTAVYYCARD PRGATLYYYY YGMDVWGQGT TVTVSSASTK
variable heavy 121 GPSVEPLAPC SRSTSESLLAA hGCLVKDYEP EPVTVSWNSG ALTSGVN
chain
SEQ ID NO:221
1 PSSLSASVGD RVTITCRASQ SINSYLDWYQ QKPGKAPKLL IYAASSLQSG VPSRFSGSGS
tremelimumab
61 GTDFTLTISS LQPEDFATYY CQQYYSTPFT FGPGTKVEIK RTVAAPSVFI FPPSDEQLKS
variable light 121 GTASVVCLLN NFYPREAKV
chain
SEQ =D NO:222 GETFSSYGMH
10
tremelimumab
heavy chain
CDR1
SEQ _D NO:223 V1WYOGSN_KY YADSV
15
tremelimumab
heavy chain
CDR2
SEQ =D NO:224 DPRGATLYYY YYGMDV
16
tremelimumab
heavy chain
CDR3
SEQ =D NO:225 RASQSINSYL D
11
tremelimumab
light chain
CDR1
SEQ =D NO:226 AASSLQS
7
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Identifier Sequence (One-Letter Amino Acid Symbols)
tremelimumab
light chain
CDR2
SEQ ID NO:227 QQYYSTPFT 9
Lremelimumab
light chain
CDR3
10015331 In some embodiments, the CTLA-4 inhibitor is tremelimumab
or a biosimilar thereof,
and the tremelimumab is administered at a dose of about 0.5 mg/kg to about 10
mg/kg. In some
embodiments, the CTLA-4 inhibitor is tremelimumab or a biosimilar thereof, and
the tremelimumab
is administered at a dose of about 0.5 mg/kg, about 1 mg/kg, about 1.5 mg/kg,
about 2 mg/kg, about
2.5 mg/kg, about 3 mg/kg, about 3.5 mg/kg, about 4 mg/kg, about 4.5 mg/kg,
about 5 mg/kg, about
5.5 mg/kg, about 6 mg/kg, about 6.5 mg/kg, about 7 mg/kg, about 7.5 mg/kg,
about 8 mg/kg, about
8.5 mg/kg, about 9 mg/kg, about 9.5 mg/kg, or about 10 mg/kg.
10015341 In some embodiments, the CTLA-4 inhibitor is tremelimumab
or a biosimilar thereof,
and the tremelimumab is administered at a dose of about 200 mg to about 500
mg. In some
embodiments, the CTLA-4 inhibitor is tremelimumab or a biosimilar thereof, and
the tremelimumab
is administered at a dose of about 200 mg, about 220 mg, about 240 mg, about
260 mg, about 280 mg,
about 300 mg, about 320 mg, about 340 mg, about 360 mg, about 380 mg, about
400 mg, about 420
mg, about 440 mg, about 460 mg, about 480 mg, or about 500 mg.
10015351 In some embodiments, the CTLA-4 inhibitor is tremelimumab
or a biosimilar thereof,
and the tremelimumab is administered every 2 weeks, every 3 weeks, every 4
weeks, every 5 weeks,
or every 6 weeks.
10015361 In some embodiments, the CTLA-4 inhibitor is zalifrelimab
from Agenus, or
biosimilars, antigen-binding fragments, conjugates, or variants thereof.
Zalifrelimab is a fully human
monoclonal antibody. Zalifrelimab is assigned Chemical Abstracts Service (CAS)
registry number
2148321-69-9 and is also known as also known as AGEN1884. The preparation and
properties of
zalifrelimab are described in U.S. Patent No. 10,144,779 and US Patent
Application Publication No.
US2020/0024350 Al, the disclosures of which arc incorporated by reference
herein.
10015371 In some embodiments, a CTLA-4 inhibitor comprises a heavy chain given
by SEQ ID
NO.228 and a light chain given by SEQ ID NO.229. In some embodiments, a CTLA-4
inhibitor
comprises heavy and light chains having the sequences shown in SEQ ID NO:228
and SEQ ID
NO:229, respectively, or antigen binding fragments, Fab fragments, single-
chain variable fragments
(scFv), variants, or conjugates thereof. In some embodiments, a CTLA-4
inhibitor comprises heavy
and light chains that are each at least 99% identical to the sequences shown
in SEQ ID NO:228 and
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SEQ ID NO:229, respectively. In some embodiments, a CTLA-4 inhibitor comprises
heavy and light
chains that are each at least 98% identical to the sequences shown in SEQ ID
NO:228 and SEQ ID
NO:229, respectively. in some embodiments, a CTLA-4 inhibitor comprises heavy
and light chains
that are each at least 97% identical to the sequences shown in SEQ ID NO:228
and SEQ ID NO:229,
respectively. In some embodiments, a CTLA-4 inhibitor comprises heavy and
light chains that are
each at least 96% identical to the sequences shown in SEQ Ill NO:228 and SEQ
Ill NO:229,
respectively. In some embodiments, a CTLA-4 inhibitor comprises heavy and
light chains that are
each at least 95% identical to the sequences shown in SEQ ID NO:228 and SEQ ID
NO:229,
respectively.
100153811n some embodiments, the CTLA-4 inhibitor comprises the heavy and
light chain CDRs or
variable regions (VRs) of zalifrelimab. In some embodiments, the CTLA-4
inhibitor heavy chain
variable region (VH) comprises the sequence shown in SEQ ID NO:230, and the
CTLA-4 inhibitor
light chain variable region (VI) comprises the sequence shown in SEQ ID
NO:231, or conservative
amino acid substitutions thereof. In some embodiments, a CTLA-4 inhibitor
comprises VH and VL
regions that are each at least 99% identical to the sequences shown in SEQ ID
NO:230 and SEQ ID
NO:231, respectively. In some embodiments, a CTLA-4 inhibitor comprises VH and
VL regions that
are each at least 98% identical to the sequences shown in SEQ ID NO:230 and
SEQ ID NO:231,
respectively. In some embodiments, a CTLA-4 inhibitor comprises Vrt and VL
regions that are each at
least 97% identical to the sequences shown in SEQ ID NO:230 and SEQ ID NO:231,
respectively. In
some embodiments, a CTLA-4 inhibitor comprises VII and VL regions that are
each at least 96%
identical to the sequences shown in SEQ ID NO:230 and SEQ ID NO:231,
respectively. In some
embodiments, a CTLA-4 inhibitor comprises VH and Vt. regions that are each at
least 95% identical to
the sequences shown in SEQ ID NO:230 and SEQ ID NO:231, respectively.
100153911n some embodiments, a CTLA-4 inhibitor comprises the heavy chain
CDR1, CDR2 and
CDR_3 domains having the sequences set forth in SEQ ID NO:231, SEQ ID NO:233,
and SEQ ID
NO:234, respectively, or conservative amino acid substitutions thereof, and
light chain CDR1, CDR2
and CDR3 domains having the sequences set forth in SEQ ID NO:235, SEQ ID
NO:236, and SEQ ID
NO:237, respectively, or conservative amino acid substitutions thereof. In
some embodiments, the
antibody competes for binding with, and/or binds to the same epitope on CTLA-4
as any of the
aforementioned antibodies.
100154011n some embodiments, the CTLA-4 inhibitor is a CTLA-4 biosimilar
monoclonal antibody
approved by drug regulatory authorities with reference to zalifrelimab. In
some embodiments, the
biosimilar comprises an anti-CTLA-4 antibody comprising an amino acid sequence
which has at least
97% sequence identity, e.g., 97%, 98%, 99% or 100% sequence identity, to the
amino acid sequence
of a reference medicinal product or reference biological product and which
comprises one or more
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post-translational modifications as compared to the reference medicinal
product or reference
biological product, wherein the reference medicinal product or reference
biological product is
zalifrelimab. in some embodiments, the one or more post-translational
modifications are selected
from one or more of: glycosylation, oxidation, deamidation, and truncation.
The amino acid sequences
of zalifrelimab are set forth in Table 25. In some embodiments, the biosimilar
is an anti-CTLA-4
antibody authorized or submitted for authorization, wherein the anti-CTLA-4
antibody is provided in
a formulation which differs from the formulations of a reference medicinal
product or reference
biological product, wherein the reference medicinal product or reference
biological product is
zalifrelimab. The anti-CTLA-4 antibody may be authorized by a drug regulatory
authority such as the
U.S. FDA and/or the European Union's EMA. In some embodiments, the biosimilar
is provided as a
composition which further comprises one or more excipients, wherein the one or
more excipients are
the same or different to the excipients comprised in a reference medicinal
product or reference
biological product, wherein the reference medicinal product or reference
biological product is
zalifrelimab. In some embodiments, the biosimilar is provided as a composition
which further
comprises one or more excipients, wherein the one or more excipients are the
same or different to the
excipients comprised in a reference medicinal product or reference biological
product, wherein the
reference medicinal product or reference biological product is zalifrelimab.
TABLE 25. Amino acid sequences for zalifrelimab.
Identifier Sequence (One-Letter Amino Acid Symbols)
SEQ DD NO:228
1 EVQLVESGGG LVKPGGSLRL SCAASGFTFS SYSMNWVRQA PGKGLEWVSS ISSSSSYIYY
calif relimab
61 ADSVEGRFTI SRDNAENSLY LQMNSLRAED TAVYYCARVG LMGPFDIWGQ GTMVTVSSAS
heavy chain
121 TKGPSVFELA PSSKSTSGGT AALGCLVKDY FFEEVTVSWN SGALTSGVHT FPAVIQSSGL
181 YSLSSVVTVP SSSLGTQTYI CEVNEEPSNT AVEKKVEPES CDK1HTC2PC PAPELLGGPS
211 VFLFPPKPXD TLMISRTPEV TCVVVDVSHE DPEVKFNWYV DGVEVHNAKT KPREEQYNST
301 YRVV2VITVL HQDWLNGKEY KCKVSNKALP APIEKTISKA KGQPREPQVY TLPFSREEMT
361 KNQVSLTCLV KGFYPSDIAV EWESNGQPFN NYKTTPPVLD SDGSFFLYSK LTV7KSRWQQ
421 GNVFSCSVMH EALHNHYTQK SLSLSPGH
SEQ DD NO:229
1 EIVLTQSPGT LSLSFGERAT LSCRASQSVS RYLGWYQQHF GQAPRLLIYG ASTRATGIFD
zalifrelimab
61 RFSGSGSGTD FTLTITRLEF EDFAVYYCQQ YGSSFWTFGQ GTHVEIKRTV AAPSVFIFFF
light chain
121 SDEQLKSGTA SVVCLLNNFY PREAKVQWKV DNALQSGNSQ ESVTEQDSKD STYSLSSTLT
181 LSHADYEKHK VYACEVTHQG LSSPVTKSFN RGEC
SEQ DD NO:230
1 EVQLVESGGG LVKPGGSLRL SCAASGFTFS SYSMNWVRQA FGHGLEWVSS ISSSSSYIYY
calif reliinab 61 ADSVEGRFTI SRDNAENSLY LQMNSLRAED TAVYYCARVG
LMGPFDIWGQ GTMVTVSS
variable heavy
chain
SEQ =D NO:231
1 EIVLTQSPGT LSLSPGERAT LSCRASQSVS RYLGWYQQKP GQAPRLLIYG ASTRATGIPD
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Identifier Sequence (One-Letter Amino Acid Symbols)
zalifrelimab 61 RFSGEGSGTD FTLTITRLE2 EDEAVYYCQQ YGSS2WTFGQ GTHVEIK
variable light
chain
SEQ _D NO:232 G_H_LESSYS
8
zalifrelimab
heavy chain
CDR1
SEQ =D NO:233 ISSSSSYI
8
zalifrelimab
heavy chain
CDR2
SEQ =D NO:234 ARVGLMGPFD I
11
zallfrelimab
heavy chain
CDR3
SEQ =D NO:235 QSVSRY
6
zalifrelimab
lighb chain
CDR1
SEQ =D NO:236 GAS
3
zalifrelimab
light chain
CDR2
SEQ =D NO:237 QQYGSSPWT
9
zalifrelimab
light chain
CDR3
10015411Examples of additional anti-CTLA-4 antibodies includes, but are not
limited to:
AGEN1181, BMS-986218, BCD-145, ONC-392, CS1002, REGN4659, and ADG116, which
are
known to one of ordinary skill in the art.
[001542] In some embodiments, the anti -CTLA-4 antibody is an anti-CTLA-4
antibody disclosed in
any of the following patent publications (incorporated herein by reference):
1JS2019/0048096A1;
US2020/0223907; US2019/0201334; US2019/0201334; US2005/0201994; EP 1212422 Bl;
W02018204760; W02018204760; W02001014424; W02004035607; W02003086459;
W02012120125; W02000037504; W02009100140; W0200609649; W02005092380;
W02007123737; W02006029219; W020100979597; W0200612168; and W01997020574.
Additional CTLA-4 antibodies are described in U.S. Pat. Nos. 5,811,097,
5,855,887, 6,051,227, and
6,984,720; in PCT Publication Nos. WO 01/14424 and WO 00/37504; and in U.S.
Publication Nos.
2002/0039581 and 2002/086014; and/or U.S. Patent Nos. 5,977,318, 6,682,736,
7,109,003, and
7,132,281, incorporated herein by reference). In some embodiments, the anti-
CTLA-4 antibody is an,
for example, those disclosed in: WO 98/42752; U.S. Pat. Nos. 6,682,736 and
6,207,156; Hurwitz et
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al., Proc. Natl. Acad. Sci. USA, 95(17): 10067-10071 (1998); Camacho et al., I
Clin. Oncol.,
22(145): Abstract No. 2505 (2004) (antibody CP-675206); Mokyr et al., Cancer
Res., 58:5301-5304
(1998) (incorporated herein by reference).
100154311n some embodiments, the CTLA-4 inhibitor is a CTLA-4 ligand as
disclosed in
W01996040915 (incorporated herein by reference).
100154411n some embodiments, the CTLA-4 inhibitor is a nucleic acid inhibitor
of CTLA-4
expression. For example. anti-CTLA-4 RNAi molecules may take the form of the
molecules described
by Mello and Fire in PCT Publication Nos. WO 1999/032619 and WO 2001/029058;
U.S. Publication
Nos. 2003/0051263, 2003/0055020, 2003/0056235, 2004/265839, 2005/0100913,
2006/0024798,
2008/0050342, 2008/0081373, 2008/0248576, and 2008/055443; and/or U.S. Pat.
Nos. 6,506,559,
7,282,564, 7,538,095, and 7,560,438 (incorporated herein by reference). In
some instances, the anti-
CTLA-4 RNAi molecules take the form of double stranded RNAi molecules
described by Tuschl in
European Patent No. EP 1309726 (incorporated herein by reference). In some
instances, the anti-
CTLA-4 RNAi molecules take the form of double stranded RNAi molecules
described by Tuschl in
U.S. Pat. Nos. 7,056,704 and 7,078,196 (incorporated herein by reference). In
some embodiments,
the CTLA-4 inhibitor is an aptamer described in PCT Publication No.
W02004081021 (incorporated
herein by reference).
10015451In other embodiments, the anti-CTLA-4 RNAi molecules of the present
invention are RNA
molecules described by Crooke in U.S. Patent Nos. 5,898,031, 6,107,094,
7,432,249, and 7,432,250,
and European Application No. EP 0928290 (incorporated herein by reference).
S. Combinations with VEGF-A Inhibitors
10015461 In some embodiments, TILs and a VEGF-A inhibitor are administered as
a combination
therapy or co-therapy for the treatment of NSCLC.
10015471ln some embodiments, the NSCLC has undergone no prior therapy. In some
embodiments,
the VEGF-A inhibitor is administered as a front-line therapy or initial
therapy. In some embodiments,
the VEGF-A inhibitor is administered as a front-line therapy or initial
therapy in combination with the
TILs as described herein.
10015481VEGF-A (polynucleotide and polypeptide sequences shown in SEQ ID NOs:
1 and 2,
respectively) is a secreted, disulfide-linked homodimeric glycoprotein
belonging to the VEGF/PDGF
(platelet-derived growth factor) group of the cystine -knot superfamily of
hormones and extracellular
signaling molecules (see Vitt et al., Mol. Endocrinol., 15:681-694, 2001),
which are all characterized
by the presence of eight conserved cysteine residues forming the typical
cystine -knot structure (named
after cystine, a dimer of two cysteines linked by a disulfide bond). Five
human VEGF-A isoforms of
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121, 145, 165, 189 or 206 amino acids in length (VEGF-A121-206), encoded by
distinct mRNA
splice variants, have been described, all of which are capable of stimulating
mitogenesis in endothelial
cells. These isofonns differ in biological activity, receptor specificity, and
affinity for cell surface-
and extracellular matrix-associated heparan-sulfate proteoglycans, which
behave as low affinity
receptors for VEGF-A: VEGF-A121 does not bind to either heparin or heparan-
sulfate; VEGF-A145
and VEGF-A165 (Genl3ank Acc. No. M32977) arc both capable of binding to
heparin; and VEGF-
A189 and VEGF-A206 show the strongest affinity for heparin and heparan-
sulfates. VEGF-A121,
VEGF-A145, and VEGF-A165 are secreted in a soluble form, although most of VEGF-
A165 is
confined to cell surface and extracellular matrix proteoglycans, whereas VEGF-
A189 and VEGF-
A206 remain associated with extracellular matrix. Both VEGF-A189 and VEGF-A206
can be
released by treatment with heparin or heparinase, indicating that these
isoforms are bound to
extracellular matrix via proteoglycans. Cell-bound VEGF-A180 can also be
cleaved by proteases such
as plasmin, resulting in release of an active soluble VEGF-A110.
10015491 VEGF-A-driven angiogenesis has a major role in the pathogenesis of
diverse human
diseases, including cancer, eye disorders, and rheumatoid arthritis. See
Carmeliet et al., Nature
407:249-257, 2000. Recognition of the importance of VEGF-A for the development
of several
important classes of cancer recently culminated in the approval of AVASTINIm,
a humanized
monoclonal antibody to VEGF-A, for the treatment of metastatic colorectal
cancer. See Ferrara et al.,
Nat. Rev. Drug Discov. 2004, 3:391-400, 2004. Similarly, the importance of
VEGF-A in the
pathogenesis of neovascular ocular disorders is reflected in the recent
approval of LUCENTISTm, a
humanized monoclonal antibody fragment, for the treatment of neovascular (wet)
age-related macular
degeneration (AMD).
10015501 VEGF-A inhibitors for use within the present invention include
molecules that bind to
VEGF-A or a VEGF-A receptor and thereby reduce the activity of VEGF-A on cells
that express the
receptor such as, e.g., VEGFR-1, VEGFR-2, neuropilin-1, and/or neuropilin-2.
In particular, VEGF-A
inhibitors include anti-VEGF-A antibodies. Other suitable VEGF-A inhibitors
include soluble VEGF-
A receptors comprising a VEGFR extracellular domain, as well as small molecule
antagonists capable
of inhibiting the interaction of VEGF-A with its receptor or otherwise capable
in inhibiting VEGF-A-
induced intracellular signaling through a VEGF-A receptor. In addition,
binding proteins based on
non-antibody scaffolds may be employed. (See, e.g., Koide et al., J. Mol.
Biol. 284:1141-1151, 1998;
Hosse et al. Protein Sci. 15:14-27, 2006, and references therein.) Preferred
VEGF-A inhibitors for use
within the invention include antibodies that specifically bind to VEGF-A,
including bispecific
antibodies that also comprise a binding site for PDGFRI3. Antibodies that arc
specific for VEGF-A
bind at least the soluble secreted forms of VEGF-A, and preferably also bind
cell surface-associated
forms.
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10015511111 some embodiments, the VEGF-A inhibitor may be any VEGF-A inhibitor
or VEGF-A
blocker known in the art. In particular, it is one of the VEGF-A inhibitors or
blockers described in
more detail in the following paragraphs. The terms "inhibitor," "antagonist,"
and "blocker" are used
interchangeably herein in reference to VEGF-A inhibitors. For avoidance of
doubt, references herein
to a VEGF-A inhibitor that is an antibody may refer to a compound or antigen-
binding fragments,
variants, conjugates, or biosimilars thereof. For avoidance of doubt,
references herein to a VEGF-A
inhibitor may also refer to a small molecule compound or a pharmaceutically
acceptable salt, ester,
solvate, hydrate, cocrystal, or prodrug thereof.
10015521In some embodiments, the VEGF-A inhibitor may be any VEGF-A inhibitor
or VEGF-A
blocker known in the art. In particular, it is one of the VEGF-A inhibitors or
blockers described in
more detail in the following paragraphs. The terms "inhibitor," "antagonist,"
and "blocker" are used
interchangeably herein in reference to VEGF-A inhibitors. For avoidance of
doubt, references herein
to a VEGF-A inhibitor that is an antibody may refer to a compound or antigen-
binding fragments,
variants, conjugates, or biosimilars thereof. For avoidance of doubt,
references herein to a VEGF-A
inhibitor may also refer to a small molecule compound or a pharmaceutically
acceptable salt, ester,
solvate, hydrate, cocrystal, or prodrug thereof.
10015531In some embodiments, the VEGF-A inhibitor is an antibody (i.e., an
anti-VEGF-A
antibody), a fragment thereof, including Fab fragments, or a single-chain
variable fragment (scFv)
thereof In some embodiments the VEGF-A inhibitor is a polyclonal antibody. In
some embodiments,
the VEGF-A inhibitor is a monoclonal antibody. In some embodiments, the VEGF-A
inhibitor
competes for binding with VEGF-A, and/or binds to an epitope on VEGF-A. In
some embodiments,
the antibody competes for binding with VEGF-A, and/or binds to an epitope on
VEGF-A.
10015541In some embodiments, the VEGF-A inhibitor is one that binds human VEGF-
A with a KD
of about 100 pM or lower, binds human VEGF-A with a KD of about 90 pM or
lower, binds human
VEGF-A with a KD of about 80 pM or lower, binds human VEGF-A with a KD of
about 70 pM or
lower, binds human VEGF-A with a KD of about 60 pM or lower, binds human VEGF-
A with a KD of
about 50 pM or lower, binds human VEGF-A with a KD of about 40 pM or lower,
binds human
VEGF-A with a KD of about 30 pM or lower, binds human VEGF-A with a KD of
about 20 pM or
lower, binds human VEGF-A with a KD of about 10 pM or lower, or binds human
VEGF-A with a KD
of about 1 pM or lower.
10015551In some embodiments, the VEGF-A inhibitor is one that binds to human
VEGF-A with a
kaõoc of about 7.5 x 1051/M- s or faster, binds to human VEGF-A with a kaõoc
of about 7.5 x 105 1/M- s
or faster, binds to human VEGF-A with a Icassoc of about 8 x 105 1/M- s or
faster, binds to human
VEGF-A with a kassoc of about 8.5 x 105 1/Ms or faster, binds to human VEGF-A
with a kas,oc of
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about 9 x 105 1/M- s or faster, binds to human VEGF-A with a lc. of about 9.5
x 1051/M- s or faster,
or binds to human VEGF-A with a kassoc of about 1 x 106 1/M- s or faster.
10015561 In some embodiments, the VEGF-A inhibitor is one that binds to human
VEGF-A with a
kdissoc of about 2 x 10-5 1/s or slower, binds to human VEGF-A with a kdissoc
of about 2.1 x 10-5 1/s or
slower , binds to human VEGF-A with a kdiõoc of about 2.2 x 10-5 1/s or
slower, binds to human
VEGF-A with a kaisso, of about 2.3>< 10-5 1/s or slower, binds to human VEGF-A
with a kaissoc of about
2.4 x 10-5 1/s or slower, binds to human VEGF-A with a kaissoc of about 2.5 x
10-5 1/s or slower, binds
to human VEGF-A with a kaissoc of about 2.6 x 10-5 1/s or slower or binds to
human VEGF-A with a
kdissoc of about 2.7 x 10-5 1/s or slower, binds to human VEGF-A with a
kdissoc of about 2.8 x 10-5 1/s or
slower, binds to human VEGF-A with a kaissoc of about 2.9 x 10-5 1/s or
slower, or binds to human
VEGF-A with a kdissoc of about 3 x 10-5 1/s or slower.
10015571 In some embodiments, the VEGF-A inhibitor is one that blocks or
inhibits binding of
human VEGFR-1 receptor or human VEGFR-2 receptor to human VEGF-A with an ICso
of about 10
nM or lower, blocks or inhibits binding of human VEGFR-1 receptor or human
VEGFR-2 receptor to
human VEGF-A with an 1Cso of about 9 nM or lower, blocks or inhibits binding
of human VEGFR-1
receptor or human VEGFR-2 receptor to human VEGF-A with an ICso of about 8 nM
or lower, blocks
or inhibits binding of human VEGFR-1 receptor or human VEGFR-2 receptor to
human VEGF-A
with an ICso of about 7 nM or lower, blocks or inhibits binding of human VEGFR-
1 receptor or
human VEGFR-2 receptor to human VEGF-A with an ICso of about 6 nM or lower,
blocks or inhibits
binding of human VEGFR-1 receptor or human VEGFR-2 receptor to human VEGF-A
with an ICso
of about 5 nM or lower, blocks or inhibits binding of human VEGFR-1 receptor
or human VEGFR-2
receptor to human VEGF-A with an ICso of about 4 nM or lower, blocks or
inhibits binding of human
VEGFR-1 receptor or human VEGFR-2 receptor to human VEGF-A with an ICso of
about 3 nM or
lower, blocks or inhibits binding of human VEGFR-1 receptor or human VEGFR-2
receptor to human
VEGF-A with an ICso of about 2 nM or lower, or blocks or inhibits binding of
human VEGFR-1
receptor or human VEGFR-2 receptor to human VEGF-A with an ICso of about 1 nM
or lower.
10015581 In some embodiments, the VEGF-A inhibitor is bcvacizumab, or
biosimilars, antigen-
binding fragments, conjugates, or variants thereof. Bevacizumab (CAS registry
number 216974-75-3,
AVASTIN , Genentech) is an anti-VEGF monoclonal antibody against vascular
endothelial growth
factor used in cancer treatment (US 7227004; US 6884879; US 7060269; US
7169901; US 7297334),
which inhibits tumor growth by blocking neovascularization. Bevacizumab is the
first clinically
available angiogenesis inhibitor in the United States. It was approved by the
FDA in 2004 in
combination with standard chemotherapy for the treatment of metastatic colon
cancer and most forms
of metastatic non-small cell lung cancer. Several post-clinical studies are
underway to determine its
safety and effectiveness in patients with the following diseases: auxiliary /
non-metastatic colon
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cancer, metastatic breast cancer, metastatic renal cell carcinoma, metastatic
polymorphic glioblastoma
Neoplasms, metastatic ovarian cancer, metastatic hormone refractory prostate
cancer and metastasis
Metastatic pancreatic cancer or unresectable locally advanced pancreatic
cancer.
1001559] Bevacizumab includes a mutant human IgG1 framework region and an
antigen-binding
complementarity determining region from a murine anti-hVEGF monoclonal
antibody A.4.6.1 that
blocks the binding of human VEGF to its receptor. About 93% of the bevacizumab
amino acid
sequences (including most framework regions) are derived from human IgG1 and
about 7% of the
sequences are derived from the murine antibody A4.6.1. Bevacizumab has a
molecular mass of about
149,000 daltons and is glycosylated. Bevacizumab and other humanized anti-VEGF
antibodies are
further described in US 6884879. Additional anti-VEGF antibodies include G6 or
B20 series
antibodies (eg G6-31, B20-4.1) as described in any of Figures 27-29 of
International Patent
Publication No. WO 2005/012359. In one embodiment, the B20 series antibodies
bind to functional
epitopes on human VEGF containing residues F17, M18, D19, Y21, Y25, Q89, 191,
K101, E103, and
C104. Additional VEGF antibodies are in are described in International Patent
Publication No.
W02010148223.
10015601in some embodiments, a VEGF-A inhibitor comprises a heavy chain given
by SEQ ID
NO:207 and a light chain given by SEQ ID NO:208. In some embodiments, a VEGF-A
inhibitor
comprises heavy and light chains having the sequences shown in SEQ ID NO:207
and SEQ ID
NO:208, respectively, or antigen binding fragments, Fab fragments, single-
chain variable fragments
(scFv), variants, or conjugates thereof. In some embodiments, a VEGF-A
inhibitor comprises heavy
and light chains that arc each at least 99% identical to the sequences shown
in SEQ ID NO:207 and
SEQ ID NO:208, respectively. In some embodiments, a VEGF-A inhibitor comprises
heavy and light
chains that are each at least 98% identical to the sequences shown in SEQ ID
NO:207 and SEQ ID
NO:208, respectively. In some embodiments, a VEGF-A inhibitor comprises heavy
and light chains
that are each at least 97% identical to the sequences shown in SEQ ID NO:207
and SEQ ID NO:208,
respectively. In some embodiments, a VEGF-A inhibitor comprises heavy and
light chains that are
each at least 96% identical to the sequences shown in SEQ ID NO:207 and SEQ ID
NO:208,
respectively. In some embodiments, a VEGF-A inhibitor comprises heavy and
light chains that are
each at least 95% identical to the sequences shown in SEQ ID NO:207 and SEQ ID
NO:208,
respectively.
100156111n some embodiments, the VEGF-A inhibitor comprises the heavy and
light chain CDRs or
variable regions (VRs) of bevacizumab. In some embodiments, the VEGF-A
inhibitor heavy chain
variable region (VII) comprises the sequence shown in SEQ ID NO:209, and the
VEGF-A inhibitor
light chain variable region (VI) comprises the sequence shown in SEQ ID
NO:210, and conservative
amino acid substitutions thereof. In some embodiments, a VEGF-A inhibitor
comprises VII and VL
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regions that are each at least 99% identical to the sequences shown in SEQ ID
NO:209 and SEQ ID
NO:210, respectively. In some embodiments, a VEGF-A inhibitor comprises VH and
VL regions that
are each at least 98% identical to the sequences shown in SEQ ID NO:209 and
SEQ ID NO:210,
respectively. In some embodiments, a VEGF-A inhibitor comprises VH and VL
regions that are each at
least 97% identical to the sequences shown in SEQ ID NO:209 and SEQ ID NO:210,
respectively. In
some embodiments, a VEGF-A inhibitor comprises V H and VI, regions that are
each at least 96%
identical to the sequences shown in SEQ ID NO:209 and SEQ ID NO:210,
respectively. In some
embodiments, a VEGF-A inhibitor comprises Vn and VL regions that are each at
least 95% identical to
the sequences shown in SEQ ID NO:209 and SEQ ID NO:210, respectively.
10015621ln some embodiments, a VEGF-A inhibitor comprises the heavy chain
CDR1, CDR2 and
CDR3 domains having the sequences set forth in SEQ ID NO:211, SEQ ID NO:212,
and SEQ ID
NO:213, respectively, and conservative amino acid substitutions thereof, and
light chain CDR1,
CDR2 and CDR3 domains having the sequences set forth in SEQ TD NO:214, SEQ ID
NO:215, and
SEQ ID NO:216, respectively, and conservative amino acid substitutions
thereof. In some
embodiments, the antibody competes for binding with, and/or binds to the same
epitope on VEGF-A
as any of the aforementioned antibodies.
10015631 In some embodiments, the VEGF-A inhibitor is a VEGF-A biosimilar
monoclonal antibody
approved by drug regulatory authorities with reference to bevacizumab. In some
embodiments, the
biosimilar comprises an anti-VEGF-A antibody comprising an amino acid sequence
which has at least
97% sequence identity, e.g., 97%, 98%, 99% or 100% sequence identity, to the
amino acid sequence
of a reference medicinal product or reference biological product and which
comprises one or more
post-translational modifications as compared to the reference medicinal
product or reference
biological product, wherein the reference medicinal product or reference
biological product is
bevacizumab. In some embodiments, the one or more post-translational
modifications are selected
from one or more of: glycosylation, oxidation, deamidation, and truncation. In
some embodiments,
the biosimilar is an anti-VEGF-A antibody authorized or submitted for
authorization, wherein the
anti-VEGF-A antibody is provided in a formulation which differs from the
formulations of a reference
medicinal product or reference biological product, wherein the reference
medicinal product or
reference biological product is bevacizumab. The anti-VEGF-A antibody may be
authorized by a drug
regulatory authority such as the U.S. FDA and/or the European Union's EMA. In
some embodiments,
the biosimilar is provided as a composition which further comprises one or
more excipients, wherein
the one or more excipients are the same or different to the excipients
comprised in a reference
medicinal product or reference biological product, wherein the reference
medicinal product or
reference biological product is bevacizumab. In some embodiments, the
biosimilar is provided as a
composition which further comprises one or more excipients, wherein the one or
more excipients are
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the same or different to the excipients comprised in a reference medicinal
product or reference
biological product, wherein the reference medicinal product or reference
biological product is
bevacizumab.
TABLE 26. Amino acid sequences for bevacizumab.
Identifier Sequence (One-Letter Amino Acid Symbols)
SEQ _D
1 EVQLVESGGG LVQPGGSLRL SCAASGYTJ'l NYGMNWVRQA PGKGLWVGW INTYTGEPTY
NO:207
61 AADFKRRFTF SLDTSKSTAY LQMNSLRAED TAVYYCAHYP HYYGSSHWYF DVWGQGTLVT
bevacizumab 121 VSSASTKGPS VFPLAPSSKS TSGGTAALGC LVKDYFE'EPV
TVSWNSGALT SGVHTFPAVL
heavy chain 181 QSSGLYSLSS VVTVPSSSLG TQTYICNVNH KPSNTKVDKK
VEPKSCDKTH TCPPCPAPEL
241 LGGPSVFLFP PKPKDTLMIS RTPEVTCVVV DVSHEDPEVK FNWYVDGVEV HNAKTKPREE
301 QYNSTYRVVS VLTVLHQDWL NGKEYKCKVS NKALPAPIEK TISKAKGQPR EPQVYTLPPS
361 REEMTHNQVS LTCLVKGYYP SDIAMEWESN GQPENNYHTT PPVLDSDGSF FLYSKLTVPH
421 SRWQQGNVFS CSVMHEALHN HYTQHSLSLS PGK
SEQ _D
1 DIUUQSPSS LSASVGDRVT ITCSASQD_LS NYLNWYQQKP GKAPKVLIYM TSSLHSGVPS
NO:208
61 RFSGSGSGTD FTLTISSLQP EDFATYYCQQ YSTVPWTFGQ GTKVEIKRTV AAPSVFIFPP
bevacizumab 121 SDEOLKSGTA SVVCLLNNFY PREAKVQWKV DNALQSGNSQ
ESVTEQDSHD STYSLSSTLT
light chain 181 LSKADYEKHK VYACEVTHQG LSSPVTKSYN RGEC
SEQ DD
1 EVQLVESGGG LVQPGGSLRL SCAASGYTYT NYGMNWVRQA PGKGLEWVGW INTYTGEPTY
NO:209
61 AADFKRRFTE SLDTSKSTAY LQMNSLRAED TAVYY-CAKYP HYYGSSHWYF DVTAIGQGTLVT
bevacizumab 121 VSS
variable
heavy chain
SEQ _D
1 21QNL'QS2SS LSASVG2rWT 1TCSASQ21S NYLNWYQQK2 GKA_PKVLIYJ2 TSSLHSGV2S
NO:210 61 RFSGSGSGTD FTLTISSLQP EDFATYYCQQ YSTVPWTFGQ GTKVEIKR
bevacizumab
variable
light chain
SEQ ID GYTFTNYGMN
10
NO:211
bevacizumab
heavy chain
CDR1
SEQ DD WINTYTGEPT YAADFK
16
NO: 212
bevacizumab
heavy chain
CDR2
SEQ =0 YPHYYGSSHW YFDV
14
NO; 213
bevacizumab
heavy chain
CDR3
SEQ DD SASQDISNYL N
11
NO: 214
bevacizumab
light chain
CDR1
SEQ =D FTSSLHS
7
NO; 215
bevacizumab
light chain
CDR2
SEQ DD QQYSTVPWT
9
NO:216
bevacizumab
light chain
CDR3
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10015641ln some embodiments, the VEGF-A inhibitor is ranibizumab, or
biosimilars, antigen-
binding fragments, conjugates, or variants thereof. Ranibizumab (CAS registry
number 347396-82-1,
Lucentis ) is a monoclonal antibody fragment (Fab) created from the same
parent mouse antibody as
bevacizumab. It is an anti-angiogenic that has been approved to treat age-
related macular degeneration
(AMD, also ARMD), a common form of age-related vision loss. Its rates of side
effects is similar to
that of bevacizumab. However, ranibizumab typically costs $2,000 a dose, while
the equivalent dose
of bevacizumab typically costs $50.
10015651ln some embodiments, a VEGF-A inhibitor comprises a heavy chain given
by SEQ ID
NO:217 and a light chain given by SEQ ID NO:218. In some embodiments, a VEGF-A
inhibitor
comprises heavy and light chains having the sequences shown in SEQ ID NO:217
and SEQ ID
NO:218, respectively, or antigen binding fragments, Fab fragments, single-
chain variable fragments
(scFv), variants, or conjugates thereof. In some embodiments, a VEGF-A
inhibitor comprises heavy
and light chains that are each at least 99% identical to the sequences shown
in SEQ ID NO:217 and
SEQ ID NO:218, respectively. In some embodiments, a VEGF-A inhibitor comprises
heavy and light
chains that are each at least 98% identical to the sequences shown in SEQ ID
NO:217 and SEQ ID
NO:218, respectively. In some embodiments, a VEGF-A inhibitor comprises heavy
and light chains
that are each at least 97% identical to the sequences shown in SEQ ID NO:217
and SEQ ID NO:218,
respectively. In some embodiments, a VEGF-A inhibitor comprises heavy and
light chains that are
each at least 96% identical to the sequences shown in SEQ ID NO:217 and SEQ ID
NO:218.
respectively. In some embodiments, a VEGF-A inhibitor comprises heavy and
light chains that are
each at least 95% identical to the sequences shown in SEQ ID NO:217 and SEQ ID
NO:218,
respectively.
10015661ln some embodiments, the VEGF-A inhibitor comprises the heavy and
light chain CDRs or
variable regions (VRs) of ranibizumab. In some embodiments, the VEGF-A
inhibitor heavy chain
variable region (VH) comprises the sequence shown in SEQ ID NO:219, and the
VEGF-A inhibitor
light chain variable region (VL) comprises the sequence shown in SEQ ID
NO:220, and conservative
amino acid substitutions thereof. In some embodiments, a VEGF-A inhibitor
comprises VH and VL
regions that are each at least 99% identical to the sequences shown in SEQ ID
NO:219 and SEQ ID
NO:220, respectively. In some embodiments, a VEGF-A inhibitor comprises VH and
VL regions that
are each at least 98% identical to the sequences shown in SEQ ID NO:219 and
SEQ ID NO:220,
respectively. In some embodiments, a VEGF-A inhibitor comprises VH and VL
regions that are each at
least 97% identical to the sequences shown in SEQ ID NO:219 and SEQ ID NO:220,
respectively. In
some embodiments, a VEGF-A inhibitor comprises VH and VL regions that are each
at least 96%
identical to the sequences shown in SEQ ID NO:219 and SEQ ID NO:220,
respectively. In some
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embodiments, a VEGF-A inhibitor comprises VE and VL regions that are each at
least 95% identical to
the sequences shown in SEQ ID NO:219 and SEQ ID NO:220, respectively.
10015671ln some embodiments, a VEGF-A inhibitor comprises the heavy chain
CDR1, CDR2 and
CDR3 domains having the sequences set forth in SEQ ID NO:221, SEQ ID NO:222,
and SEQ ID
NO:223, respectively, and conservative amino acid substitutions thereof, and
light chain CDR1,
CDR2 and CDR3 domains having the sequences set forth in SEQ ID NO:224, SEQ ID
NO:225, and
SEQ ID NO:226, respectively, and conservative amino acid substitutions
thereof. In some
embodiments, the antibody competes for binding with, and/or binds to the same
epitope on VEGF-A
as any of the aforementioned antibodies.
10015681ln some embodiments, the VEGF-A inhibitor is a VEGF-A biosimilar
monoclonal antibody
approved by drug regulatory authorities with reference to ranibizumab. In some
embodiments, the
biosimilar comprises an anti-VEGF-A antibody comprising an amino acid sequence
which has at least
97% sequence identity, e.g., 97%, 98%, 99% or 100% sequence identity, to the
amino acid sequence
of a reference medicinal product or reference biological product and which
comprises one or more
post-translational modifications as compared to the reference medicinal
product or reference
biological product, wherein the reference medicinal product or reference
biological product is
ranibizumab. In some embodiments, the one or more post-translational
modifications are selected
from one or more of: glycosylation, oxidation, deamidation, and truncation. In
some embodiments,
the biosimilar is an anti-VEGF-A antibody authorized or submitted for
authorization, wherein the
anti-VEGF-A antibody is provided in a formulation which differs from the
formulations of a reference
medicinal product or reference biological product, wherein the reference
medicinal product or
reference biological product is ranibizumab. The anti-VEGF-A antibody may be
authorized by a drug
regulatory authority such as the U.S. FDA and/or the European Union's EMA. In
some embodiments,
the biosimilar is provided as a composition which further comprises one or
more excipients, wherein
the one or more excipients are the same or different to the excipients
comprised in a reference
medicinal product or reference biological product, wherein the reference
medicinal product or
reference biological product is ranibizumab. In some embodiments, the
biosimilar is provided as a
composition which further comprises one or more excipients, wherein the one or
more excipients are
the same or different to the excipients comprised in a reference medicinal
product or reference
biological product, wherein the reference medicinal product or reference
biological product is
ranibizumab.
TABLE 27. Amino acid sequences for ranibizumab.
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Identifier Sequence (One-Letter Amino Acid Symbols)
SEQ DD NO:217
1 EVQLVESGGG LVQPGGSLRL SCAASGYDFT HYGMNWVRQA PGHGLEWVGW INTYTGEPTY
ranibizumab
61 AAJJFKRRFTE SL2TSKSTAY LQMNSLRAE2 TAVYYCAKYY YYYGTSNWYF IWWGQGTLVT
heavy chain
121 VSSASTKGPS VFPLAPSSKS TSGGTAALGC LV7DYFPEPV TVSWNSGALT SGVHTFPAVL
181 QSSGLYSLSS VVTVPSSSLG TQTYICNVNH KPSNTKVDHK VEPKSCDHTH L
SEQ =D NO:218
1 DIQLTQSPSS LSASVGDRVT ITCSASQDIS NYLNWYQQHP GKAPKVLIYF TSSLHSGVPS
ranibizumab
61 RFSGSGSGTD FTLTISSLQP EDFATYYCOO YSTVPWTFGQ GTKVEIKRTV AAPSVFIFPP
light chain
121 SDEQIKSGTA SVVCLLNNEY PREAKVQWKV DNALQSGNSQ ESVTEQDSKD STYSLSSTLT
181 LSHADYEKHK VYACEVTHQG LSSPVTKSFN RGEC
SEQ ID NO:219
1 EVQLVESGGG LVQPGGSLRI SCAASGYDFT HYGMNWVLQA PGKGLEWVGW INTYTGEPTY
ranibizumab
61 AADFERRFTF SLDTSESTAY LQMNSLRAED TAVYYCAEYP YYYGTSHWYF DVWGQGTLVT
variable heavy 121 VS5
chain
SEQ =D NO:220
1 DIQLTQSPSS LSASVGDRVT ITCSASQDIS NYLNWYQQHP GKAPKVLIYY TSSLHSGVPS
ranibizumab 61 RFSGSGSGTD FTLTISSLQP EDFATYYCQQ YSTVPWTFGQ
GTHVEIKR
variable light
chain
SEQ DD NO:221
GYDFTHYGMN 10
ranibizumab
heavy chain
CDR1
SEQ =D NO:222
WINTYTGEPT YAADFHR 17
ranibizumab
heavy chain
CDR2
SEQ =D NO:223
YPYYYGTSHW YFDV 14
ranibizumab
heavy chain
CDR3
SEQ DD NO:224
SASQDISNYL N 11
ranibizumab
light chain
CDR1
SEQ DD NO:225
FTSSLHS 7
ranibizumab
light chain
CDR2
SEQ ID NO:226
QQYSTVPWT 9
ranibizumab
light chain
COR3
1001569] In some embodiments, the VEGF-A inhibitor is icrucumab, or
biosimilars, antigen-binding
fragments, conjugates, or variants thereof Icrucumab (CAS registry number
1024603-92-6, also
known as IMC-18F1) is a human monoclonal antibody designed for the treatment
of solid tumors. A
fully human IgG1 monoclonal antibody directed against human vascular
endothelial growth factor
receptor 1 (VEGFR-1/FLT-1) with potential antiangiogenesis and antineoplastic
activities. Icrucumab
specifically binds to and inhibits the activity of VEGFR-1, which may prevent
the activation of
downstream signaling pathways and so inhibit tumor angiogenesis; the
subsequent reduction in tumor
nutrient supply may inhibit tumor cell proliferation. Tumor cell
overexpression of VEGFR-1 may be
associated with tumor angiogenesis and tumor cell proliferation and invasion;
VECiFR-1 may
modulate VEGFR-2 signaling.
10015701in some embodiments, a VEGF-A inhibitor comprises a heavy chain given
by SEQ ID
NO:227 and a light chain given by SEQ ID NO:228. In some embodiments, a VEGF-A
inhibitor
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comprises heavy and light chains having the sequences shown in SEQ ID NO:227
and SEQ ID NO:
564, respectively, or antigen binding fragments, Fab fragments, single-chain
variable fragments
(scFv), variants, or conjugates thereof. In some embodiments, a VEGF-A
inhibitor comprises heavy
and light chains that are each at least 99% identical to the sequences shown
in SEQ ID NO:227 and
SEQ ID NO:228, respectively. In some embodiments, a VEGF-A inhibitor comprises
heavy and light
chains that arc each at least 98% identical to the sequences shown in SEQ Ill
NO:227 and SEQ Ill
NO:228, respectively. In some embodiments, a VEGF-A inhibitor comprises heavy
and light chains
that are each at least 97% identical to the sequences shown in SEQ ID NO:227
and SEQ ID NO:228,
respectively. In some embodiments, a VEGF-A inhibitor comprises heavy and
light chains that are
each at least 96% identical to the sequences shown in SEQ ID NO:227 and SEQ ID
NO:228,
respectively. In some embodiments, a VEGF-A inhibitor comprises heavy and
light chains that are
each at least 95% identical to the sequences shown in SEQ ID NO:227 and SEQ ID
NO:228,
respectively.
10015711ln some embodiments, the VEGF-A inhibitor comprises the heavy and
light chain CDRs or
variable regions (VRs) of icrucumab. In some embodiments, the VEGF-A inhibitor
heavy chain
variable region (VH) comprises the sequence shown in SEQ ID NO:229, and the
VEGF-A inhibitor
light chain variable region (VL) comprises the sequence shown in SEQ ID
NO:230, and conservative
amino acid substitutions thereof. In some embodiments, a VEGF-A inhibitor
comprises VH and VL
regions that are each at least 99% identical to the sequences shown in SEQ ID
NO:229 and SEQ ID
NO:230, respectively. in some embodiments, a VEGF-A inhibitor comprises VH and
VL regions that
are each at least 98% identical to the sequences shown in SEQ ID NO:229 and
SEQ ID NO:230,
respectively. In some embodiments, a VEGF-A inhibitor comprises VH and VL
regions that are each at
least 97% identical to the sequences shown in SEQ ID NO:229 and SEQ ID NO:230,
respectively. In
some embodiments, a VEGF-A inhibitor comprises VH and VL regions that are each
at least 96%
identical to the sequences shown in SEQ ID NO:229 and SEQ ID NO:230,
respectively. In some
embodiments, a VEGF-A inhibitor comprises VE and VL regions that are each at
least 95% identical to
the sequences shown in SEQ ID NO:229 and SEQ ID NO:230, respectively.
10015721In some embodiments, a VEGF-A inhibitor comprises the heavy chain
CDR1, CDR2 and
CDR3 domains having the sequences set forth in SEQ ID NO:231, SEQ ID NO:232,
and SEQ ID
NO:233, respectively, and conservative amino acid substitutions thereof, and
light chain CDR1,
CDR2 and CDR3 domains having the sequences set forth in SEQ ID NO:234, SEQ ID
NO:235, and
SEQ ID NO:236, respectively, and conservative amino acid substitutions
thereof. In some
embodiments, the antibody competes for binding with, and/or binds to the same
cpitopc on VEGF-A
as any of the aforementioned antibodies.
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[0015731M some embodiments, the VEGF-A inhibitor is a VEGF-A biosimilar
monoclonal antibody
approved by drug regulatory authorities with reference to icrucumab. In some
embodiments, the
biosimilar comprises an anti-VEGF-A antibody comprising an amino acid sequence
which has at least
97% sequence identity, e.g., 97%, 98%, 99% or 100% sequence identity, to the
amino acid sequence
of a reference medicinal product or reference biological product and which
comprises one or more
post-translational modifications as compared to the reference medicinal
product or reference
biological product, wherein the reference medicinal product or reference
biological product is
icrucumab. In some embodiments, the one or more post-translational
modifications are selected from
one or more of: glycosylation, oxidation, deamidation, and truncation. In some
embodiments, the
biosimilar is an anti-VEGF-A antibody authorized or submitted for
authorization, wherein the anti-
VEGF-A antibody is provided in a formulation which differs from the
formulations of a reference
medicinal product or reference biological product, wherein the reference
medicinal product or
reference biological product is icrucumab. The anti-VEGF-A antibody may be
authorized by a drug
regulatory authority such as the U.S. FDA and/or the European Union's EMA. In
some embodiments,
the biosimilar is provided as a composition which further comprises one or
more excipients, wherein
the one or more excipients are the same or different to the excipients
comprised in a reference
medicinal product or reference biological product, wherein the reference
medicinal product or
reference biological product is icrucumab. In some embodiments, the biosimilar
is provided as a
composition which further comprises one or more excipients, wherein the one or
more excipients are
the same or different to the excipients comprised in a reference medicinal
product or reference
biological product, wherein the reference medicinal product or reference
biological product is
icrucumab.
TABLE 28. Amino acid sequences for icrucumab.
Identifier Sequence (One-Letter Amino Acid
Symbols)
SEQ ED NO:227
1 QAQVVESCCG VVQSGRSLRL SCAASGFAFS SYGMHWVRQA PCKGLEWVAV IWYEGSNKYY
icrucumab heavy chain
61 ADSVRGRFTI SRDNSENTLY LQMNSLRAED TAVYYCARDH YGSGVHHYFY YGLEVWGQGT
121 TVTVSSASTK GPSVF'PLAPS SKSTSGGiAA LGCLVKDYFP EPVTVSWNSG ALfSGVHP
181 AVLQSSGLYS LSSVVTVPSS SLGTQTYECN VNHKPSNTKV DKPVEPKSCD KTHTCPPCPA
PELLGGPSVF LFPPhPKDTL MISRTPEVTC VVVDVSHEDP EVEFNWYVDG VEVHNAKTKP
REEQYNSTYR VVSVLTVLHQ DWLNGKEYKC KVSNYALPAP IEKTISKAKG QPREPQVYTL
PPSREEMTXN QVSLTCLVKG FYPSDIAVEW ESNGQPENNY HTTPPVLDSD GSFELYSHLT
VDKSRWCQGN VFSCSVMHEA LHNHYTQKSL SLSPGh
SEQ ED NO:228
1 EIVLTQSPGT LSLSPGERAT LSCRASQSVS SSYLAWYQQK PGQAPRLLIY GASSRATGIP
icrucumab light chain
61 DRFSGSGSGT DFTLTISRLE PEDFAVYYCQ QYGSSPLTFG GGIKVEIKRT VAAPSVFIFP
121 PSNEQIKSGT ASVVCLLNI\Lb YPREAKVQWK VDNALQSGNS QESVTEQDSK DS_LYSLSSTL
181 TLSKADYEEN HVYACEVTHQ GLSSEWTHSF NRGEC
SEQ ED NO :229
QAQWESGGGVVQSGPSLRLECAASGFAFSSYGMNWVRQAPGKGLEWVAVIWYDGSNKYYADSVRGRYTISRDNSEN
icrucumab variable
TLYLQMNSLRAEDTAVYYCARDHYGSGVHHYFYYGLDVWGQGTTVTVSS
heavy chain
SEQ ED NO:230
EIVLTQSPGTLSLSPGERATLSCRASQSVSSSYLAWYQQKPGQAPRLLIYaASSRATGIPDRFSGSGSGTDFTLTI
icrucumab variable SRLEPEDFAVYYCQQYGSSPLTFGGGTHVEIK
light chain
SEQ ED NO:231 VVQSGRS
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Identifier Sequence (One-Letter Amino Acid
Symbols)
icrucumab heavy chain
C2Ri
SEQ =D NO:232 SGFAESSYG
icrucumab heavy chain
CDR2
SEQ =D NO:233 WVAVIWYDGSNKYYADS
icrucumab heavy chain
CDR3
SEQ =D NO:234 LSPGERA
icrucumab light chain
CDRI
SEQ =D NO:235 QSV
icrucumab light chain
CDR2
SEQ =D NO:236 APRLLIYGAS
icrucumab light chain
CDR3
10015741In some embodiments, the VEGF-A inhibitor is a decoy VEGF receptor
(also known as
VEGF trap), such as Aflibercept (Eylea, Zaltrap), which is disclosed in US
Patent Application
Publication No. US2019/0298801 and International Patent Application
Publication No.
W020140061 13A1 (disclosure incorporated herein by reference) or Conbercept,
which is disclosed in
US Patent Application Publication Nos. US2019/0002546 and US2019/0343918
(disclosure
incorporated herein by reference). Additional disclosure and examples of decoy
VEGF receptor is
provided in US Patent Nos. 6,383,486, 6,375,929, 6,348,333, 6,100,071, and
9,777,261, the disclosure
of which are incorporated herein by reference in their entireties.
10015751In some embodiments, the VEGF-A inhibitor is a small molecule tyrosine
kinases inhibitor.
Small molecule tyrosine kinases are known to those of ordinary skill in the
art. Examples of small
molecule tyrosine kinases inhibitor include, but are not limited to,
Pegaptanib, Pazopanib, lapatinib,
Sunitinib, sorafcnib, regorafenib, Ponatinib, lenvatinib, axitinib (AG-
013736), Cediranib (AZD2171),
vatalanib, and/or Lucitanib.
10015761In some embodiments, the VEGF-A inhibitor is an anti-VEGFR ribozyme,
an anti-VEGFR
antisense, and an siRNA that inhibits a VEGFR that are disclosed in US Patent
No. 7,148,342 and
International Patent Application No. W02010058426 (disclosure incorporated
herein by reference in
their entireties).
6. Lymphodepletion Preconditioning of Patients
100157711n some embodiments, the invention includes a method of treating a
cancer with a
population of TILs, wherein a patient is pre-treated with non-myeloablative
chemotherapy prior to an
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infusion of TILs according to the present disclosure. In some embodiments, the
invention includes a
population of TILs for use in the treatment of cancer in a patient which has
been pre-treated with non-
myeloablative chemotherapy. in some embodiments, the population of TILs is for
administration by
infusion. In some embodiments, the non-myeloablative chemotherapy is
cyclophosphamide 60
mg/kg/d for 2 days (days 27 and 26 prior to TIL infusion) and fludarabine 25
mg/m2/d for 5 days
(days 27 to 23 prior to TIL infusion). In some embodiments, after non-
myeloablativc chemotherapy
and TIL infusion (at day 0) according to the present disclosure, the patient
receives an intravenous
infusion of IL-2 (aldesleukin, commercially available as PROLEUKIN)
intravenously at 720,000
IU/kg every 8 hours to physiologic tolerance. In certain embodiments, the
population of TILs is for
use in treating cancer in combination with IL-2, wherein the IL-2 is
administered after the population
of TILs.
10015781 Experimental findings indicate that lymphodepletion prior to adoptive
transfer of tumor-
specific T lymphocytes plays a key role in enhancing treatment efficacy by
eliminating regulatory T
cells and competing elements of the immune system ('cytokine sinks').
Accordingly, some
embodiments of the invention utilize a lymphodepletion step (sometimes also
referred to as
"immunosuppressive conditioning") on the patient prior to the introduction of
the TILs of the
invention.
[001579] In general, lymphodepletion is achieved using administration of
fludarabine or
cyclophosphamide (the active form being referred to as mafosfamide) and
combinations thereof. Such
methods are described in Gassner, et al., Cancer Immunol.ltnnmnother. 2011,
60, 75-85, Muranski,
etal., Nat. Cl/n. Pract. Oncol., 2006, 3, 668-681, Dudley, et al.,J. Cl/n.
Oncol. 2008, 26, 5233-5239,
and Dudley, et al., J. Cl/n. Oncol. 2005, 23, 2346-2357, all of which are
incorporated by reference
herein in their entireties.
[001580] In some embodiments, the fludarabine is administered at a
concentration of 0.5 ng/mL to 10
itig/mL fludarabine. In some embodiments, the fludarabine is administered at a
concentration of 1
ttg/mL fludarabine. In some embodiments, the fludarabine treatment is
administered for 1 day, 2 days,
3 days, 4 days, 5 days, 6 days, or 7 days or more. In some embodiments, the
fludarabine is
administered at a dosage of 10 mg/kg/day, 15 mg/kg/day, 20 mg/kg/days 25
mg/kg/day-,
30 mg/kg/day, 35 mg/kg/day, 40 mg/kg/day, or 45 mg/kg/day. In some
embodiments, the fludarabine
treatment is administered for 2-7 days at 35 mg/kg/day. In some embodiments,
the fludarabine
treatment is administered for 4-5 days at 35 mg/kg/day. In some embodiments,
the fludarabine
treatment is administered for 4-5 days at 25 mg/kg/day.
[001581] In some embodiments, the mafosfamide, the active form of
cyclophosphamide, is obtained
at a concentration of 0.5 ,g/mL to 10 Rg/mL by administration of
cyclophosphamide. In some
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embodiments, mafosfamide, the active form of cyclophosphamide, is obtained at
a concentration of 1
lig/mL by administration of cyclophosphamide. In some embodiments, the
cyclophosphamide
treatment is administered for 1 day, 2 days, 3 days, 4 days, 5 days, 6 days,
or 7 days or more. In some
embodiments, the cyclophosphamide is administered at a dosage of 100
mg/m2/day, 150 mg/m2/day,
175 mg/m2/day% 200 mg/m2/day, 225 mg/m2/day, 250 mg/m2/day, 275 mg/m2/day, or
300 mg/m2/day.
In some embodiments, the cyclophosphamide is administered intravenously (i.e.,
i.v.). In some
embodiments, the cyclophosphamide treatment is administered for 2-7 days at 35
mg/kg/day. In some
embodiments, the cyclophosphamide treatment is administered for 4-5 days at
250 mg/m2/day i.v. In
some embodiments, the cyclophosphamide treatment is administered for 4 days at
250 mg/m2/day i.v.
1001582] In some embodiments, lymphodepletion is performed by administering
the fludarabine and
the cyclophosphamide together to a patient. In some embodiments, fludarabine
is administered at
25 mg/m2/day i.v. and cyclophosphamide is administered at 250 mg/m2/day i.v.
over 4 days.
1001583] In some embodiments, the lymphodepletion is performed by
administration of
cyclophosphamide at a dose of 60 mg/m2/day for two days followed by
administration of fludarabine
at a dose of 25 mg/m2/day for five days.
1001584] In some embodiments, the lymphodepletion is performed by
administration of
cyclophosphamide at a dose of 60 mg/m2/day for two days and administration of
fludarabine at a dose
of 25 mg/m2/day for five days, wherein cyclophosphamide and fludarabine arc
both administered on
the first two days, and wherein the lymphodepletion is performed in five days
in total.
1001585] In some embodiments, the lymphodepletion is performed by
administration of
cyclophosphamide at a dose of about 50 mg/m2/day for two days and
administration of fludarabine at
a dose of about 25 mg/m2/day for five days, wherein cyclophosphamide and
fludarabine arc both
administered on the first two days, and wherein the lymphodepletion is
performed in five days in
total.
1001586] In some embodiments, the lymphodepletion is performed by
administration of
cyclophosphamide at a dose of about 50 mg/m2/day for two days and
administration of fludarabine at
a dose of about 20 mg/m2/day for five days, wherein cyclophosphamide and
fludarabine are both
administered on the first two days, and wherein the lymphodepletion is
performed in five days in
total.
100158711n some embodiments, the lymphodepletion is performed by
administration of
cyclophosphamide at a dose of about 40 mg/m2/day for two days and
administration of fludarabine at
a dose of about 20 mg/m2/day for five days, wherein cyclophosphamide and
fludarabine are both
administered on the first two days, and wherein the lymphodepletion is
performed in five days in
total.
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10015881111 some embodiments, the lymphodepletion is performed by
administration of
cyclophosphamide at a dose of about 40 mg/m2/day for two days and
administration of fludarabine at
a dose of about 15 mg/m2/day for five days, wherein cyclophosphamide and
fludarabine are both
administered on the first two days, and wherein the lymphodepletion is
performed in five days in
total.
[0015891M some embodiments, the lymphodepletion is performed by administration
of
cyclophosphamide at a dose of 60 mg/m2/day and fludarabine at a dose of 25
mg/m2/day for two days
followed by administration of fludarabine at a dose of 25 mg/m2/day for three
days.
10015901111 some embodiments, the cyclophosphamide is administered with mesna.
In some
embodiments, mesna is administered at 15 mg/kg. In some embodiments where
mesna is infused, and
if infused continuously, mesna can be infused over approximately 2 hours with
cyclophosphamide (on
Days -5 and/or -4), then at a rate of 3 mg/kg/hour for the remaining 22 hours
over the 24 hours
starting concomitantly with each cyclophosphamide dose.
10015911111 some embodiments, the lymphodepletion comprises the step of
treating the patient with
an IL-2 regimen starting on the day after administration of the third
population of TILs to the patient.
10015921111 some embodiments, the lymphodepletion comprises the step of
treating the patient with
an 1L-2 regimen starting on the same day as administration of the third
population of TILs to the
patient.
10015931In some embodiments, the lymphodeplete comprises 5 days of
preconditioning treatment. In
some embodiments, the days are indicated as days -5 through -1, or Day 0
through Day 4. In some
embodiments, the regimen comprises cyclophosphamide on days -5 and -4 (i.e.,
days 0 and 1). In
some embodiments, the regimen comprises intravenous cyclophosphamide on days -
5 and -4 (i.e.,
days 0 and 1). In some embodiments, the regimen comprises 60 mg/kg intravenous
cyclophosphamide
on days -5 and -4 (i.e., days 0 and 1). In some embodiments, the
cyclophosphamidc is administered
with mesna. In some embodiments, the regimen further comprises fludarabine. In
some embodiments,
the regimen further comprises intravenous fludarabine. In some embodiments,
the regimen further
comprises 25 mg/m2 intravenous fludarabine. In some embodiments, the regimen
further comprises 25
mg/m2 intravenous fludarabine on days -5 and -1 (i.e., days 0 through 4). In
some embodiments, the
regimen further comprises 25 mg/m2 intravenous fludarabine on days -5 and -1
(i.e., days 0 through
4).
10015941111 some embodiments, the non-myeloablative lymphodepletion regimen
comprises the steps
of administration of cyclophosphamide at a dose of 60 mg/m2/day and
fludarabine at a dose of 25
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mg/m2/day for two days followed by administration of fludarabine at a dose of
25 mg/m2/day for five
days.
10015951 In some embodiments, the non-myeloablative lymphodepletion regimen
comprises the steps
of administration of cyclophosphamide at a dose of 60 mg/m2/day for two days
followed by
administration of fludarabine at a dose of 25 mg/m2/day for five days.
10015961 In some embodiments, the non-myeloablative lymphodepletion regimen
comprises the steps
of administration of cyclophosphamide at a dose of 60 mg/m2/day for two days
followed by
administration of fludarabine at a dose of 25 mg/m2/day for three days.
10015971 In some embodiments, the non-myeloablative lymphodepletion regimen
comprises the steps
of administration of cyclophosphamide at a dose of 60 mg/m2/day and
fludarabine at a dose of 25
mg/m2/day for two days followed by administration of fludarabine at a dose of
25 mg/m2/day for three
days.
10015981 In some embodiments, the non-myeloablative lymphodepletion regimen
comprises the steps
of administration of cyclophosphamide at a dose of 60 mg/m2/day and
fludarabine at a dose of 25
mg/m2/day for two days followed by administration of fludarabine at a dose of
25 mg/m2/day for one
day.
10015991
In some embodiments, the non-myeloablative lymphodepletion regimen
comprises
the steps of administration of cyclophosphamide at a dose of 60 mg/m2/day for
two days followed by
administration of fludarabine at a dose of 25 mg/m2/day for three days.
10016001 In some embodiments, the non-myeloablative lymphodepletion regimen
comprises the
steps of administration of cyclophosphamide at a dose of 60 mg/m2/day and
fludarabine at a dose of
25 mg/m2/day for two days followed by administration of fludarabine at a dose
of 25 mg/m2/day for
three days.
10016011 In some embodiments, the non-myeloablative lymphodepletion regimen is
administered
according to Table 29.
TABLE 29. Exemplary lymphodepletion and treatment regimen.
Day -5 -4 -3 -2 -1 0 1 2 3 4
Cyclophosphamide 60 mg/kg X X
Mesna (as needed) X X
Fludarabine 25 mg/m2/day X X X X X
TIL infusion X
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10016021 In some embodiments, the non-myeloablative lymphodepletion regimen is
administered
according to Table 30.
TABLE 30. Exemplary lymphodepletion and treatment regimen.
Day -4 -3 -2 -1 0 1 2 3 4
Cyclophosphamide 60 mg/kg X X
Mesna (as needed) X X
Fludarabine 25 mg/m2/clay X X X X
TIL infusion X
10016031 In some embodiments, the non-myeloablative lymphodepletion regimen is
administered
according to Table 31.
TABLE 31. Exemplary lymphodepletion and treatment regimen.
Day -3 -2 -1 0 1 2 3 4
Cyclophosphamide 60 mg/kg X X
Mesna (as needed) X X
Fludarabine 25 mg/m2/day X X X
TIL infusion X
10016041 In some embodiments, the non-myeloablative lymphodepletion regimen is
administered
according to Table 32.
TABLE 32. Exemplary lymphodepletion and treatment regimen.
Day -5 -4 -3 -2 -1 0 1 2 3 4
Cyclophosphamide 60 mg/kg X X
Mesna (as needed) X X
Fludarabine 25 mg/m2/day X X X
TIL infusion X
100160511n some embodiments, the non-myeloablative lymphodepletion regimen is
administered
according to Table 33.
TABLE 33. Exemplary lymphodepletion and treatment regimen.
Day -5 -4 -3 -2 -1 0 1 2 3 4
Cyclophosphamide 300 mg/kg X X
Mesna (as needed) X X
Fludarabine 30 mg/m2/day X X X X X
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Day -5 -4 -3 -2 -1 0 1 2 3 4
TIL infusion X
10016061111 some embodiments, the non-myeloablative lymphodepletion regimen is
administered
according to Table 34.
TABLE 34. Exemplary lymphodepletion and treatment regimen.
Day -4 -3 -2 -1 0 1 2 3 4
Cyclophosphamide 300 mg/kg X X
Mesna (as needed) X X
Fludarabine 30 mg/m2/day X X X X
TIL infusion X
100160711n some embodiments, the non-mycloablative lymphodepletion regimen is
administered
according to Table 35.
TABLE 35. Exemplary lymphodepletion and treatment regimen.
Day -3 -2 -1 0 1 2 3 4
Cyclophosphamide 300 mg/kg X X
Mesna (as needed) X X
Fludarabine 30 mg/m2/day X X X
TIL infusion X
100160811n some embodiments, the non-myeloablative lymphodepletion regimen is
administered
according to Table 36.
TABLE 36. Exemplary lymphodepletion and treatment regimen.
Day -5 -4 -3 -2 -1 0 1 2 3 4
Cyclophosphamide 300 mg/kg X X
Mesna (as needed) X X
Fludarabine 30 mg/m2/day X X X
TIL infusion X
10016091 In some embodiments, the TIL infusion used with the foregoing
embodiments of
myeloablative lymphodepletion regimens may be any TIL composition described
herein, as well as
the addition of IL-2 regimens and administration of co-therapies (such as PD-1
and PD-Li inhibitors)
as described herein.
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7. IL-2 Regimens
[001610] In some embodiments, the 1L-2 regimen comprises a high-dose IL-2
regimen, wherein the
high-dose IL-2 regimen comprises aldesleukin, or a biosimilar or variant
thereof, administered
intravenously starting on the day after administering a therapeutically
effective portion of the
therapeutic population of TILs, wherein the aldesleukin or a biosimilar or
variant thereof is
administered at a dose of 0.037 mg/kg or 0.044 mg/kg IU/kg (patient body mass)
using 15-minute
bolus intravenous infusions every eight hours until tolerance, for a maximum
of 14 doses. Following 9
days of rest, this schedule may be repeated for another 14 doses, for a
maximum of 28 doses in total.
In some embodiments, 1L-2 is administered in 1, 2, 3, 4, 5, or 6 doses. In
some embodiments, 1L-2 is
administered at a maximum dosage of up to 6 doses.
10016111 In some embodiments, the IL-2 regimen comprises a decrescendo IL-2
regimen.
Decrescendo IL-2 regimens have been described in O'Day, et at., I Chn. Oncol.
1999, 17, 2752-61
and Eton, et al., Cancer 2000, 88, 1703-9, the disclosures of which are
incorporated herein by
reference. In some embodiments, a decrescendo 1L-2 regimen comprises 18 x 106
IU/m2 aldesleukin,
or a biosimilar or variant thereof, administered intravenously over 6 hours,
followed by 18 x 106
IU/m2 administered intravenously over 12 hours, followed by 18 x 106 IU/m2
administered
intravenously over 24 hours, followed by 4.5 x 106 IU/m2 administered
intravenously over 72 hours.
This treatment cycle may be repeated every 28 days for a maximum of four
cycles. In some
embodiments, a decrescendo IL-2 regimen comprises 18,000,000 IU/m2 on day 1,
9,000,000 IU/m2 on
day 2, and 4,500,000 IU/m2 on days 3 and 4.
[001612] In some embodiments, the IL-2 regimen comprises a low-dose IL-2
regimen. Any low-dose
IL-2 regimen known in the art may be used, including the low-dose IL-2
regimens described in
Dominguez-Villar and Hafler, Nat. Immunology 2000, 19, 665-673; Hartemann, et
al., Lancet
Diabetes Enclocrinol . 2013, /, 295-305; and Rosenzwaig, etal., Ann. Rheum.
Dis. 2019, 78, 209-217,
the disclosures of which are incorporated herein by reference. In some
embodiments, a low-dose IL-2
regimen comprises 18 x 106 III per m2 of aldesleukin, or a biosimilar or
variant thereof, per 24 hours,
administered as a continuous infusion for 5 days, followed by 2-6 days without
IL-2 therapy,
optionally followed by an an additional 5 days of intravenous aldesleukin or a
biosimilar or variant
thereof, as a continuous infusion of 18 x 10' IU per m2 per 24 hours,
optionally followed by 3 weeks
without IL-2 therapy, after which additional cycles may be administered.
10016131 In some embodiments, IL-2 is administered at a maximum dosage of up
to 6 doses. In some
embodiments, the high-dose IL-2 regimen is adapted for pediatric use. In some
embodiments, a dose
of 600,000 international units (1U)/kg of aldesleukin every 8-12 hours for up
to a maximum of 6
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doses is used. In some embodiments, a dose of 500,000 international units
(IU)/kg of aldesleukin
every 8-12 hours for up to a maximum of 6 doses is used. In some embodiments,
a dose of 400,000
international units (IU)/kg of aldesleukin every 8-12 hours for up to a
maximum of 6 doses is used. In
some embodiments, a dose of 500,000 international units (IU)/kg of aldesleukin
every 8-12 hours for
up to a maximum of 6 doses is used. In some embodiments, a dose of 300,000
international units
(1U)/kg of aldesleukin every 8-12 hours for up to a maximum of 6 doses is
used. In some
embodiments, a dose of 200,000 international units (IU)/kg of aldesleukin
every 8-12 hours for up to
a maximum of 6 doses is used. In some embodiments, a dose of 100,000
international units (IU)/kg of
aldesleukin every 8-12 hours for up to a maximum of 6 doses is used.
10016141 In some embodiments, the IL-2 regimen comprises administration of
pegylated IL-2 every
1, 2, 4, 6, 7, 14 or 21 days at a dose of 0.10 mg/day to 50 mg/day. In some
embodiments, the IL-2
regimen comprises administration of bempegaldesleukin, or a fragment, variant,
or biosimilar thereof,
every 1, 2, 4, 6, 7, 14 or 21 days at a dose of 0.10 mg/day to 50 mg/day.
1001615] In some embodiments, the IL-2 regimen comprises administration of
THOR-707, or a
fragment, variant, or biosimilar thereof, every 1, 2, 4, 6, 7, 14 or 21 days
at a dose of 0.10 mg/day to
50 mg/day.
10016161 In some embodiments, the IL-2 regimen comprises administration of
nemvaleukin alfa, or a
fragment, variant, or biosimilar thereof, following administration of TIL. In
certain embodiments, the
patient the nemvaleukin is administered every 1, 2, 4, 6, 7, 14 or 21 days at
a dose of 0.10 mg/day to
50 mg/day.
10016171 In some embodiments, the IL-2 regimen comprises administration of an
IL-2 fragment
engrafted onto an antibody backbone. In some embodiments, the IL-2 regimen
comprises
administration of an antibody-cytokine engrafted protein that binds the IL-2
low affinity receptor. In
some embodiments, the antibody cytokine engrafted protein comprises a heavy
chain variable region
(VII), comprising complementarity determining regions HCDR1, HCDR2, HCDR3; a
light chain
variable region (VL), comprising LCDR1, LCDR2, LCDR3; and an 1L-2 molecule or
a fragment
thereof engrafted into a CDR of the V1 or the VL, wherein the antibody
cytokine engrafted protein
preferentially expands T effector cells over regulatory T cells. In some
embodiments, the antibody
cytokine engrafted protein comprises a heavy chain variable region (VH),
comprising complementarity
determining regions HCDR1, HCDR2, HCDR3; a light chain variable region (VL),
comprising
LCDR1, LCDR2, LCDR3; and an IL-2 molecule or a fragment thereof engrafted into
a CDR of the
VII or the VL, wherein the IL-2 molecule is a mutein, and wherein the antibody
cytokine engrafted
protein preferentially expands T effector cells over regulatory T cells. In
some embodiments, the IL-2
regimen comprises administration of an antibody comprising a heavy chain
selected from the group
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consisting of SEQ ID NO:29 and SEQ ID NO:38 and a light chain selected from
the group consisting
of SEQ ID NO:37 and SEQ ID NO:39, or a fragment, variant, or biosimilar
thereof, every 1, 2, 4, 6, 7,
14 or 21 days at a dose of 0.10 mg/day to 50 mg/day.
10016181 In some embodiments, the antibody cytokine engrafted
protein described herein has a
longer serum half-life that a wild-type IL-2 molecule such as, but not limited
to, aldesleukin
(Proleukink) or a comparable molecule.
10016191ln some embodiments, the TIL infusion used with the foregoing
embodiments of
myeloablative lymphodepletion regimens may be any TIL composition described
herein and may also
include infusions of MILs and PBLs in place of the TIL infusion, as well as
the addition of IL-2
regimens and administration of co-therapies (such as PD-1 and/or PD-Li
inhibitors and/or CTLA-4
inhibitors) as described herein.
8. Additional Methods of Treatment
10016201ln some embodiments, the invention provides a method of treating non-
small cell lung
carcinoma (NSCLC) by administering a population of tumor infiltrating
lymphocytes (TILs) to a
subject or patient in need thereof, wherein the subject or patient has at
least one of:
i. a predetermined tumor proportion score (TPS) of of PD-L1 <1%,
a TPS score of of PD-Li of 1%-49%, or
a predetermined absence of one or more driver mutations, wherein the driver
mutation is
selected from the group consisting of an EGFR mutation, an EGFR insertion, an
EGFR exon 20
mutation, a KRAS mutation, a BRAF mutation, an ALK mutation, a c-ROS mutation
(ROS1
mutation), a ROS1 fusion, a RET mutation, a RET fusion, an ERBB2 mutation, an
ERBB2
amplification, a BRCA mutation, a MAP2K1 mutation, PIK3CA, CDKN2A, a PTEN
mutation, an
UMD mutation, an NRAS mutation; a KRAS mutation, an NF1 mutation,a MET
mutation, a MET
splice and/or altered MET signaling, a TP53 mutation, a CREBBP mutation, a
KMT2C mutation, a
KMT2D mutation, an ARID 1A mutation, a RB1 mutation, an ATM mutation, a SETD2
mutation, a
FLT3 mutation, a PTPN11 mutation, a FGFR1 mutation, an EP300 mutation, a MYC
mutation, an
EZH2 mutation, a JAK2 mutation, a FBXW7 mutation, a CCND3 mutation, and a
GNAll mutation,
and wherein the method comprises:
(a) obtaining and/or receiving a first population of TILs from a tumor
resected from the
subject or patient by processing a tumor sample obtained from the subject into
multiple
tumor fragments;
(b) adding the first population of TILs into a closed system;
(c) performing a first expansion by culturing the first population of TILs in
a cell culture
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medium comprising IL-2 to produce a second population of TILs, wherein the
first
expansion is performed in a closed container providing a first gas-permeable
surface area;
wherein the first expansion is performed for about 3-14 days to obtain the
second
population of TILs, and wherein the transition from step (b) to step (c)
occurs without
opening the system;
(d) performing a second expansion by supplementing the cell culture medium of
the second
population of TILs with additional IL-2, OKT-3, and antigen presenting cells
(APCs), to
produce a third population of TILs, wherein the second expansion is performed
for about
7-14 days to obtain the third population of TILs, wherein the third population
of TILs is a
therapeutic population of TILs, wherein the second expansion is performed in a
closed
container providing a second gas-permeable surface area, and wherein the
transition from
step (c) to step (d) occurs without opening the system;
(c) harvesting therapeutic population of TILs obtained from step (d), wherein
the transition
from step (d) to step (e) occurs without opening the system; and
(0 transferring the harvested TIL population from step (e) to an infusion bag,
wherein the
transfer from step (e) to (f) occurs without opening the system;
(g) cryopreserving the infusion bag comprising the harvested TIL population
from step (0
using a cryopreservation process; and
(h) administering a therapeutically effective dosage of the third population
of TILs from the
infusion bag in step (g) to the subject or patient.
10016211ln some embodiments, the invention provides a method of treating non-
small cell lung
carcinoma (NSCLC) by administering a population of tumor infiltrating
lymphocytes (T1Ls) to a
patient in need thereof, wherein the method comprises:
(a) testing the patient's tumor for PD-Li expression and tumor proportion
score (TPS) of
PD-L1,
(b) testing the patient for the absence of one or more driver mutations,
wherein the driver
mutation is selected from the group consisting of an EGFR mutation, an EGFR
insertion,
an EGFR exon 20 mutation, a KRAS mutation, a BRAF mutation, an ALK mutation, a
c-
ROS mutation (ROS1 mutation), a ROS1 fusion, a RET mutation, a RET fusion, an
ERBB2 mutation, an ERBB2 amplification, a BRCA mutation, a MAP2K1 mutation,
PIK3CA, CDKN2A, a PTEN mutation, an UMD mutation, an NRAS mutation, a KRAS
mutation, an NF1 mutation,a MET mutation, a MET splice and/or altered MET
signaling,
a TP53 mutation, a CREBBP mutation, a KMT2C mutation, a 1(1VIT2D mutation, an
ARID1A mutation, a RB1 mutation, an ATM mutation, a SETD2 mutation, a FLT3
mutation, a PTPN11 mutation, a FGFR1 mutation, an EP300 mutation, a MYC
mutation,
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an EZH2 mutation, a JAK2 mutation, a FBXW7 mutation, a CCND3 mutation, and a
GNAll mutation,
(c) determining that the patient has a TPS score for PD-Li of about 1% to
about 49% and
determining that the patient also has no driver mutations,
(d) obtaining and/or receiving a first population of TILs from a tumor
resected from the
subject or patient by processing a tumor sample obtained from the subject into
multiple
tumor fragments;
(e) adding the first population of TILs into a closed system;
(f) performing a first expansion by culturing the first population of TILs in
a cell culture
medium comprising IL-2 to produce a second population of TILs, wherein the
first
expansion is performed in a closed container providing a first gas-permeable
surface area,
wherein the first expansion is performed for about 3-14 days to obtain the
second
population of TILs, and wherein the transition from step (e) to step (f)
occurs without
opening the system;
(g) performing a second expansion by supplementing the cell culture medium of
the second
population of TILs with additional IL-2, OKT-3, and antigen presenting cells
(APCs), to
produce a third population of TILs, wherein the second expansion is performed
for about
7-14 days to obtain the third population of TILs, wherein the third population
of TILs is a
therapeutic population of TILs; wherein the second expansion is performed in a
closed
container providing a second gas-permeable surface area, and wherein the
transition from
step (f) to step (g) occurs without opening the system;
(h) harvesting therapeutic population of TILs obtained from step (d), wherein
the transition
from step (d) to step (e) occurs without opening the system; and
(i) transferring the harvested TIL population from step (e) to an infusion
bag, wherein the
transfer from step (e) to (f) occurs without opening the system;
(j) cryopreserving the infusion bag comprising the harvested TIL population
from step (f)
using a cryopreservation process; and
(k) administering a therapeutically effective dosage of the third population
of TILs from the
infusion bag in step (g) to the subject or patient.
10016221 In some embodiments, the invention provides a method of treating non-
small cell lung
carcinoma (NSCLC) by administering a population of tumor infiltrating
lymphocytes (TILs) to a
patient in need thereof, wherein the method comprises:
(a) testing the patient's tumor for PD-Li expression and tumor proportion
score (TPS) of
PD-L1,
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(b) testing the patient for the absence of one or more driver mutations,
wherein the driver
mutation is selected from the group consisting of an EGFR mutation, an EGFR
insertion,
an EGFR exon 20 mutation, a KRAS mutation, a BRAF mutation, an ALK mutation, a
c-
ROS mutation (ROS1 mutation), a ROS1 fusion, a RET mutation, a RET fusion, an
ERBB2 mutation, an ERBB2 amplification, a BRCA mutation, a MAP2K1 mutation,
P1K3CA, CDKN2A, a PTEN mutation, an UMD mutation, an NRAS mutation, a KRAS
mutation, an NF1 mutation,a MET mutation, a MET splice and/or altered MET
signaling,
a TP53 mutation, a CREBBP mutation, a KMT2C mutation, a KMT2D mutation, an
ARID1A mutation, a RB1 mutation, an ATM mutation, a SETD2 mutation, a FLT3
mutation, a PTPN11 mutation, a FGFR1 mutation, an EP300 mutation, a MYC
mutation,
an EZH2 mutation, a JAK2 mutation, a FBXW7 mutation, a CCND3 mutation, and a
GNAll mutation,
(c) determining that the patient has a TPS score for PD-Li of less than about
1% and
determining that the patient also has no driver mutations,
(d) obtaining and/or receiving a first population of TILs from a tumor
resected from the
subject or patient by processing a tumor sample obtained from the subject into
multiple
tumor fragments;
(e) adding the first population of TILs into a closed system;
(f) performing a first expansion by culturing the first population of TILs in
a cell culture
medium comprising IL-2 to produce a second population of TILs, wherein the
first
expansion is performed in a closed container providing a first gas-permeable
surface area,
wherein the first expansion is performed for about 3-14 days to obtain the
second
population of TILs, and wherein the transition from step (e) to step (f)
occurs without
opening the system;
(g) performing a second expansion by supplementing the cell culture medium of
the second
population of TILs with additional IL-2, OKT-3, and antigen presenting cells
(APCs), to
produce a third population of TILs, wherein the second expansion is performed
for about
7-14 days to obtain the third population of TILs, wherein the third population
of TILs is a
therapeutic population of TILs, wherein the second expansion is performed in a
closed
container providing a second gas-pemieable surface area, and wherein the
transition from
step (f) to step (g) occurs without opening the system;
(h) harvesting therapeutic population of TILs obtained from step (d), wherein
the transition
from step (d) to step (e) occurs without opening the system; and
(i) transferring the harvested TIL population from step (e) to an infusion
bag, wherein the
transfer from step (e) to (f) occurs without opening the system;
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(j) cryopreserving the infusion bag comprising the harvested TIL population
from step (f)
using a cryopreservation process; and
(k) administering a therapeutically effective dosage of the third population
of TILs from the
infusion bag in step (g) to the subject or patient.
10016231 In some embodiments, the invention provides a method of treating non-
small cell lung
carcinoma (NSCLC) by administering a population of tumor infiltrating
lymphocytes (TILs) to a
patient in need thereof, wherein the method comprises:
(a) testing the patient's tumor for PD-Li expression and tumor proportion
score (TPS) of
PD-Li,
(b) testing the patient for the absence of one or more driver mutations,
wherein the driver
mutation is selected from the group consisting of an EGFR mutation, an EGFR
insertion,
a KRAS mutation, a BRAF mutation, an ALK mutation, a c-ROS mutation (ROS1
mutation), a ROS1 fusion, a RET mutation, or a RET fusion,
(c) determining that the patient has a TPS score for PD-L1 of about 1% to
about 49% and
determining that the patient also has no driver mutations,
(d) obtaining and/or receiving a first population of TILs from a tumor
resected from the
subject or patient by processing a tumor sample obtained from the subject into
multiple
tumor fragments;
(e) adding the first population of TILs into a closed system;
(0 performing a first expansion by culturing the first population of TILs in a
cell culture
medium comprising IL-2 to produce a second population of TILs, wherein the
first
expansion is performed in a closed container providing a first gas-permeable
surface area;
wherein the first expansion is performed for about 3-14 days to obtain the
second
population of TILs, and wherein the transition from step (e) to step (f)
occurs without
opening the system;
(g) performing a second expansion by supplementing the cell culture medium of
the second
population of TILs with additional IL-2, OKT-3, and antigen presenting cells
(APCs), to
produce a third population of TILs, wherein the second expansion is performed
for about
7-14 days to obtain the third population of TILs, wherein the third population
of TILs is a
therapeutic population of TTI,s, wherein the second expansion is performed in
a closed
container providing a second gas-permeable surface area, and wherein the
transition from
step (0 to step (g) occurs without opening the system;
(h) harvesting therapeutic population of TILs obtained from step (d), wherein
the transition
from step (d) to step (e) occurs without opening the system; and
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(i) transferring the harvested TIL population from step (e) to an infusion
bag, wherein the
transfer from step (e) to (0 occurs without opening the system;
(j) cryopreserving the infusion bag comprising the harvested TIL population
from step (0
using a cryopreservation process; and
(k) administering a therapeutically effective dosage of the third population
of TILs from the
infusion bag in step (g) to the subject or patient.
[001624] In some embodiments, the invention provides a method of treating non-
small cell lung
carcinoma (NSCLC) by administering a population of tumor infiltrating
lymphocytes (TILs) to a
patient in need thereof, wherein the method comprises:
(a) testing the patient's tumor for PD-Li expression and tumor proportion
score (TPS) of
PD-L1,
(b) testing the patient for the absence of one or more driver mutations,
wherein the driver
mutation is selected from the group consisting of an EGFR mutation, an EGFR
insertion,
a KRAS mutation, a BRAF mutation, an ALK mutation, a c-ROS mutation (ROS1
mutation), a ROS1 fusion, a RET mutation, or a RET fusion,
(c) determining that the patient has a TPS score for PD-Ll of less than about
1% and
determining that the patient also has no driver mutations,
(d) obtaining and/or receiving a first population of TILs from a tumor
resected from the
subject or patient by processing a tumor sample obtained from the subject into
multiple
tumor fragments;
(e) adding the first population of TILs into a closed system;
(0 performing a first expansion by culturing the first population of TILs in a
cell culture
medium comprising IL-2 to produce a second population of TILs, wherein the
first
expansion is performed in a closed container providing a first gas-permeable
surface area,
wherein the first expansion is performed for about 3-14 days to obtain the
second
population of TILs, and wherein the transition from step (e) to step (0 occurs
without
opening the system;
(g) performing a second expansion by supplementing the cell culture medium of
the second
population of TILs with additional IL-2, OKT-3, and antigen presenting cells
(APCs), to
produce a third population of TILs, wherein the second expansion is performed
for about
7-14 days to obtain the third population of TILs, wherein the third population
of TILs is a
therapeutic population of TILs, wherein the second expansion is performed in a
closed
container providing a second gas-permeable surface area, and wherein the
transition from
step (0 to step (g) occurs without opening the system;
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(h) harvesting therapeutic population of TILs obtained from step (d), wherein
the transition
from step (d) to step (e) occurs without opening the system; and
(i) transfen-ing the harvested TIL population from step (e) to an infusion
bag, wherein the
transfer from step (e) to (f) occurs without opening the system;
(j) cryopreserving the infusion bag comprising the harvested TIL population
from step (f)
using a cryoprescrvation process; and
(k) administering a therapeutically effective dosage of the third population
of TILs from the
infusion bag in step (g) to the subject or patient.
[0016251ln some embodiments, the second population of TILs is at least 50-fold
greater in number
than the first population of TILs,
100162611n some embodiments, the invention provides a method for treating a
subject with cancer
comprising administering to the subject a therapeutically effective dosage of
the therapeutic TIL
population described in any of the preceding paragraphs above.
10016271 In some embodiments, the invention provides a method for treating a
subject with cancer
comprising administering to the subject a therapeutically effective dosage of
the TIL composition
described in any of the preceding paragraphs above.
[0016281ln some embodiments, the invention provides the method for treating a
subject with cancer
described in any of the preceding paragraphs above modified such that prior to
administering the
therapeutically effective dosage of the therapeutic TIL population and the TIL
composition described
in any of the preceding paragraphs above, respectively, a non-myeloablative
lymphodepletion
regimen has been administered to the subject.
10016291ln some embodiments, the invention provides the method for treating a
subject with cancer
described in any of the preceding paragraphs above modified such that the non-
myeloablative
lymphodepletion regimen comprises the steps of administration of
cyclophosphamide at a dose of 60
mg/m2/day for two days followed by administration of fludarabine at a dose of
25 mg/m2/day for five
days.
[001630] In some embodiments, the invention provides the method for treating a
subject with cancer
described in any of the preceding paragraphs above modified to further
comprise the step of treating
the subject with a high-dose IL-2 regimen starting on the day after
administration of the TIL cells to
thc subject.
[001631] In some embodiments, the invention provides the method for treating a
subject with cancer
described in any of the preceding paragraphs above modified such that the high-
dose IL-2 regimen
comprises 600,000 or 720,000 I1J/kg administered as a 15-minute bolus
intravenous infusion every
eight hours until tolerance.
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[001632] In some embodiments, the invention provides the method for treating a
subject with cancer
described in any of the preceding paragraphs above modified such that the
cancer is a solid tumor.
[001633] In some embodiments, the invention provides the method for treating a
subject with cancer
described in any of the preceding paragraphs above modified such that the
cancer is melanoma,
ovarian cancer, cervical cancer, non-small-cell lung cancer (NSCLC), lung
cancer, bladder cancer,
breast cancer, cancer caused by human papilloma virus, head and neck cancer
(including head and
neck squamous cell carcinoma (HNSCC)), glioblastoma (including GBM),
gastrointestinal cancer,
renal cancer, or renal cell carcinoma.
[001634] In some embodiments, the invention provides the method for treating a
subject with cancer
described in any of the preceding paragraphs above modified such that the
cancer is melanoma,
HNSCC, cervical cancers, NSCLC, glioblastoma (including GBM), and
gastrointestinal cancer.
[001635] In some embodiments, the invention provides the method for treating a
subject with cancer
described in any of the preceding paragraphs above modified such that the
cancer is melanoma.
[001636] In some embodiments, the invention provides the method for treating a
subject with cancer
described in any of the preceding paragraphs above modified such that the
cancer is HNSCC.
[001637] In some embodiments, the invention provides the method for treating a
subject with cancer
described in any of the preceding paragraphs above modified such that the
cancer is a cervical cancer.
[001638] In some embodiments, the invention provides the method for treating a
subject with cancer
described in any of the preceding paragraphs above modified such that the
cancer is NSCLC.
[001639] In some embodiments, the invention provides the method for treating a
subject with cancer
described in any of the preceding paragraphs above modified such that the
cancer is glioblastoma
(including GBM).
[001640] In some embodiments, the invention provides the method for treating a
subject with cancer
described in any of the preceding paragraphs above modified such that the
cancer is gastrointestinal
cancer.
[001641] In some embodiments, the invention provides the method for treating a
subject with cancer
described in any of the preceding paragraphs above modified such that the
cancer is a hypermutated
cancer.
[001642] In some embodiments, the invention provides the method for treating a
subject with cancer
described in any of the preceding paragraphs above modified such that the
cancer is a pediatric
hypermutated cancer.
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10016431 In some embodiments, the invention provides the therapeutic TIL
population described in
any of the preceding paragraphs above for use in a method for treating a
subject with cancer
comprising administering to the subject a therapeutically effective dosage of
the therapeutic TIL
population.
1001644] In some embodiments, the invention provides the TIL composition
described in any of the
preceding paragraphs above for use in a method for treating a subject with
cancer comprising
administering to the subject a therapeutically effective dosage of the T1L
composition.
10016451 In some embodiments, the invention provides the therapeutic TIL
population described in
any of the preceding paragraphs above or the TIL composition described in any
of the preceding
paragraphs above modified such that prior to administering to the subject the
therapeutically effective
dosage of the therapeutic TIL population described in any of the preceding
paragraphs above or the
TIL composition described in any of the preceding paragraphs above, a non-
myeloablative
lympho depletion regimen has been administered to the subject.
10016461 In some embodiments, the invention provides the therapeutic TIL
population or the TIL
composition described in any of the preceding paragraphs above modified such
that the non-
myeloablative lymphodepletion regimen comprises the steps of administration of
cyclophosphamide
at a dose of 60 mg/m2/day for two days followed by administration of
fludarabine at a dose of 25
mg/m2/day for five days.
10016471 In some embodiments, the invention provides the therapeutic TIL
population or the TIL
composition described in any of the preceding paragraphs above modified to
further comprise the step
of treating patient with a high-dose 1L-2 regimen starting on the day after
administration of the TIL
cells to the patient.
10016481 In some embodiments, the invention provides the therapeutic TIL
population or the TIL
composition described in any of the preceding paragraphs above modified such
that the high-dose IL-
2 regimen comprises 600,000 or 720,000 I1J/kg administered as a 15-minute
bolus intravenous
infusion every eight hours until tolerance.
10016491 In some embodiments, the invention provides the therapeutic TIL
population or the TIL
composition described in any of the preceding paragraphs above modified such
that the cancer is a
solid tumor.
10016501 In some embodiments, the invention provides the therapeutic TIL
population or the TIL
composition described in any of the preceding paragraphs above modified such
that the cancer is
melanoma, ovarian cancer, cervical cancer, non-small-cell lung cancer (NSCLC),
lung cancer, bladder
cancer, breast cancer, cancer caused by human papilloma virus, head and neck
cancer (including head
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and neck squamous cell carcinoma (HNSCC)), glioblastoma (including GBM),
gastrointestinal
cancer, renal cancer, or renal cell carcinoma.
[00165111n some embodiments, the invention provides the therapeutic TIL
population or the TIL
composition described in any of the preceding paragraphs above modified such
that the cancer is
melanoma, HNSCC, cervical cancers, NSCLC, glioblastoma (including GBM), and
gastrointestinal
cancer.
[0016521M some embodiments, the invention provides the therapeutic TIL
population or the TIL
composition described in any of the preceding paragraphs above modified such
that the cancer is
melanoma.
[0016531ln some embodiments, the invention provides the therapeutic TIL
population or the TIL
composition described in any of the preceding paragraphs above modified such
that the cancer is
HNSCC.
[0016541ln some embodiments, the invention provides the therapeutic TIL
population or the TIL
composition described in any of the preceding paragraphs above modified such
that the cancer is a
cervical cancer.
[0016551ln some embodiments, the invention provides the therapeutic TIL
population or the TIL
composition described in any of the preceding paragraphs above modified such
that the cancer is
NSCLC.
[001656] In some embodiments, the invention provides the therapeutic TEL
population or the TIL
composition described in any of the preceding paragraphs above modified such
that the cancer is
glioblastoma (including GBM).
[00165711n some embodiments, the invention provides the therapeutic TIL
population or the T1L
composition described in any of the preceding paragraphs above modified such
that the cancer is
gastrointestinal cancer.
[0016581ln some embodiments, the invention provides the therapeutic TIL
population or the TIL
composition described in any of the preceding paragraphs above modified such
that the cancer is a
hypermutated cancer.
[0016591ln some embodiments, the invention provides the therapeutic TIL
population or the TIL
composition described in any of the preceding paragraphs above modified such
that the cancer is a
pediatric hypermutated cancer.
[001660] In some embodiments, the invention provides the use of the
therapeutic TIL population
described in any of any of the preceding paragraphs above in a method of
treating cancer in a subject
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comprising administering to the subject a therapeutically effective dosage of
the therapeutic TIL
population.
10016611 In some embodiments, the invention provides the use of the TIL
composition described in
any of the preceding paragraphs above in a method of treating cancer in a
subject comprising
administering to the subject a therapeutically effective dosage of the TIL
composition.
1001662] In some embodiments, the invention provides the use of the
therapeutic TIL population
described in any of the preceding paragraphs above or the TIL composition
described in any of the
preceding paragraphs above in a method of treating cancer in a subject
comprising administering to
the subject a non-myeloablative lymphodepletion regimen and then administering
to the subject the
therapeutically effective dosage of the therapeutic TIL population described
in any of the preceding
paragraphs above or the therapeutically effective dosage of the TIL
composition described in any of
the preceding paragraphs above.
EXAMPLES
10016631 The embodiments encompassed herein arc now described with reference
to the following
examples. These examples are provided for the purpose of illustration only and
the disclosure
encompassed herein should in no way be construed as being limited to these
examples, but rather
should be construed to encompass any and all variations which become evident
as a result of the
teachings provided herein.
EXAMPLE 1: PREPARATION OF MEDIA FOR PRE-REP AND REP PROCESSES
10016641 This Example describes the procedure for the preparation of tissue
culture media for use in
protocols involving the culture of tumor infiltrating lymphocytes (TIL)
derived from various tumor
types including non-small cell lung carcinoma (NSCLC). This media can be used
for preparation of
any of the TILs described in the present application and Examples.
Preparation of CM1
10016651 Removed the following reagents from cold storage and warmed them in a
37 C water bath:
(RPMI1640, Human AB serum, 200mM L-glutamine). Prepared CM1 medium according
to Table 37
below by adding each of the ingredients into the top section of a 0.2um filter
unit appropriate to the
volume to be filtered. Store at 4 C.
TABLE 37: Preparation of CM1
Ingredient Final concentration Final Volume 500 ml
Final Volume IL
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RPMI1640 NA 450 ml 900 ml
Human AB serum, 50 ml 100 ml
heat-inactivated 10%
200mM L-glutamine 2 mM 5 nil 10 ml
55mM BME 55 hM 0.5 ml 1 ml
50mg/mL gcntamicin 50 hg/mL 0.5 ml 1 ml
sulfate
10016661011 the day of use, prewarmed required amount of CM1 in 37 C water
bath and add 6000
IU/ml IL-2.
10016671Additional supplementation - as needed according to Table 38.
TABLE 38: Additional supplementation of CM1, as needed.
Supplement Stock concentration Dilution Final
concentration
GlutaMAX' 200mM 1:100 2mM
Penicillin/streptomycin 10,000 U/mL penicillin 1:100 100 U/mL
penicillin
10,000 hg/mL 100 hg/mL
streptomycin
streptomycin
Amphotericin B 250h1g/mL 1:100 2.5hg/mL
Preparation of CM2
19016681 Removed prepared CM1 from refrigerator or prepare fresh CM1 as per
Section 7.3 above.
Removed AIM-V from refrigerator and prepared the amount of CM2 needed by
mixing prepared
CM I with an equal volume of AIM-V in a sterile media bottle. Added 3000 I
U/ni L IL-2 to CM2
medium on the day of usage. Made sufficient amount of CM2 with 3000 IU/mL IL-2
on the day of
usage. Labeled the CM2 media bottle with its name, the initials of the
preparer, the date it was
filtered/prepared, the two-week expiration date and store at 4 C until needed
for tissue culture.
Preparation of CM3
10016691Prepared CM3 on the day it was required for use. CM3 was the same as
AIM-V medium,
supplemented with 3000 IU/mL IL-2 on the day of use. Prepared an amount of CM3
sufficient to
experimental needs by adding IL-2 stock solution directly to the bottle or bag
of AIM-V. Mixed well
by gentle shaking. Label bottle with "3000 IU/mL IL-2" immediately after
adding to the AIM-V. If
there was excess CM3, stored it in bottles at 4 C labeled with the media name,
the initials of the
preparer, the date the media was prepared, and its expiration date (7 days
after preparation). Discarded
media supplemented with IL-2 after 7 days storage at 4 C.
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Preparation of CM4
10016701 CM4 was the same as CM3, with the additional supplement of 2mM
GlutaMAX' (final
concentration). For every IL of CM3, added 10m1 of 200mM GlutaMAXTm. Prepared
an amount of
CM4 sufficient to experimental needs by adding IL-2 stock solution and
GlutaMAX' stock solution
directly to the bottle or bag of AIM-V. Mixed well by gentle shaking. Labeled
bottle with "3000
IL/nil IL-2 and GlutaMAX- immediately after adding to the AIM-V. If there was
excess CM4, stored
it in bottles at 4 C labeled with the media name, "GlutaMAX", and its
expiration date (7 days after
preparation). Discarded media supplemented with IL-2 after 7-days storage at 4
C.
EXAMPLE 2: USE OF IL-2, IL-15, AND IL-21 CYTOKINE COCKTAIL
10016711 This example describes the use of IL-2, IL-15, and IL-21 cytokincs,
which serve as
additional T cell growth factors, in combination with the TIL process of
Examples A to G.
10016721Using the processes described herein, TILs can be grown from non-small
cell lung
carcinoma (NSCLC) tumors in presence of IL-2 in one arm of the experiment and,
in place of IL-2, a
combination of IL-2, IL-15, and IL-21 in another arm at the initiation of
culture. At the completion of
the pre-REP, cultures were assessed for expansion, phenotype, function (CD107a-
F and IFN-y) and
TCR V13 repertoire. 1L-15 and 1L-21 are described elsewhere herein and in
Gruijl, et al., 1L-21
promotes the expansion of CD27+CD28+ tumor infiltrating lymphocytes with high
cytotoxic potential
and low collateral expansion of regulatory T cells, Santegoets, S I, J Transl
Med., 2013, 11:37
(https://www.ncbi.nlm.nih.gov/pmearticles/PMC3626797/).
10016731 The results can show that enhanced TIL expansion (>20%), in both CD4
and CD8+ cells in
the 1L-2, 1L-15, and 1L-21 treated conditions can observed relative to the 1L-
2 only conditions. There
was a skewing towards a predominantly CD8' population with a skewed TCR VI3
repertoire in the
TILs obtained from the IL-2. IL-15, and IL-21 treated cultures relative to the
IL-2 only cultures. IFN-y
and CD107a were elevated in the IL-2, IL-15, and IL-21 treated TILs, in
comparison to TILs treated
only IL-2.
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EXAMPLE 3: QUALIFYING INDIVIDUAL LOTS OF GAMMA-IRRADIATED
PERIPHERAL MONONUCLEAR CELLS
10016741 This Example describes an abbreviated procedure for
qualifying individual lots of
gamma-irradiated peripheral mononuclear cells (PBMCs, also known as
mononuclear cells or MNCs)
for use as allogeneic feeder cells in the exemplary methods described herein.
10016751 Each irradiated MNC feeder lot was prepared from an
individual donor. Each lot or
donor was screened individually for its ability to expand TIL in the REP in
the presence of purified
anti-CD3 (clone OKT3) antibody and interleukin-2 (IL-2). In addition, each lot
of feeder cells was
tested without the addition of TIL to verify that the received dose of gamma
radiation was sufficient
to render them replication incompetent.
10016761 Gamma-irradiated, growth-arrested MNC feeder cells are
required for REP of TILs.
Membrane receptors on the feeder MNCs bind to anti-CD3 (clone OKT3) antibody
and crosslink to
TILs in the REP flask, stimulating the TIL to expand. Feeder lots were
prepared from the
leukapheresis of whole blood taken from individual donors. The leukapheresis
product was subjected
to centrifugation over Ficoll-Hypaque, washed, irradiated, and cryopreserved
under GMP conditions.
10016771 It is important that patients who received TIL therapy
not be infused with viable
feeder cells as this can result in graft-versus-host disease (GVHD). Feeder
cells are therefore growth-
arrested by dosing the cells with gamma-irradiation, resulting in double
strand DNA breaks and the
loss of cell viability of the MNC cells upon re-culture.
10016781 Feeder lots were evaluated on two criteria: (1) their
ability to expand TILs in co-
culture >100-fold and (2) their replication incompetency.
10016791 Feeder lots were tested in mini-REP format utilizing two
primary pre-REP TIL lines
grown in upright T25 tissue culture flasks. Feeder lots were tested against
two distinct TIL lines, as
each TIL line is unique in its ability to proliferate in response to
activation in a REP. As a control, a
lot of irradiated MNC feeder cells which has historically been shown to meet
the criteria above was
run alongside the test lots.
10016801 To ensure that all lots tested in a single experiment
receive equivalent testing,
sufficient stocks of the same pre-REP TIL lines were available to test all
conditions and all feeder
lots.
10016811 For each lot of feeder cells tested, there was a total of
six T25 flasks: Pre-REP TIL
line #1(2 flasks); Pre-REP TIL line #2 (2 flasks); and feeder control (2
flasks). Flasks containing TIL
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lines #1 and #2 evaluated the ability of the feeder lot to expand TIL. The
feeder control flasks
evaluated the replication incompetence of the feeder lot.
A. Experimental Protocol
10016821 Day -2/3, Thaw of TIL lines. Prepare CM2 medium and warm
CM2 in 37 C water
bath. Prepare 40 mL of CM2 supplemented with 3000 IU/mL IL-2. Keep warm until
use. Place 20 mL
of pre-warmed CM2 without IL-2 into each of two 50 mL conical tubes labeled
with names of the TIL
lines used. Removed the two designated pre-REP TIL lines from LN2 storage and
transferred the vials
to the tissue culture room. Thawed vials by placing them inside a sealed
zipper storage bag in a 37 C
water bath until a small amount of ice remains.
10016831 Using a sterile transfer pipet, the contents of each vial
were immediately transferred
into the 20 mL of CM2 in the prepared, labeled 50 mL conical tube. QS to 40 mL
using CM2 without
IL-2 to wash cells and centrifuged at 400 >< CF for 5 minutes. Aspirated the
supernatant and resuspend
in 5 mL warm CM2 supplemented with 3000 IU/mL IL-2.
10016841 A small aliquot (20 iiL) was removed in duplicate for
cell counting using an
automated cell counter. The counts were recorded. While counting, the 50 mL
conical tube with T1L
cells was placed into a humidified 37 C, 5% CO? incubator, with the cap
loosened to allow for gas
exchange. The cell concentration was determined, and the TILs were diluted to
1 106 cells/mL in
CM2 supplemented with IL-2 at 3000 IU/mL.
10016851 Cultured in 2 mL/well of a 24-well tissue culture plate
in as many wells as needed in
a humidified 37 C incubator until Day 0 of the mini-REP. The different TIL
lines were cultured in
separate 24-well tissue culture plates to avoid confusion arid potential cross-
contamination.
10016861 Day 0, initiate Mini-REP. Prepared enough CM2 medium for
the number of feeder
lots to be tested. (e.g., for testing 4 feeder lots at one time, prepared 800
mL of CM2 medium).
Aliquoted a portion of the CM2 prepared above and supplemented it with 3000
IU/mL IL-2 for the
culturing of the cells. (e.g., for testing 4 feeder lots at one time, prepare
500 mL of CM2 medium with
3000 IU/mL IL-2).
10016871 Working with each TIL line separately to prevent cross-
contamination, the 24-well
plate with TIL culture was removed from the incubator and transferred to the
BSC.
10016881 Using a sterile transfer pipet or 100-1000 p.1_, pipettor
and tip, about 1 mL of medium
was removed from each well of TILs to be used and placed in an unused well of
the 24-well tissue
culture plate.
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[001689] Using a fresh sterile transfer pipet or 100-1000 1i1_,
pipettor and tip, the remaining
medium was mixed with TILs in wells to resuspend the cells and then
transferred the cell suspension
to a 50 mL conical tube labeled with the TIL lot name and recorded the volume.
[001690] Washed the wells with the reserved media and transferred
that volume to the same 50
mL conical tube. Spun the cells at 400 x CF to collect the cell pellet.
Aspirated off the media
supernatant and resuspend the cell pellet in 2-5 mL of CM2 medium containing
3000 IU/mL IL-2,
volume to be used based on the number of wells harvested and the size of the
pellet ¨ volume should
be sufficient to ensure a concentration of >1.3 x 106 cells/mL.
[001691] Using a serological pipet, the cell suspension was mixed
thoroughly and the volume
was recorded. Removed 2001AL for a cell count using an automated cell counter.
While counting,
placed the 50 mL conical tube with TIL cells into a humidified, 5% CO2, 37 C
incubator, with the cap
loosened to allow gas exchange. Recorded the counts.
[001692] Removed the 50 mL conical tube containing the TIL cells
from the incubator and
resuspend them cells at a concentration of 1.3 x106 cells/mL in warm CM2
supplemented with 3000
IU/mL 1L-2. Returned the 50 mL conical tube to the incubator with a loosened
cap.
[001693] The steps above were repeated for the second TIL line.
[001694] Just prior to plating the TIL into the T25 flasks for the
experiment, TIL were diluted
1:10 for a final concentration of 1.3 x 105 cclls/mL as per below.
[001695] Prepare MACS GMP CD3 pure (OKT3) working solution. Took
out stock solution of
OKT3 (1 mg/mL) from 4 C refrigerator and placed in BSC. A final concentration
of 30 ng/mL OKT3
was used in the media of the mini-REP.
[001696] 600 ng of OKT3 were needed for 20 mL in each T25 flask of
the experiment; this was
the equivalent of 60 juL of a 10 jug/mL solution for each 20 mL, or 360 uL for
all 6 flasks tested for
each feeder lot.
[001697] For each feeder lot tested, made 4001AL of a 1:100
dilution of 1 mg/mL OKT3 for a
working concentration of 101Ag/mL (e.g., for testing 4 feeder lots at one
time, make 1600 1_, of a
1:100 dilution of 1 mg/mL OKT3: 16 !IL of 1 mg/mL OKT3 + 1.584 mL of CM2
medium with 3000
IU/mL IL-2.)
[001698] Prepare T25 flasks. Labeled each flask and filled flask
with the CM2 medium prior to
preparing the feeder cells. Placed flasks into 37 C humidified 5% CO2
incubator to keep media warm
while waiting to add the remaining components. Once feeder cells were
prepared, the components
will be added to the CM2 in each flask.
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100021 Further information is provided in Table 39.
TABLE 39. Solution information.
Component Volume in co- Volume in
culture flasks control
(feeder
only) flasks
CM2 + 3000 IU/mL IL-2 18 mL 19 mL
MNC: 1.3 x 107/mL in CM2 + 3000 IU
1 niL 1 niL
IL-2
(final concentration 1.3 x 107/flask)
OKT3: 10 laL/mL in CM2 = 3000 IU IL- 60 ttL 60 1_,
2
T1L: 1.3 x 105/mL in CM2 with 3000 IU
1 mL 0
of IL-2
(final concentration 1.3 x 105/flask)
10016991 Prepare Feeder Cells. A minimum of 78 x 106 feeder cells
were needed per lot tested
for this protocol. Each 1 mL vial frozen by SDBB had 100 x 106 viable cells
upon freezing. Assuming
a 50% recovery upon thaw from liquid N2 storage, it was recommended to thaw at
least two 1 mL
vials of feeder cells per lot giving an estimated 100 x 106 viable cells for
each REP. Alternately, if
supplied in 1.8 mL vials, only one vial provided enough feeder cells.
10017001 Before thawing feeder cells, approximately 50 mL of CM2
without IL-2 was pre-
warmed for each feeder lot to be tested. The designated feeder lot vials were
removed from LN2
storage, placed in zipper storage bag, and placed on ice. Vials were thawed
inside closed zipper
storage bag by immersing in a 37 C water bath. Vials were removed from zipper
bag, sprayed or
wiped with 70% Et0H, and transferred to a BSC.
10017011 Using a transfer pipet, the contents of feeder vials were
immediately transferred into
30 mL of warm CM2 in a 50 mL conical tube. The vial was washed with a small
volume of CM2 to
remove any residual cells in the vial and centrifuged at 400 x CF for 5
minutes. Aspirated the
supernatant and resuspended in 4 mL warm CM2 plus 3000 IU/mL IL-2. Removed 200
[IL for cell
counting using the automated cell counter. Recorded the counts.
10017021 Resuspended cells at 1.3 x 107 cals/mL in warm CM2 plus
3000 IU/mL IL-2. Diluted
TIL cells from 1.3 x 106 cells/mL to 1.3 x 105 cells/mL.
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10017031 Setup Co-Culture. Diluted TIL cells from 1.3 x 106
cells/mL to 1.3 x 105 cells/mL.
Added 4.5 mL of CM2 medium to a 15 mL conical tube. Removed TIL cells from
incubator and
resuspended well using a 10 mL serological pipet. Removed 0.5 mL of cells from
the 1.3 X 106
cells/mL TIL suspension and added to the 4.5 mL of medium in the 15 mL conical
tube. Returned TIL
stock vial to incubator. Mixed well. Repeated for the second TIL line.
10017041 Transferred flasks with pre-warmed media for a single
feeder lot from the incubator
to the BSC. Mixed feeder cells by pipetting up and down several times with a 1
mL pipet tip and
transferred 1 mL (1.3 >< 107 cells) to each flask for that feeder lot. Added
60 viL of OKT3 working
stock (10 vig/mL) to each flask. Returned the two control flasks to the
incubator.
10017051 Transferred 1 mL (1.3 x 105) of each TIL lot to the
correspondingly labeled T25
flask. Returned flasks to the incubator and incubate upright. Did not disturb
until Day 5. This
procedure was repeated for all feeder lots tested.
10017061 Day 5, Media change. Prepared CM2 with 3000 IU/mL IL-2.
10 mL is needed for
each flask. With a 10 mL pipette, transferred 10 mL warm CM2 with 3000 IU/mL
IL-2 to each flask.
Returned flasks to the incubator and incubated upright until day 7. Repeated
for all feeder lots tested.
10017071 Day 7, Harvest. Removed flasks from the incubator and
transfer to the BSC, care as
taken not to disturb the cell layer on the bottom of the flask. Without
disturbing the cells growing on
the bottom of the flasks, 10 mL of medium was removed from each test flask and
15 mL of medium
from each of the control flasks.
10017081 Using a 10 mL serological pipet, the cells were
resuspended in the remaining medium
and mix well to break up any clumps of cells. After thoroughly mixing cell
suspension by pipetting,
removed 200 [it for cell counting. Counted the TIL using the appropriate
standard operating
procedure in conjunction with the automatic cell counter equipment. Recorded
counts in day 7. This
procedure was repeated for all feeder lots tested.
10017091 Feeder control flasks were evaluated for replication
incompetence and flasks
containing TIL were evaluated for fold expansion from day 0.
10017101 Day 7, Continuation of Feeder Control Flasks to Day 14.
After completing the day 7
counts of the feeder control flasks, 15 mL of fresh CM2 medium containing 3000
IU/mL IL-2 was
added to each of the control flasks. The control flasks were returned to the
incubator and incubated in
an upright position until day 14.
10017111 Day 14, Extended Non-proliferation of Feeder Control
Flasks. Removed flasks from
the incubator and transfer to the BSC, care was taken not to disturb the cell
layer on the bottom of the
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flask. Without disturbing the cells growing on the bottom of the flasks,
approximately 17 mL of
medium was removed from each control flasks. Using a 5 mL serological pipet,
the cells were
resuspended in the remaining medium and mixed well to break up any clumps of
cells. The volumes
were recorded for each flask.
10017121 After thoroughly mixing the cell suspension by pipetting,
200 [it was removed for
cell counting. The TIL were counted using the appropriate standard operating
procedure in
conjunction with the automatic cell counter equipment and the counts were
recorded. This procedure
was repeated for all feeder lots tested.
B. Results and Acceptance Criteria Protocol
10017131 Results. The dose of gamma irradiation was sufficient to
render the feeder cells
replication incompetent. All lots were expected to meet the evaluation
criteria and also demonstrated a
reduction in the total viable number of feeder cells remaining on day 7 of the
REP culture compared
to day 0. All feeder lots were expected to meet the evaluation criteria of 100-
fold expansion of TIL
growth by day 7 of the REP culture. Day 14 counts of Feeder Control flasks
were expected to
continue the non-proliferative trend seen on day 7.
10017141 Acceptance Criteria. The following acceptance criteria
were met for each replicate
TIL line tested for each lot of feeder cells. Acceptance criteria were two-
fold, as shown in Table 40
below.
TABLE 40. Embodiments of acceptance criteria.
Test Acceptance criteria
Irradiation of MNC and Replication No growth observed at 7 and 14 days
Incompetence
At least a 100-fold expansion of each
TIL expansion
TIL (minimum of 1.3 >< 107 viable cells)
10017151 The dose of radiation was evaluated for its sufficiency
to render the MNC feeder cells
replication incompetent when cultured in the presence of 30 ng/mL OKT3
antibody and 3000 IU/mL
IL-2. Replication incompetence was evaluated by total viable cell count (TVC)
as determined by
automated cell counting on day 7 and day 14 of the REP.
10017161 The acceptance criteria was "No Growth," meaning the
total viable cell number has
not increased on day 7 and day 14 from the initial viable cell number put into
culture on Day 0 of the
REP.
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10017171 The ability of the feeder cells to support TIL expansion
was evaluated. TIL growth
was measured in terms of fold expansion of viable cells from the onset of
culture on day 0 of the REP
to day 7 of the REP. On day 7, TIL cultures achieved a minimum of 100-fold
expansion, (i.e., greater
than 100 times the number of total viable TIL cells put into culture on REP
day 0), as evaluated by
automated cell counting.
10017181 Contingency Testing of MNC Feeder Lots that do not meet
acceptance criteria. In the
event that an MNC feeder lot did not meet the either of the acceptance
criteria outlined above, the
following steps will be taken to retest the lot to rule out simple
experimenter error as its cause.
10017191 If there are two or more remaining satellite testing
vials of the lot, then the lot was
retested. If there were one or no remaining satellite testing vials of the
lot, then the lot was failed
according to the acceptance criteria listed above.
10017201 In order to be qualified, the lot in question and the
control lot had to achieve the
acceptance criteria above. Upon meeting these criteria, the lot is released
for use.
EXAMPLE 4: PREPARATION OF IL-2 STOCK SOLUTION (CELLGENIX)
10017211 This Example describes the process of dissolving purified,
lyophilized recombinant human
interleukin-2 into stock samples suitable for use in further tissue culture
protocols, including all of
those described in the present application and Examples, including those that
involve using rhIL-2.
Procedure
10017221Prepared 0.2% Acetic Acid solution (HAc). Transferred 29mL sterile
water to a 50mL
conical tube. Added lmL IN acetic acid to the 50mL conical tube. Mixed well by
inverting tube 2-3
times. Sterilized the HAc solution by filtration using a Steriflip filter
10017231Prepare 1% HSA in PBS. Added 4mL of 25% HSA stock solution to 96mL PBS
in a
150mL sterile filter unit. Filtered solution. Stored at 4 C. For each vial of
rhIL-2 prepared, fill out
forms
10017241Prepared rhIL-2 stock solution (6>< 106 IU/mL final concentration).
Each lot of rhIL-2 was
different and required information found in the manufacturer's Certificate of
Analysis (COA), such as:
1) Mass of rhIL-2 per vial (mg), 2) Specific activity of rhIL-2 (IU/mg) and 3)
Recommended 0.2%
HAc reconstitution volume (mL).
10017251 Calculated the volume of 1% HSA required for rhIL-2 lot by using the
equation below:
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( Vtal MOSS (ng) x Biological Activity
l'U
6x106 ----g.,
7111_, ing-
Hitc voi (tvL). =1% .1- ISA vol (mL)
.
10017261 For example, according to CellGenix's rhIL-2 lot 10200121 COA, the
specific activity for
the lmg vial is 25x10 IU/mg. It recommends reconstituting the rhIL-2 in 2mL
0.2% HAc.
/ill \
.1m..,g- x 25x106
.=
(
, -,=?- =
6xi 0.6 - li=,.1, - ¨ Ant .--- 2,1677m1 HSA.
\ nil,
10017271 Wiped rubber stopper of IL-2 vial with alcohol wipe. Using a 16G
needle attached to a 3mL
syringe, injected recommended volume of 0.2% HAc into vial. Took care to not
dislodge the stopper
as the needle is withdrawn. Inverted vial 3 times and swirled until all powder
is dissolved. Carefully
removed the stopper and set aside on an alcohol wipe. Added the calculated
volume of 1% HSA to the
vial.
10017281 Storage of rhIL-2 solution. For short-term storage (<72hrs), stored
vial at 4 C. For long-
term storage (>72hrs), aliquoted vial into smaller volumes and stored in
cryovials at -20 C until ready
to use. Avoided freeze/thaw cycles. Expired 6 months after date of
preparation. Rh-IL-2 labels
included vendor and catalog number, lot number, expiration date, operator
initials, concentration and
volume of aliquot.
EXAMPLE 5: CRYOPRESERVATION PROCESS
10017291 This example describes a cryopreservation process method for TILs
prepared with the
procedure described herein using the CryoMed Controlled Rate Freezer, Model
7454 (Thermo
Scientific).
10017301 The equipment used was as follows: aluminum cassette holder rack
(compatible with
CS750 freezer bags), cryostorage cassettes for 750 mL bags, low pressure (22
psi) liquid nitrogen
tank, refrigerator, thermocouple sensor (ribbon type for bags), and CryoStore
CS750 freezing bags
(OriGen Scientific).
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10017311The freezing process provides for a 0.5 C rate from nucleation to -20
C and 1 C per
minute cooling rate to -80 C end temperature. The program parameters are as
follows: Step 1 - wait
at 4 C; Step 2: 1.0 C/min (sample temperature) to -4 C; Step 3: 20.0 C/min
(chamber temperature)
to -45 'V; Step 4: 10.0 C/min (chamber temperature) to -10.0 "V; Step 5: 0.5
C/min (chamber
temperature) to -20 C; and Step 6: 1.0 C/min (sample temperature) to -80 'C.
EXAMPLE 6: TUMOR EXPANSION PROCESSES WITH DEFINED MEDIUM
10017321 The processes disclosed above may be performed
substituting the CM1 and CM2
media with a defined medium according (e.g., CTSTm OpTmizerTm T-Cell Expansion
SFM,
'ThermoFisher, including for example DM1 and DM2).
EXAMPLE 7: NSCLC TREATMENT WITH ANTI-PD-1 ANTIBODIES
Patient population:
10017331Treatment naive NSCLC or post chemotherapy but anti-PD-1/PD-L1 naive
Treatment schedules:
10017341Tumor fragment, treat with up to 4 doses of anti-PD-1/PD-Li, treat the
primary refractory
patients with TIL product which is cryo-preserved and ready for use upon
immediate progression.
Primary refractory patients may have progressed after 2 doses.
10017351Relapse patients can also be treated upon progression (the timing may
vary from months to
years later).
10017361Full strength IL-2 up to 6 doses.
10017371Patient populations to further consider with the same manufacturing
permutations noted
earlier:
= Treatment naive NSCLC or post chemotherapy but anti-PD-1/PD-L1 naive
= Treatment naive NSCLC or post chemotherapy but anti-PD-1/PD-L1 naive who
have low
expression of PD-Li
= Treatment naive NSCLC or post chemotherapy but anti-PD-1/PD-Li naive who
have low
expression of PD-Li and/or have bulky disease at baseline- (for example, bulky
disease is
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indicated where the maximal tumor diameter is greater than 7 cm measured in
either the
transverse or coronal plane or swollen lymph nodes with a short-axis diameter
of 20 mm or
greater on CT were defined as bulky; see for example, Samejima, J., Japanese
Journal of
Clinical Oncology, 45(11): 1050-10541 2015, incorporated herein by referece)
EXAMPLE 8: EXEMPLARY GEN 2 PRODUCTION OF A CRYOPRESERVED TIL CELL
THERAPY
19017381 This example describes the the cGMP manufacture of
Iovance Biotherapeutics, Inc.
TIL Cell Therapy Process in G-REX Flasks according to current Good Tissue
Practices and current
Good Manufacturing Practices. This example describes an exemplary cGMP
manufacture of TIL Cell
Therapy Process in G-REX Flasks according to current Good Tissue Practices and
current Good
Manufacturing Practices.
TABLE 41. Process Expansion Exemplary Plan.
Estimated Day
Estimated Total
(post-seed) Activity Target Criteria
Anticipated Vessels
Volume (mL)
< 50 desirable tumor fragments
0 Tumor Dissection per G-REX-100MCS
G-REX-100MCS 1 flask <1000
¨ 200>< 106viab1e cells per
11 REP Seed G-REX-500MCS 1
flasks <5000
G-REX-500MCS
1 x 109viab1e cells per
16 REP Split G-REX-500MCS <5
flasks <25000
G-REX-500MCS
22 Harvest Total available cells 3-4 CS-750 bags
<530
TABLE 42. Flask Volumes.
Working
Flask Type
Volume/Flask
G-REX-100MCS 1000
G-R_EX-500MCS 5000
10017391 Day 0 CM1 Media Preparation. In the BSC added reagents to
RPMI 1640 Media
bottle. Added the following reagents t Added per bottle: Heat Inactivated
Human AB Scrum (100.0
mL); GlutaMaxTm (10.0 mL); Gentamicin sulfate, 50 mg/mL (1.0 mL); 2-
mereaptoethanol (1.0 mL)
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10017401 Removed unnecessary materials from BSC. Passed out media
reagents from BSC, left
Gentamicin Sulfate and HBSS in BSC for Formulated Wash Media preparation.
10017411 Thawed IL-2 aliquot. Thawed one 1.1 mL IL-2 aliquot
(6x106 IU/mL) (BR71424)
until all ice had melted. Recorded IL-2: Lot # and Expiry
10017421 Transferred IL-2 stock solution to media. In the BSC,
transferred 1.0 mL of IL-2
stock solution to the CM1 Day 0 Media Bottle prepared. Added CM1 Day 0 Media 1
bottle and IL-2
(6x106 IU/mL) 1.0 mL.
10017431 Passed G-REXIOOMCS into BSC. Aseptically passed G-
REXIOOMCS (W3013130)
into the BSC.
10017441 Pumped all Complete CM1 Day 0 Media into G-REX100MCS
flask. Tissue
Fragments Conical or GRex100MCS .
10017451 Day 0 Tumor Wash Media Preparation. In the BSC, added 5.0
mL Gentamicin
(W3009832 or W3012735) to lx 500 mL HBSS Media (W3013128) bottle. Added per
bottle: HBSS
(500.0 mL), Gentamicin sulfate, 50 mg/mL (5.0 mL). Filtered HBSS containing
gentamicin prepared
through a 1L 0.22-micron filter unit (W1218810).
10017461 Day 0 Tumor Processing. Obtained tumor specimen and
transferred into suite at 2-8
C immediately for processing. Aliquoted tumor wash media. Tumor wash 1 is
performed using 8"
forceps (W3009771). The tumor is removed from the specimen bottle and
transferred to the "Wash 1"
dish prepared. This is followed by tumor wash 2 and tumor wash 3. Measured and
assessed tumor.
Assessed whether > 30% of entire tumor area observed to be necrotic and/or
fatty tissue. Clean up
dissection if applicable. If tumor was large and >30% of tissue exterior was
observed to be
necrotic/fatty, performed "clean up dissection" by removing necrotic/fatty
tissue while preserving
tumor inner structure using a combination of scalpel and/or forceps. Dissect
tumor. Using a
combination of scalpel and/or forceps, cut the tumor specimen into even,
appropriately sized
fragments (up to 6 intermediate fragments). Transferred intermediate tumor
fragments. Dissected
tumor fragments into pieces approximately 3x3x3mm in size. Stored Intermediate
Fragments to
prevent drying. Repeated intermediate fragment dissection. Determined number
of pieces collected. If
desirable tissue remains, selected additional favorable tumor pieces from the -
favorable intermediate
fragments- 6-well plate to fill the drops for a maximum of 50 pieces.
10017471 Prepared conical tube. Transferred tumor pieces to 50 mL
conical tube. Prepared BSC
for G-REX100MCS. Removed G-REX100MCS from incubator. Aseptically passed G-
REX100MCS
flask into the BSC. Added tumor fragments to G-REX100MCS flask. Evenly
distributed pieces.
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10017481 Incubated G-REX100MCS at the following parameters:
Incubated G-REX flask:
Temperature LED Display: 37.0+2.0 C; CO2 Percentage: 5.0+1.5 %CO2.
Calculations: Time of
incubation; lower limit = time of incubation + 252 hours; upper limit = time
of incubation + 276
hours.
10017491 After process was complete, discarded any remaining
warmed media and thawed
aliquots of IL-2.
10017501 Day 11 ¨ Media Preparation. Monitored incubator.
Incubator parameters:
Temperature LED Display: 37.0+2.0 C; CO2 Percentage: 5.0+1.5 %CO2.
10017511 Warmed 3x 1000 mL RPMI 1640 Media (W3013112) bottles and
3x 1000 mL AIM-
V (W3009501) bottles in an incubator fork 30 minutes. Removed RPM! 1640 Media
from incubator.
Prepared RPM! 1640 Media. Filter Media. Thawed 3 x 1.1 mL aliquots of IL-2
(6x106 IU/mL)
(BR71424). Removed AIM-V Media from the incubator. Add IL-2 to AIM-V.
Aseptically transferred
a 10 L Labtainer Bag and a repeater pump transfer set into the BSC.
10017521 Prepared 10 L Labtainer media bag. Prepared Baxa pump.
Prepared 10L Labtainer
media bag. Pumped media into 10 L Labtainer. Removed pumpmatic from Labtainer
bag.
10017531 Mixed media. Gently massaged the bag to mix. Sample media
per sample plan.
Removed 20.0 mL of media and place in a 50 mL conical tube. Prepared cell
count dilution tubes. In
thc BSC, added 4.5 mL of AIM-V Mcdia that had been labelled with -For Cell
Count Dilutions" and
lot number to four 15 mL conical tubes. Transferred reagents from the BSC to 2-
8 C. Prepared 1 L
Transfer Pack. Outside of the BSC weld (per Process Note 5.11) a 1L Transfer
Pack to the transfer set
attached to the "Complete CM2 Day 11 Media" bag prepared. Prepared feeder cell
transfer pack.
Incubated Complete CM2 Day 11 Media.
10017541 Day 11 - TIL Harvest. Preprocessing table. Incubator
parameters: Temperature LED
display: 37.0+2.0 C; CO2 Percentage: 5.0+1.5 % CO2. Removed G-REX100MCS from
incubator.
Prepared 300 mL Transfer Pack. Welded transfer packs to G-REX100MCS.
10017551 Prepare flask for TIL Harvest and initiation of TIL
Harvest. TIL Harvested. Using the
GatheRex, transferred the cell suspension through the blood filter into the
300 mL transfer pack.
Inspect membrane for adherent cells.
10017561 Rinsed flask membrane. Closed clamps on G-REX100MCS.
Ensured all clamps are
closed. Heat sealed the TIL and the -Supernatant" transfer pack. Calculated
volume of TIL
suspension. Prepared Supernatant Transfer Pack for Sampling.
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10017571 Pulled Bac-T Sample. In the BSC, draw up approximately
20.0 mL of supernatant
from the IL "Supernatant" transfer pack and dispense into a sterile 50 mL
conical tube.
10017581 Inoculated BacT per Sample Plan. Removed a 1.0 mL sample
from the 50 mL conical
labeled BacT prepared using an appropriately sized syringe and inoculated the
anaerobic bottle.
10017591 Incubated TIL. Placed TIL transfer pack in incubator
until needed. Performed cell
counts and calculations. Determined the Average of Viable Cell Concentration
and Viability of the
cell counts performed. Viability 2. Viable Cell Concentration 2.
Determined Upper and Lower
Limit for counts. Lower Limit: Average of Viable Cell Concentration x 0.9.
Upper Limit: Average of
Viable Cell Concentration x 1.1. Confirmed both counts within acceptable
limits. Determined an
average Viable Cell Concentration from all four counts performed.
10017601 Adjusted Volume of TIL Suspension: Calculate the adjusted
volume of TIL
suspension after removal of cell count samples. Total TIL Cell Volume (A).
Volume of Cell Count
Sample Removed (4.0 mL) (B) Adjusted Total TIL Cell Volume C=A-B.
10017611 Calculated Total Viable TIL Cells. Average Viable Cell
Concentration*: Total
Volume; Total Viable Cells: C = A x B.
10017621 Calculation for flow cytometry: if the Total Viable TIL
Cell count from was >
4.0x107, calculated the volume to obtain 1.0x107ce11s for the flow cytometry
sample.
10017631 Total viable cells required for flow cytometry:
1.0x107ce11s. Volume of cells required
for flow cytometry: Viable cell concentration divided by 1.0x107cells A.
10017641 Calculated the volume of TIL suspension equal to
2.0x108viable cells. As needed,
calculated the excess volume of TIL cells to remove and removed excess TIL and
placed T1L in
incubator as needed. Calculated total excess TIL removed, as needed.
10017651 Calculated amount of CS-10 media to add to excess TIL
cells with the target cell
concentration for freezing is 1.0x 108 cells/mL. Centrifuged excess TILs, as
needed. Observed conical
tube and added CS-10.
10017661 Filled Vials. Aliquoted 1.0 mL cell suspension, into
appropriately sized cryovials.
Aliquoted residual volume into appropriately sized cryovial. If volume is <0.5
mL, add CS10 to vial
until volume is 0.5 mL.
10017671 Calculated the volume of cells required to obtain
1x107ce11s for cryopreservation.
Removed sample for cryopreservation. Placed TIL in incubator.
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10017681 Cryopreservation of sample. Observed conical tube and
added CS-10 slowly and
record volume of 0.5 mL of CSIO added.
10017691 Day 11 - Feeder Cells. Obtained feeder cells. Obtained 3
bags of feeder cells with at
least two different lot numbers from LN2 freezer. Kept cells on dry ice until
ready to thaw. Prepared
water bath or cryotherm. Thawed feeder cells at 37.0 2.0 C in the water bath
or cytotherm for ¨3-5
minutes or until ice has just disappeared. Removed media from incubator.
Pooled thawed feeder cells.
Added feeder cells to transfer pack. Dispensed the feeder cells from the
syringe into the transfer pack.
Mixed pooled feeder cells and labeled transfer pack.
10017701 Calculated total volume of feeder cell suspension in
transfer pack. Removed cell
count samples. Using a separate 3 mL syringe for each sample, pulled 4x1.0 mL
cell count samples
from Feeder Cell Suspension Transfer Pack using the needless injection port.
Aliquoted each sample
into the cryovials labeled. Performed cell counts and determine multiplication
factors, elected
protocols and entered multiplication factors. Determined the average of viable
cell concentration and
viability of the cell counts performed. Determined upper and lower limit for
counts and confirm
within limits.
10017711 Adjusted volume of feeder cell suspension. Calculated the
adjusted volume of feeder
cell suspension after removal of cell count samples. Calculated total viable
feeder cells. Obtained
additional feeder cells as needed. Thawed additional feeder cells as needed.
Placed the 4th feeder cell
bag into a zip top bag and thaw in a 37.0 2.0 C water bath or cytotherm for
¨3-5 minutes and
pooled additional feeder cells. Measured volume. Measured the volume of the
feeder cells in the
syringe and recorded below (B). Calculated the new total volume of feeder
cells. Added feeder cells to
transfer pack.
10017721 Prepared dilutions as needed, adding 4.5 mL of AIM-V
Media to four 15 mL conical
tubes. Prepared cell counts. Using a separate 3 mL syringe for each sample,
removed 4 x 1.0 mL cell
count samples from Feeder Cell Suspension transfer pack, using the needless
injection port.
Performed cell counts and calculations. Determined an average viable cell
concentration from all four
counts performed. Adjusted volume of feeder cell suspension and calculated the
adjusted volume of
feeder cell suspension after removal of cell count samples. Total Feeder Cell
Volume minues 4.0 mL
removed. Calculated the volume of Feeder Cell Suspension that was required to
obtain 5x109viable
feeder cells. Calculated excess feeder cell volume. Calculated the volume of
excess feeder cells to
remove. Removed excess feeder cells.
10017731 Using a 1.0 mL syringe and 16G needle, drew up 0.15 mL of
OKT3 and added
OKT3. Heat sealed the feeder cell suspension transfer pack.
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10017741 Day 11 G-REX Fill and Seed Set up G-REX500MCS. Removed
"Complete CM2
Day 11 Media", from incubator and pumped media into G-REX500MCS. Pumped 4.5L
of media into
the G-REX500MCS, filling to the line marked on the flask. Heat sealed and
incubated flask as
needed. Welded the Feeder Cell suspension transfer pack to the G-REX500MCS.
Added Feeder Cells
to G-REX500MCS. Heat sealed. Welded the TIL Suspension transfer pack to the
flask. Added TIL to
G-REX500MCS. Heat sealed. Incubated G-REX500MCS at 37.0 2.0 C, CO2
Percentage: 5.0 1.5
%CO2.
10017751 Calculated incubation window. Performed calculations to
determine the proper time
to remove G-REX500MCS from incubator on Day 16. Lower limit: Time of
incubation -1 108 hours.
Upper limit: Time of incubation + 132 hours.
10017761 Day 11 Excess TIL Cryopreservation. Applicable: Froze
Excess TIL Vials. Verified
the CRF has been set up prior to freeze. Perform Cryopreservation. Transferred
vials from Controlled
Rate Freezer to the appropriate storage. Upon completion of freeze, transfer
vials from CRF to the
appropriate storage container. Transferred vials to appropriate storage.
Recorded storage location in
LN2.
10017771 Day 16 Media Preparation. Pre-warmed AIM-V Media.
Calculated time Media was
warmed for media bags 1, 2, and 3. Ensured all bags have been warmed for a
duration between 12 and
24 hours. Setup 10L Labtainer for Supernatant. Attached the larger diameter
end of a fluid pump
transfer set to one of the female ports of a 10L Labtainer bag using the Luer
connectors. Setup 10L
Labtainer for Supernatant and label. Setup 10L Labtainer for Supernatant.
Ensure all clamps were
closed prior to removing from the BSC. NOTE: Supernatant bag was used during
TIL Harvest, which
may be performed concurrently with media preparation.
10017781 Thawed IL-2. Thawed 5x1.1 mL aliquots of IL-2
(6x106IU/mL) (BR71424) per bag
of CTS AIM V media until all ice had melted. Aliquoted 100.0 mL GlutaMaxTm.
Added IL-2 to
GlutaMaxTm. Prepared CTS AIM V media bag for formulation. Prepared CTS AIM V
media bag for
formulation. Stage Baxa Pump. Prepared to formulate media. Pumped GlutaMaxTm
+IL-2 into bag.
Monitored parameters: Temperature LED Display: 37.0 2.0 'V, CO2 Percentage:
5.0 1.5% CO2.
Warmed Complete CM4 Day 16 Media. Prepared Dilutions.
10017791 Day 16 REP Spilt. Monitored Incubator parameters:
Temperature LED display:
37.0 2.0 "C, CO2 Percentage: 5.0+1.5 %CO2. Removed G-REX500MCS from the
incubator. Prepared
and labeled 1 L Transfer Pack as TIL Suspension and weighed 1L.
10017801 Volume Reduction of G-REX500MCS. Transferred ¨4.5L of
culture supernatant
from the G-REX500MCS to the IOL Labtainer.
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10017811 Prepared flask for TIL harvest. After removal of the
supernatant, closed all clamps to
the red line.
10017821 Initiation of TIL Harvest. Vigorously tap flask and swirl
media to release cells and
ensure all cells have detached.
10017831 TIL Harvest. Released all clamps leading to the TIL
suspension transfer pack. Using
the GatheRex transferred the cell suspension into the TIL Suspension transfer
pack. NOTE: Be sure to
maintain the tilted edge until all cells and media are collected. Inspected
membrane for adherent cells.
Rinsed flask membrane. Closed clamps on G-REX500MCS. Heat sealed the Transfer
Pack containing
the TIL. Heat sealed the 10L Labtainer containing the supernatant. Recorded
weight of Transfer Pack
with cell suspension and calculate the volume suspension. Prepared transfer
pack for sample removal.
Removed testing samples from cell supernatant.
10017841 Sterility 8z BacT testing sampling. Removed a 1.0 mL
sample from the 15 mL conical
labeled BacT prepared. Removed Cell Count Samples. In the BSC, using separate
3 mL syringes for
each sample, removed 4x1.0 mL cell count samples from "TIL Suspension"
transfer pack.
10017851 Removed mycoplasma samples. Using a 3 mL syringe, removed
1.0 mL from TIL
Suspension transfer pack and place into 15 mL conical labeled "Mycoplasma
diluent" prepared.
10017861 Prepared transfer pack for seeding. Placed TIL in
incubator. Removed cell suspension
from the BSC and place in incubator until needed. Performed cell counts and
calculations. Diluted cell
count samples initially by adding 0.5 mL of cell suspension into 4.5 mL of AIM-
V media prepared
which gave a 1:10 dilution. Determined the average of viable cell
concentration and viability of the
cell counts performed. Determined upper and lower limit for counts. Note:
dilution may be adjusted
according based off the expected concentration of cells. Determined an average
viable cell
concentration from all four counts performed. Adjusted volume of TIL
suspension. Calculated the
adjusted volume of TIL suspension after removal of cell count samples. Total
TIL cell volume minus
5.0 mL removed for testing.
10017871 Calculated total viable TIL cells. Calculated the total
number of flasks to seed.
NOTE: The maximum number of G-REX500MCS flasks to seed was five. If the
calculated number of
flasks to seed exceeded five, only five were seeded using the entire volume of
cell suspension
available.
10017881 Calculate number of flasks for subculture. Calculated the
number of media bags
required in addition to the bag prepared. Prepared one 10L bag of "CM4 Day 16
Media" for every two
G-REX-500M flask needed as calculated. Proceeded to seed the first GREX-500M
flask(s) while
additional media is prepared and warmed. Prepared and warmed the calculated
number of additional
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media bags determined. Filled G-REX500MCS. Prepared to pump media and pumped
4.5L of media
into G-REX500MCS. Heat Sealed. Repeated Fill. Incubated flask. Calculated the
target volume of
TIL suspension to add to the new G-REX500MCS flasks. if the calculated number
of flasks exceeds
five only five will be seeded, USING THE ENTIRE VOLUME OF CELL SUSPENSION.
Prepared
Flasks for Seeding. Removed G-REX500MCS from the incubator. Prepared G-
REX500MCS for
pumping. Closed all clamps on except large filter line. Removed IlL from
incubator. Prepared cell
suspension for seeding. Sterile welded (per Process Note 5.11) "TIL
Suspension" transfer pack to
pump inlet line. Placed TIL suspension bag on a scale.
10017891 Seeded flask with TIL Suspension. Pump the volume of TIL
suspension calculated
into flask. Heat sealed. Filled remaining flasks.
10017901 Monitored Incubator. Incubator parameters: Temperature
LED Display: 37.0 2.0 C,
CO2 Percentage: 5.0+1.5 % CO2. Incubated Flasks.
10017911 Determined the time range to remove G-REX500MCS from
incubator on Day 22.
10017921 Day 22 Wash Buffer Preparation. Prepared 10 L Labtainer
Bag. In BSC, attach a 4"
plasma transfer set to a 10L Labtainer Bag via luer connection. Prepared 10 L
Labtainer Bag. Closed
all clamps before transferring out of the BSC. NOTE: Prepared one 10L
Labtainer Bag for every two
G-REX500MCS flasks to be harvested. Pumped Plasmalyte into 3000 mL bag and
removed air from
3000 mL Origen bag by reversing the pump and manipulating the position of the
bag. Added human
albumin 25% to 3000 mL Bag. Obtain a final volumeof 120.0 mL of human albumin
25%.
10017931 Prepared IL-2 diluent. Using a 10 mL syringe, removed 5.0
mL of LOVO Wash
Buffer using the needleless injection port on the LOVO Wash Buffer bag.
Dispensed LOVO wash
buffer into a 50 mL conical tube.
10017941 CRF blank bag LOVO wash buffer aliquotted. Using a 100 mL
syringe, drew up 70.0
mL of LOVO Wash Buffer from the needleless injection port.
10017951 Thawed one 1.1 mL of IL-2 (6x106 IU/mL), until all ice
has melted. Added 50 jiL IL-
2 stock (6)<106 IU/mL) to the 50 mL conical tube labeled "IL-2 Diluent."
10017961 Cryopreservation preparation. Placed 5 cryo-cassettes at
2-8 C to precondition them
for final product cryopreservation.
10017971 Prepared cell count dilutions. In the BSC, added 4.5 mL
of AIM-V Media that has
been labelled with lot number and "For Cell Count Dilutions" to 4 separate 15
mL conical tubes.
Prepared cell counts. Labeled 4 cryovials with vial number (1-4). Kept vials
under BSC to be used.
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10017981 Day 22 TIL Harvest. Monitored Incubator. Incubator
Parameters Temperature LED
display: 37 2.0 C, CO2 Percentage: 5% 1.5%. Removed G-REX500MCS Flasks from
Incubator.
Prepared TIL collection bag and labeled. Sealed off extra connections. Volume
Reduction:
Transferred -4.5L of supernatant from the G-REX500MCS to the Supernatant bag.
10017991 Prepared flask for TIL harvest. Initiated collection of
TIL. Vigorously tap flask and
swirl media to release cells. Ensure all cells have detached. Initiated
collection of TIL. Released all
clamps leading to the TIL suspension collection bag. TIL Harvest. Using the
GatheRex, transferred
the TIL suspension into the 3000 mL collection bag. Inspect membrane for
adherent cells. Rinsed
flask membrane. Closed clamps on G- Rex500MCS and ensured all clamps are
closed. Transferred
cell suspension into LOVO source bag. Closed all clamps. Heat Sealed. Removed
4x1.0 mL Cell
Counts Samples
10018001 Performed Cell Counts. Performed cell counts and
calculations utilizing NC-200 and
Process Note 5.14. Diluted cell count samples initially by adding 0.5 mL of
cell suspension into 4.5
mL of AIM-V media prepared. This gave a 1:10 dilution. Determined the average
viability, viable cell
concentration, and total nucleated cell concentration of the cell counts
performed. Determined Upper
and Lower Limit for counts. Determined the average viability, viable cell
concentration, and total
nucleated cell concentration of the cell counts performed. Weighed LOVO source
bag. Calculated
total viable TIL Cells. Calculated total nucleated cells.
10018011 Prepared Mycoplasma Diluent. Removed 10.0 mL from one
supernatant bag via luer
sample port and placed in a 15 mL conical.
10018021 Performed "TIL G-REX Harvest- protocol and determined the
final product target
volume. Loaded disposable kit. Removed filtrate bag. Entered Filtrate
capacity. Placed Filtrate
container on benchtop. Attached PlasmaLyte. Verified that the PlasmaLyte was
attached and observed
that the PlasmaLyte is moving. Attached Source container to tubing and
verified Source container was
attached. Confirmed PlasmaLyte was moving.
10018031 Final Formulation and Fill. Target volume/bag
calculation. Calculated volume of CS-
and LOVO wash buffer to formulate blank bag. Prepared CRF Blank.
10018041 Calculated the volume of IL-2 to add to the Final
Product. Final 1L-2 Concentration
desired (IU/mL) - 3001U/mL. IL-2 working stock: 6 x 104 IU/mL. Assembled
connect apparatus.
Sterile welded a 4S-4M60 to a CC2 cell connection. Sterile welded the CS750
crvobags to the harness
prepared. Welded CS-10 bags to spikes of the 45-4M60. Prepared TIL with IL-2.
Using an
appropriately sized syringe, removed amount of IL-2 determined from the "IL-2
6x104" aliquot.
Labeled forumlated TIL Bag. Added the formulated TIL bag to the apparatus.
Added CSIO. Switched
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Syringes. Drew ¨10 mL of air into a 100 mL syringe and replaced the 60 mL
syringe on the
apparatus. Added CS10. Prepared CS-750 bags. Dispensed cells.
10018051 Removed air from final product bags and take retain. Once
the last final product bag
was filled, closed all clamps. Drew 10 mL of air into anew 100 mL syringe and
replace the syringe
on the apparatus. Dispensed retain into a 50 mL conical tube and
label tube as "Retain- and lot
number. Repeat air removal step for each bag.
10018061 Prepared final product for cryopreservation, including
visual inspection. Held the
cryobags on cold pack or at 2-8 C until cryopreseryation.
10018071 Removed cell count sample. Using an appropriately sized
pipette, remove 2.0 mL of
retain and place in a 15 mL conical tube to be used for cell counts. Performed
cell counts and
calculations. NOTE: Diluted only one sample to appropriate dilution to verify
dilution is sufficient.
Diluted additional samples to appropriate dilution factor and proceed with
counts. Determined the
Average of Viable Cell Concentration and Viability of the cell counts
performed. Determined Upper
and Lower Limit for counts. NOTE: Dilution may be adjusted according based off
the expected
concentration of cells. Determined the Average of Viable Cell Concentration
and Viability.
Determined IJpper and Lower Limit for counts. Calculated IFN-y. Heat Sealed
Final Product bags_
10018081 Labeled and collected samples per exemplary sample plan
below.
TABLE 43. Sample plan.
Sample
Number of Volume to
Container
Sample
Containers Add to Type
Each
15 mL
*Myeoplasma 1 1.0 mL
Conical
Endotoxin 2 1.0 inL 2
iinL Cryovial
Gram Stain 1 1.0 mL 2 mL
Ciyovial
1FN-y 1 1.0 mL 2 mL
Cryovial
Flow Cytometry 1 1.0 mL 2 mL
Ciyovial
**Bac-T
2 1.0 mL Bae-
T Bottle
Sterility
QC Retain 4 1.0 mL 2 mL
Cryovial
Satellite Vials 10 0.5 mL 2 mL
Cryovial
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10018091 Sterility and BacT testing. Testing Sampling. In the BSC,
remove a 1.0 mL sample
from the retained cell suspension collected using an appropriately sized
syringe and inoculate the
anaerobic bottle. Repeat the above for the aerobic bottle.
10018101 Final Product Cryopreservation. Prepared controlled rate
freezer (CRF). Verified the
CRF had been set up. Set up CRF probes. Placed final product and samples in
CRF. Determined the
time needed to reach 4 C 1.5 C and proceed with the CRF run. CRF completed
and stored.
Stopped the CRF after the completion of the run. Remove cassettes and vials
from CRF. Transferred
cassettes and vials to vapor phase LN2 for storage. Recorded storage location.
10018111 Post-Processing and analysis of final drug product
included the following tests: (Day
22) Determination of CD3+ cells on Day 22 REP by flow cytometry; (Day 22) Gram
staining method
(GMP); (Day 22) Bacterial endotoxin test by Gel Clot LAL Assay (GMP); (Day 16)
BacT Sterility
Assay (GMP); (Day 16) Mycoplasma DNA detection by TD-PCR (GMP); Acceptable
appearance
attributes; (Day 22) BacT sterility assay (GMP)(Day 22); (Day 22) IFN-gamma
assay. Other potency
assay as described herein are also employed to analyze TIL products.
EXAMPLE 9: AN EXEMPLARY EMBODIMENT OF THE GEN 3 EXPANSION
PLATFORM
DAY 0
10018121 Prepared tumor wash media. Media warmed prior to start.
Added 5 mL of gcntamicin
(50mg/mL) to the 500 mL bottle of HBSS. Added 5mL of Tumor Wash Media to a
15mL conical to
be used for OKT3 dilution. Prepared feeder cell bags. Sterilely transfered
feeder cells to feeder cell
bags and stored at 37 C until use or freeze. Counted feeder cells if at 37
C. Thawed and then
counted feeder cells if frozen.
10018131 Optimal range for the feeder cell concentration is
between 5 x104 and 5>< 106 cells/mL.
Prepared four conical tubes with 4.5 mL of AIM-V. Added 0.5 mL of cell
fraction for each cell count.
If total viable feeder cell number was > 1 x 109 cells, proceeded to adjust
the feeder cell
concentration. Calculated the volume of feeder cells to remove from the first
feeder cell bag in order
to add lx 109 cells to a second feeder cell bag.
10018141 Using the p1000 micropipcttc, transferred 900 vtL of
Tumor Wash Media to the
OKT3 aliquot (100uL). Using a syringe and sterile technique, drew up 0.6 mL of
OKT3 and added
into the second feeder cell bag. Adjusted media volume to a total volume of
2L. Transferred the
second feeder cells bag to the incubator.
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10018151 OKT3 formulation details: OKT3 may be aliquoted and
frozen in original stock
concentration from the vial (1 mg/mL) in 100 ML aliquots. ¨10X aliquots per 1
mL vial. Stored at -
80C. Day 0: 15 ug/flask, i.e. 30 ng/mL in 500 mL ¨ 60 ML max ¨ 1 aliquot.
10018161 Added 5 mL of Tumor Wash Medium into all wells of the 6-
well plate labelled
Excess Tumor Pieces. Kept the Tumor Wash Medium available for further use in
keeping the tumor
hydrated during dissection. Added 50 mL of Tumor Wash Medium to each 100 mm
petri dish.
10018171 Dissected the tumor into 27 min3 fragments (3x3x3mm),
using the ruler under the
Dissection dish lid as a reference. Dissected intermediate fragment until 60
fragments were reached.
Counted total number of final fragments and prepared G-REX-100MCS flasks
according to the
number of final fragments generated (generally 60 fragments per flask).
10018181 Retained favorable tissue fragments in the conical tubes
labeled as Fragments Tube 1
through Fragments Tube 4. Calculated the number of G-REX-100MCS flasks to seed
with feeder cell
suspension according to the number of fragments tubes originated.
10018191 Removed feeder cells bag from the incubator and seed the
G-REX-100MCS. Label as
DO (Day 0).
10018201 Tumor fragment addition to culture in G-REX-100 MCS.
Under sterile conditions,
unscrewed the cap of the G-REX-100MCS labelled Tumor Fragments Culture (DO) 1
and the 50 mL
conical tube labelled Fragments Tube. Swirled the opened Fragments Tube 1 and,
at the same time,
slightly lifted the cap of the G-REX100MCS. Added the medium with the
fragments to the G-
REX100MCS while being swirled. Recorded the number of fragments transferred
into the G-
REX100MCS.
10018211 Once the fragments were located at the bottom of the GREX
flask, drew 7 mL of
media and created seven 1 mL aliquots ¨ 5 mL for extended characterization and
2 mL for sterility
samples. Stored the 5 aliquots (final fragment culture supernatant) for
extended characterization at -
20 C until needed.
10018221 Inoculated one anaerobic BacT/Alert bottle and one
aerobic BacT/Alert bottle each
with 1 mL of final fragment culture supernatant. Repeat for each flask
sampled.
AT DAY 7-8
10018231 Prepared feeder cell bags. Thawed feeder bags for 3-5
minutes in 37 C water bath
when frozen. Counted feeder cells if frozen. Optimal range for the feeder cell
concentration is
between 5> 104 and 5x 106 cells/mL. Prepared four conical tubes with 4.5 mL of
AIM-V. Added 0.5
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mL of cell fraction for each cell count into a new cryovial tube. Mixed the
samples well and
proceeded with the cell count.
10018241 If total viable feeder cell number was > 2 x109 cells,
proceeded to the next step to
adjust the feeder cell concentration. Calculated the volume of feeder cells to
remove from the first
feeder cell bag in order to add 2 x 109 cells to the second feeder cell bag.
10018251 Using the p1000 micropipette, transfer 900 uL ofFIBSS to
a 100 L OKT3 aliquot.
Mix by pipetting up and down 3 times. Prepared two aliquots.
10018261 OKT3 formulation details: OKT3 may be aliquoted and
frozen in original stock
concentration from the vial (1 mg/mL) in 100 "IL aliquots. ¨10x aliquots per 1
mL vial. Stored at -
80C. Day7/8: 30 ug/flask, i.e. 60 ng/mL in 500 mL ¨ 120 p1 max ¨ 2 aliquots.
10018271 Using a syringe and sterile technique, drew up 0.6 mL of
OKT3 and added into the
feeder cell bag, ensuring all added. Adjusted media volume to a total volume
of 2 L. Repeated with
second OKT3 aliquot and added to the feeder cell bag. Transferred the second
feeder cells bag to the
incubator.
10018281 Preparation of G-REX100MCS flask with feeder cell
suspension. Recorded the
number of G-REX-100MCS flasks to process according to the number of G-REX
flasks generated on
Day 0. Removed G-REX flask from incubator and removed second feeder cells bag
from incubator.
10018291 Removal of supernatant prior to feeder cell suspension
addition. Connected one 10
mL syringe to the G-REX100 flask and drew up 5 mL of media. Created five 1 mL
aliquots ¨ 5 mL
for extended characterization and storeed the 5 aliquots (final fragment
culture supernatant) for
extended characterization at -20 C until requested by sponsor. Labeled and
repeated for each G-
REX100 flask.
10018301 5-20>< 1 mL samples for characterization, dependeding on
number of flasks:
= 5 mL = 'flask
= 10 mL = 2 flasks
= 15 mL = 3 flasks
= 20 mL = 4 flasks
10018311 Continued seeding feeder cells into the G-REX100 MCS and
repeated for each G-
REX100 MCS flask. Using sterile transfer methods, gravity transferred 500 mL
of the second feeder
cells bag by weight (assume 1 g = 1 mL) into each G-REX-100MCS flask and
recoreded amount.
Labeled as Day 7 culture and repeated for each G-REX100 flask. Transferred G-
REX-100MCS flasks
to the incubator.
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DAY 10-11
10018321 Removed the first G-REX-100MCS flask and using sterile
conditions removed 7 mL
of pre-process culture supernatant using a 10 mL syringe. Created seven 1 mL
aliquots ¨ 5 mL for
extended characterization and 2 mL for sterility samples.
10018331 Mixed the flask carefully and using a new 10 mL syringe
remove 10 mL supernatant
and transfer to a 15 mL tube labelled as D10/11 mycoplasma supernatant.
10018341 Mixed the flask carefully and using a new syringe removed
the volume below
according to how many flasks were to be processed:
= 1 flask = 40 mL
= 2 flask = 20 mL/flask
= 3 flask = 13.3 mL/flask
= 4 flask = 10 mL/flask
10018351 A total of 40 mL should be pulled from all flasks and
pooled in a 50 mL conical tube
labeled 'Day 10/11 QC Sample' and stored in the incubator until needed.
Performed a cell count and
allocated the cells.
10018361 Stored the 5 aliquots (pre-process culture supernatant)
for extended characterization
at <-20 C until needed. Inoculated one anaerobic BacT/Alert bottle and one
aerobic BacT/Alert bottle
each with 1 mL of pre-process culture supernatant.
10018371 Continued with cell suspension transferred to the G-REX-
500MCS and repeated for
each G-REX-100MCS. Using sterile conditions, transferred the contents of each
G-REX-100MCS
into a G-REX-500MCS, monitoring about 100 mL of fluid transfer at a time.
Stopped transfer when
the volume of the G-REX-100MCS was reduced to 500 mL.
10018381 During transfer step, used 10 mL syringe and drew 10 mL
of cell suspension into the
syringe from the G-REX-100MCS. Followed the instructions according to the
number of flasks in
culture. If only 1 flask: Removed 20 mL total using two syringes. If 2 flasks:
removed 10 mL per
flask. If 3 flasks: removed 7 mL per flask. If 4 flasks: removed 5 mL per
flask. Transferred the cell
suspension to one common 50 mL conical tube. Keep in the incubator until the
cell count step and QC
sample. Total number of cells needed for QC was ¨ 20e6 cells: 4 x 0.5 mL cell
counts (cell counts
were undiluted first).
10018391 The quantities of cells needed for assays are as follows:
1. 10x 106 cells minimum for potency assays, such as those described herein,
or for an IFN-y
or granzyme B assay
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2. 1 x106 cells for mycoplasma
3. 5x106 cells for flow cytometry for CD3+/CD45+
10018401 Transferred the G-REX-500MCS flasks to the incubator.
10018411 Prepared QC Samples. At least 15 x 10' cells were needed
for the assays in this
embodiment. Assays included: Cell count and viability; Mycoplasma (1 x 106
cells/ average viable
concentration) flow (5 x 106 cells/ average viable concentration;) and IFN-g
assay (5 x 106 cells ¨ 1 x
106 cells; 8-10 x 106 cells are required for the IFN-y assay.
10018421 Calculated the volume of cells fraction for
cryopreservation at 10 x 106 cells/mL and
calculated the number of vials to prepare
DAY 16-17
10018431 Wash Buffer preparation (1% HSA Plasmalyte A).
Transferred HSA and Plasmalyte
to 5 L bag to make LOVO wash buffer. Using sterile conditions, transferred a
total volume of 125 mL
of 25% HSA to the 5L bag. Removed and transferred 10 mL or 40 mL of wash
buffer in the 'IL-2 6 x
104 IU/mL' tube (10 mL if IL-2 was prepared in advance or 40 mL if IL-2 was
prepared fresh).
10018441 Calculated volume of reconstituted IL-2 to add to
Plasmalyte + 1% HSA: volume of
reconstituted IL-2 = (Final concentration of IL-2 x Final volume)/ specific
activity of the IL-2 (based
on standard assay). The Final Concentration of IL-2 was 6>< 104 IU/mL. The
final volume was 40 mL.
10018451 Removed calculated initial volume of IL-2 needed of
reconstituted IL-2 and transfer
to the 'IL-2 6x104 IU/mL; tube. Added 100 L of 1L-2 6x106 IU/mL from the
aliquot prepared in
advance to the tube labelled 'IL-2 6x104 IU/mL' containing 10 mL of LOVO wash
buffer.
10018461 Removed about 4500 mL of supernatant from the G-REX-
500MCS flasks. Swirled
the remaining supernatant and transferred cells to the Cell Collection Pool
bag. Repeated with all G-
REX-500MCS flasks.
10018471 Removed 60 mL of supernatant and add to supernatant tubes
for quality control
assays, including mycoplasma detection. Stored at +2-8 C.
10018481 Cell collection. Counted cells. Prepare four 15 mL
conicals with 4.5 mL of AIM-V.
These may be prepared in advance. Optimal range = is between 5x 104 and 5x106
cells/mL. (1:10
dilution was recommended). For 1:10 dilution, to 4500 [1,1_, of AIM V prepared
previously, add 5000_,
of CF. Recorded dilution factor.
10018491 Calculated the TC (Total Cells) pre-LOVO (live + dead) =
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Average Total Cell
Concentration (TC cone pre LOVO)
(live + dead)
X
Volume of Source bag
10018501 Calculated the TVC (Total Viable Cells) pre-LOVO (live) =
Average Total Viable Cell
Concentration (TVC pre LOVO)
(live)
X
Volume of LOVO Source Bag
10018511 When the total cell (TC) number was >5 x 109, remove 5 x
108 cells to be
cryopreserved as MDA retention samples. 5 x 108 + avg TC concentration (step
14.44) = volume to
remove.
10018521 When the total cell (TC) number was < 5 x 109, remove 4 x
106 cells to he
cryopre served as MDA retention samples. 4 x 106 + avg TC concentration =
volume to remove.
10018531 When the total cell number was determined, the number of
cells to remove should
allow retention of 150x 109 viable cells. Confirm TVC pre-LOVO 5 x 108 or 4 x
106 or not applicable.
Calculated the volume of cells to remove.
10018541 Calculated the remaining Total Cells Remaining in Bag.
Calculated the TC (Total
Cells) pre-LOVO. [Avg. Total cell concentration X Remaining Volume = TC pre-
LOVO Remaining]
10018551 According to the total number of cells remaining, the
corresponding process in Table
44 is selected.
TABLE 44. Total number of cells.
Total cells: Retentate (FttL)
0 < Total cells < 31 x 109 115
31 x 109 < Total cells < 71 x 109 165
71 x 109 < Total Cells <110 x 109 215
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110 > 109< Total Cells < 115 x 109 265
10018561 Chose the volume of IL-2 to add corresponding to the used
process. Volume
calculated as: Retentate Volume x 2 x 300 IU/mL = IU of IL-2 required. IU of
IL-2 required / 6 x104
IU/mL = Volume of 1L-2 to add Post LOVO bag. Recorded all volumes added.
Obtained samples in
cryovial for further analyses.
10018571 Mixed the cell product well. Sealed all bags for further
processing, included
cryopreservation when applicable.
10018581 Performed endotoxin, IFN-y, sterility, and other assays
as needed on cryovial samples
obtained.
EXAMPLE 10: GEN 2 AND GEN 3 EXEMPLARY PROCESSES
10018591 This example demonstrates the Gen 2 and Gen 3 processes.
Process Gen 2 and Gen 3
TILs are generally composed of autologous TIL derived from an individual
patient through surgical
resection of a tumor and then expanded ex vivo. The priming first expansion
step of the Gen 3 process
was a cell culture in the presence of interlcukin-2 (IL-2) and the monoclonal
antibody OKT3, which
targets the T-cell co-receptor CD3 on a scaffold of irradiated peripheral
blood mononuclear cells
(PBMCs).
10018601 The manufacture of Gen 2 TIL products consists of two
phases: 1) pre-Rapid
Expansion (Pre-REP) and 2) Rapid Expansion Protocol (REP). During the Pre-REP
resected tumors
were cut up into < 50 fragments 2-3 mm in each dimension which were cultured
with serum-
containing culture medium (RPMI 1640 media containing 10% HuSAB supplemented)
and 6,000
IU/mL of Interleukin-2 (IL-2) for a period of 11 days. On day 11 TIL were
harvested and introduced
into the large-scale secondary REP expansion. The REP consists of activation
of <200 x 106 of the
viable cells from pre-REP in a co-culture of 5x109 irradiated allogeneic PBMCs
feeder cells loaded
with 150 ng of monoclonal anti-CD3 antibody (OKT3) in a 5 L volume of CM2
supplemented with
3000 IU/mL of rhIL-2 for 5 days. On day 16 the culture is volume reduced 90%
and the cell fraction
is split into multiple G-REX-500 flasks at? 1 x 109 viable lymphocytes/flask
and QS to 5L with
CM4. TIL are incubated an additional 6 days. The REP is harvested on day 22,
washed, formulated,
and cryo-preserved prior to shipping at -150 C to the clinical site for
infusion.
10018611 The manufacture of Gen 3 TIL products consists of three
phases: 1) Priming First
Expansion Protocol, 2) Rapid Second Expansion Protocol (also referred to as
rapid expansion phase
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or REP), and 3) Subculture Split. To effect the Priming First Expansion TIL
propagation, resected
tumor was cut up into < 120 fragments 2-3 mm in each dimension. On day 0 of
the Priming First
Expansion, a feeder layer of approximately 2.5 10x allogeneic irradiated PBMCs
feeder cells loaded
with OKT-3 was established on a surface area of approximately 100cm' in each
of 3 100 MCS
vessels. The tumor fragments were distributed among and cultured in the 3 100
MCS vessels each
with 500 mL serum-containing CM1 culture medium and 6,000 1U/mL of Interleukin-
2 (IL-2) and 15
ug OKT-3 for a period of 7 days. On day 7, REP was initiated by incorporating
an additional feeder
cell layer of approximately 5x108 allogeneic irradiated PBMCs feeder cells
loaded with OKT-3 into
the tumor fragmented culture phase in each of the three 100 MCS vessels and
culturing with 500 mL
CM2 culture medium and 6,000 IU/mL IL-2 and 30 ing OKT-3. The REP initiation
was enhanced by
activating the entire Priming First Expansion culture in the same vessel using
closed system fluid
transfer of OKT3 loaded feeder cells into the 100MCS vessel. For Gen 3, the
TIL scale up or split
involved process steps where the whole cell culture was scaled to a larger
vessel through closed
system fluid transfer and was transferred (from 100 M flask to a 500 M flask)
and additional 4 L of
CM4 media was added. The REP cells were harvested on day 16, washed,
formulated, and cryo-
preserved prior to shipping at -150 C to the clinical site for infusion.
10018621 Overall, the Gen 3 process is a shorter, more scalable, and easily
modifiable
expansion platform that will accommodate to fit robust manufacturing and
process comparability.
TABLE 45. Comparison of Exemplary Gen 2 and Exemplary Gen 3 manufacturing
process.
Step Process (Gen 2) Process (Gen 3)
Whole tumor up to 120 fragments divided
evenly among up to 3 flasks. 1 flask: 1-60
fragments
Up to 50 fragments, 1 G-REX- 2 flasks: 61-89 fragments
100MCS, 11 days
Pre REP- 3 flasks 90-120 fragments
day 0 In 1 L of CM1 media
7 days in 500 mL of CM1 media
+ IL-2 (6000 IU/mL)
+ IL-2 (6000 IU/mL)
2.5 x108 feeder cells/flask
15 ug OKT-3/flask
Direct to REP, Day 11, Direct to REP, Day 7, all
cells, same G-
REX-100MCS
REP <200 x106 TIL
Initiation Add 500 CM2 media
(1)G-REX-500MCS in 5L CM2
media 1L-2 (6000 1U/mL)
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IL-2 (3000 IU/mL) 5 x108 feeder cells/flask
x109 feeder cells 3Oug OKT-3/flask
15Oug OKT-3
Volume reduce and split cell fraction
in up to 5 G-REX-500MCS
Each G-REX-100MCS(1L) transfers to 1
4.5L CM4 media + IL-2 (3000
TIL G-REX-500MCS
IU/mL)
propagation
Add 4L CM4 media +IL-2 (3000 IU/mL)
or Scale up > 1 x109 TVC / flask
Split day 16 Scale up on day 9 to 11
Harvest day 22, Harvest day 16
Harvest
LOVO-automated cell washer LOVO- automated cell
washer
C
Cryopre served Product ryopreserved product
Final
formulation 300 IU/mL IL2- CS10 in LN2, 300 IU/mL IL-2-CSIO in LN
multiple aliquots multiple aliquots
Process
22 days 16 days
time
10018631 On day 0,
for both processes, the tumor was washed 3 times and the fragments were
randomized and divided into two pools; one pool per process_ For the Gen 2
Process, the fragments
were transferred to one -GREX 100MCS flask with 1 L of CM1 media containing
6,0001U/mL rh1L-
2. For the Gen 3 Process, fragments were transferred to one G-REX-100MCS flask
with 500 mL of
CM1 containing 6,000IU/mL rhIL-2, 15 ug OKT-3 and 2.5 x 108 feeder cells.
Seeding of TIL for Rep
initiation day occurred on different days according to each process. For the
Gen 2 Process, in which
the G-REX-l00MCS flask was 90% volume reduced, collected cell suspension was
transferred to a
new G-REX-500MCS to start REP initiation on day 11 in CM2 media containing IL-
2 (3000 IU/mL),
plus 5x 109 feeder cells and OKT-3 (30 ng/mL). Cells were expanded and split
on day 16 into multiple
G-REX-500 MCS flasks with CM4 media with IL-2 (3000 IU/mL) per protocol. The
culture was then
harvested and cryopreserved on day 22 per protocol. For the Gen 3 process, the
REP initiation
occurred on day 7, in which the same G-REX-100MCS used for REP initiation.
Briefly, 500 mL of
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CM2 media containing IL-2 (6000 IU/mL) and 5>< 108 feeder cells with 30ug OKT-
3 was added to
each flask. On day 9-11 the culture was scaled up. The entire volume of the G-
REX100M (1 L) was
transferred to a G-REX-500MCS and 4L of CM4 containing IL-2 (3000 IU/mL) was
added. Flasks
were incubated 5 days. Cultures were harvested and cryopreserved on Day 16.
10018641 Three different tumors were included in the comparison,
two lung tumors (L4054 and
L4055) and one melanoma tumor (M1085T).
10018651 CM I (culture media 1), CM2 (culture media 2), and CM4
(culture media 4) media
were prepared in advance and held at 4 C for L4054 and L4055. CM1 and CM2
media were prepared
without filtration to compare cell growth with and without filtration of
media.
10018661 Media was warmed at 37 C up to 24 hours in advance for
L4055 tumor on REP
initiation and scale-up.
10018671 Results. Gen 3 results fell within 30% of Gen 2 for total
viable cells achieved. Gen 3
final product exhibited higher production of IFN-y after restimulation. Gen 3
final product exhibited
increased clonal diversity as measured by total unique CDR3 sequences present.
Gen 3 final product
exhibited longer mean telomere length.
10018681 Pre-REP and REP expansion on Gen 2 and Gen 3 processes
followed the procedures
described above. For each tumor, the two pools contained equal number of
fragments. Due to the
small size of tumors, the maximum number of fragments per flask was not
achieved. Total pre-REP
cells (TVC) were harvested and counted at day 11 for the Gcn 2 process and at
day 7 for the Gen 3
process. To compare the two pre-REP arms, the cell count was divided over the
number of fragments
provided in the culture in order to calculate an average of viable cells per
fragment. As indicated in
Table 46 below, the Gen 2 process consistently grew more cells per fragment
compared to the Gen 3
Process. An extrapolated calculation of the number of TVC expected for Gen 3
process at day 11,
which was calculated dividing the pre-REP TVC by 7 and then multiply by 11.
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TABLE 46. Pre-REP cell counts
Tumor ID L4054 L4055*
M1085T
Process Gen 2 Gen 3 Gen 2 Gen 3 Gen 2
Gen 3
pre-REP TVC
1.42E+08 4.32E+07 2.68E+07 1.38E+07 1.23E+07 3.50E+06
Number of fragments 21 21 24 24 16
16
Average TVC per fragment
at pre-REP 6.65E+06 2.06E+06 1.12E+06 5.75E-
h05 7.66E+05 2.18E+05
Gen 3 extrapolated value at
pre REP day 11 N/A 6.79E+07 N/A 2.17E+07 N/A
5.49E+06
* L4055, unfiltered media.
10018691 For the Gen 2 and Gen 3 processes, TVC was counted per
process condition and
percent viable cells was generated for each day of the process. On harvest,
day 22 (Gen 2) and day 16
(Gen 3) cells were collected and the TVC count was established. The TVC was
then divided by the
number of fragments provided on day 0, to calculate an average of viable cells
per fragment. Fold
expansion was calculated by dividing harvest TVC by over the REP initiation
TVC. As exhibited in
Table 47, comparing Gen 2 and the Gen 3, fold expansions were similar for
L4054; in the case of
L4055, the fold expansion was higher for the Gen 2 process. Specifically, in
this case, the media was
warmed up 24 in advance of REP initiation day. A higher fold expansion was
also observed in Gen 3
for M1085T. An extrapolated calculation of the number of TVC expected for Gen
3 process at day 22,
which was calculated dividing the REP TVC by 16 and then multiply by 22.
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TABLE 47. Total viable cell count and fold expansion on TIL final product.
Tumor ID L4054 L4055
M1085T
Process Gen 2 Gen 3 Gen 2 Gen 3 Gen 2
Gen 3
# Fragments 21 21 24 24 16
16
TVC /fragment (at
3.18E+09 8.77E+08 2.30E+09 3.65E+08 7.09E+08 4.80E+08
Harvest)
REP initiation 1.42E+08 4.32E+07 2.68E+07
1.38E+07 1.23E+07 3.50E+06
3.36E+09 9.35E+08 3.49E+09 8.44E+08 1.99E+09 3.25E+08
Scale up
6.67E+10 1.84E+10 5.52E+10 8.76E+09 1.13E+10 7.68E+09
Harvest
Fold Expansion Harvest/
468.4 425.9 2056.8 634.6 925.0
2197.2
REP initiation
Gen 3 extrapolated value at
N/A 2.53E+10 N/A 1.20E+10 N/A
1.06E+10
REP harvest day 22
* L4055, unfiltered media.
10018701
Table 48: %Viability of TIL final product: Upon harvest, the final TIL
REP products
were compared against release criteria for % viability. All of the conditions
for the Gen 2 and Gen 3
processes surpassed the 70% viability criterion and were comparable across
processes and tumors.
10018711
Upon harvest, the final TIL REP products were compared against release
criteria for
A viability. All of the conditions for the Gen 2 and Gen 3 processes surpassed
the 70% viability
criterion and were comparable across processes and tumors.
TABLE 48. % Viability of REP (TIL Final Product)
Tumor ID L4054 L4055
M1085T
Process Gen 2 Gen 3 Gen 2 Gen 3 Gen
2 Gen 3
REP initiation 98.23% 97.97% 97.43% 92.03%
81.85% 68.27%
Scale up 94.00% 93.57% 90.50% 95.93%
78.55% 71.15%
Harvest 87.95% 89.85% 87.50% 86.70%
86.10% 87.45%
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10018721 Due to the number of fragments per flask below the
maximum required number, an
estimated cell count at harvest day was calculated for each tumor. The
estimation was based on the
expectation that clinical tumors were large enough to seed 2 or 3 flasks on
day 0.
TABLE 49. Extrapolated estimate cell count calculation to full scale 2 and 3
flask on Gen 3 Process.
Tumor ID L4054 L4055
M1085T
Gen 3 Process 2 flasks 3 Flasks 2 flasks 3 Flasks 2
flasks 3 Flasks
Estimate Harvest 3.68E+10 5.52E+10 1.75E+10 2.63E+10
1.54E+10 2.30E+10
10018731 Immunophenotyping - phenotypic marker comparisons on TIL
final product. Three
tumors L4054, L4055, and M1085T underwent TIL expansion in both the Gen 2 and
Gen 3 processes.
Upon harvest, the REP TIL final products were subjected to flow cytometry
analysis to test purity,
differentiation, and memory markers. For all the conditions the percentage of
TCR a/b+ cells was over
90%.
10018741 TIL harvested from the Gen 3 process showed a higher
expression of CD 8 and CD28
compared to TIL harvested from the Gen 2 process. The Gen 2 process showed a
higher percentage of
CD4+.
10018751 TIL harvested from the Gen 3 process showed a higher
expression on central memory
compartments compared to TIL from the Gen 2 process.
10018761 Activation and exhaustion markers were analyzed in TIL
from two, tumors L4054
and L4055 to compare the final TIL product by from the Gen 2 and Gen 3 TIL
expansion processes.
Activation and exhaustion markers were comparable between the Gen 2 and Gen 3
processes.
10018771 Interferon gamma secretion upon restimulation. On harvest
day, day 22 for Gen 2 and
day 16 for Gen 3, TIL underwent an overnight restimulation with coated anti-
CD3 plates for L4054
and L4055. The restimulation on M1085T was performed using anti-CD3, CD28, and
CD137 beads.
Supernatant was collected after 24 hours of the restimulation in all
conditions and the supernatant was
frozen. IFNy analysis by ELISA was assessed on the supematant from both
processes at the same time
using the same ELISA plate. Higher production of IFNy from the Gen 3 process
was observed in the
three tumors analyzed.
10018781 Measurement of IL-2 levels in culture media. To compare
the IL-2 consumption
between Gen 2 and Gen 3 process, cell supernatant was collected on REP
initiation, scale up, and
harvest day, on tumor L4054 and L4055. The quantity of IL-2 in cell culture
supernatant was
measured by Quantitate ELISA Kit from R&D. The general trend indicates that
the IL-2 concentration
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remains higher in the Gen 3 process when compared to the Gen 2 process. This
is likely due to the
higher concentration of IL-2 on REP initiation (6000 IU/mL) for Gen 3 coupled
with the carryover of
the media throughout the process.
10018791 Metabolic substrate and metabolite analysis. The levels
of metabolic substrates such
as D-glucose and L-glutamine were measured as surrogates of overall media
consumption. Their
reciprocal metabolites, such lactic acid and ammonia, were measured. Glucose
is a simple sugar in
media that is utilized by mitochondria to produce energy in the form of ATP.
When glucose is
oxidized, lactic acid is produced (lactate is an ester of lactic acid).
Lactate is strongly produced during
the cells exponential growth phase. High levels of lactate have a negative
impact on cell culture
processes.
10018801 Spent media for L4054 and L4055 was collected at REP
initiation, scale up, and
harvest days for both process Gen 2 and Gen 3. The spent media collection was
for Gen 2 on Day 11,
day 16 and day 22; for Gen 3 was on day 7, day 11 and day 16. Supernatant was
analyzed on a
CEDEX Bio-analyzer for concentrations of glucose, lactic acid, glutamine,
GlutaMaxTm, and
ammonia.
10018811 L-glutamine is an unstable essential amino acid required
in cell culture media
formulations. Glutamine contains an amine, and this amide structural group can
transport and deliver
nitrogen to cells. When L-glutamine oxidizes, a toxic ammonia by-product is
produced by the cell. To
counteract the degradation of L-glutaminc the media for the Gen 2 and Gen 3
processes was
supplemented with GlutaMaxTm, which is more stable in aqueous solutions and
does not
spontaneously degrade. In the two tumor lines, the Gen 3 arm showed a decrease
in L-glutamine and
GlutaMaxTm during the process and an increase in ammonia throughout the REP.
In the Gen 2 arm a
constant concentration of L-glutamine and GlittaMaxTm, and a slight increase
in the ammonia
production was observed. The Gen 2 and Gen 3 processes were comparable at
harvest day for
ammonia and showed a slight difference in L-glutamine degradation.
10018821 Telomere repeats by Flow-FISH. Flow-FISH technology was
used to measure the
average length of the telomere repeat on L4054 and L4055 under Gen 2 and Gen 3
process. The
determination of a relative telomere length (RTL) was calculated using
Telomere PNA kit/FITC for
flow cytometry analysis from DAKO. Gen 3 showed comparable telomere length to
Gen 2.
10018831 CD3 Analysis. To determine the clonal diversity of the
cell products generated in
each process, T1L final product harvested for L4054 and L4055, were sampled
and assayed for clonal
diversity analysis through sequencing of the CDR3 portion of the T-cell
receptors.
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10018841 Table 50 shows a comparison between Gen 2 and Gen 3 of
percentage shared unique
CDR3 sequences on L4054 on TIL harvested cell product. 199 sequences are
shared between Gen 3
and Gen 2 final product, corresponding to 97.07% of top 80% of unique CDR3
sequences from Gen 2
shared with Gen 3 final product.
TABLE 50. Comparison of shared uCDR3 sequences between Gen 2 and Gen 3
processes on L4054.
# uCDR3 All uCDR3's Top 80% uCDR3's
(% Overlap) Gen 2 Gen 3 Gen 2 Gen 3
Gen 2-L4054 8915 4355(48.85%) 205
199(97.07%)
Gen 3-L4054 18130 223
10018851 Table 51 shows a comparison between Gen 2 and Gen 3 of
percentage shared unique
CDR3 sequences on L4055 on TIL harvested cell product. 1833 sequences are
shared between Gen 3
and Gen 2 final product, corresponding to 99.45% of top 80% of unique CDR3
sequences from Gen 2
shared with Gen 3 final product.
TABLE 51. Comparison of shared uCDR3 sequences between Gen 2 and Gen 3
processes on L4055.
uCDR3 All uCDR3's Top 80% uCDR3's
(% Overlap) Gen 2 Gen 3 Gen 2 Gen 3
Gen 2-L4055 12996 6599 (50.77%) 1843
1833(99.45%)
Gen 3-L4055 27246 2616
10018861 CM I and CM2 media was prepared in advanced without
filtration and held at 4
degree C until use for tumor L4055 to use on Gen 2 and Gen 3 process.
10018871 Media was warmed up at 37 degree C for 24 hours in
advance for tumor L4055 on
REP initiation day for Gen 2 and Gen 3 process.
10018881 LDH was not measured in the supernatants collected on the
processes.
10018891 M1085T TIL cell count was executed with K2 cellometer
cell counter.
10018901 On tumor M1085T, samples were not available such as
supernatant for metabolic
analysis, TIL product for activation and exhaustion markers analysis, telomere
length and CD3 - TCR
vb Analysis.
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10018911 Conclusions. This example compares 3 independent donor
tumors tissue in terms of
functional quality attributes, plus extended phenotypic characterization and
media consumption
among Gen 2 and Gen 3 processes.
10018921 Gen 2 and Gen 3 pre-REP and REP expansion comparison were
evaluated in terms of
total viable cells generated and viability of the total nucleated cell
population. TVC cell doses at
harvest day was not comparable between Gen 2 (22 days) and Gen 3 (16 days).
Gen 3 cell doses were
lower than Gen 2 at around 40% of total viable cells collected at harvest.
10018931 An extrapolated cell number was calculated for Gen 3
process assuming the pre-REP
harvest occurred at day 11 instead day 7 and REP Harvest at Day 22 instead day
16. In both cases,
Gen 3 shows a closer number on TVC compared to the Gen 2 process, indicating
that the early
activation enhanced TIL growth.
10018941 In the case of extrapolated value for extra flasks (2 or
3) on Gen 3 process assuming a
bigger size of tumor processed, and reaching the maximum number of fragments
required per process
as described. It was observed that a similar dose can be reachable on TVC at
Day 16 Harvest for Gen
3 process compared to Gen 2 process at Day 22. This observation is important
and indicates an early
activation of the culture reduced TIL processing time.
10018951 Gen 2 and Gen 3 pre-REP and REP expansion comparison were
evaluated in tennis of
total viable cells generated and viability of the total nucleated cell
population. TVC cell doses at
harvest day was not comparable between Gen 2 (22 days) and Gen 3 (16 days).
Gen 3 cell doses were
lower than Gcn 2 at around 40% of total viable cells collected at harvest.
10018961 In terms of phenotypic characterization, a higher CD8+
and CD28+ expression was
observed on three tumors on Gen 3 process compared to Gen 2 process.
10018971 Gen 3 process showed slightly higher central memory
compartments compared to
Gen 2 process.
10018981 Gen 2 and Gen 3 process showed comparable activation and
exhaustion markers,
despite the shorter duration of the Gen 3 process.
10018991 IFN gamma (IFN7) production was 3 times higher on Gen 3
final product compared
to Gen 2 in the three tumors analyzed. This data indicates the Gen 3 process
generated a highly
functional and more potent TIL product as compared to the Gen 2 process,
possibly due to the higher
expression of CD8 and CD28 expression on Gen 3. Phenotypic characterization
suggested positive
trends in Gen 3 toward CD8+, CD28+ expression on three tumors compared to Gen
2 process.
10019001 Telomere length on TIL final product between Gen 2 and
Gen 3 were comparable.
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10019011 Glucose and Lactate levels were comparable between Gen 2 and Gen 3
final product,
suggesting the levels of nutrients on the media of Gen 3 process were not
affected due to the non-
execution of volume reduction removal in each day of the process and less
volume media overall in
the process, compared to Gen 2.
10019021 Overall Gen 3 process showed a reduction almost two times of the
processing time
compared to Gen 2 process, which would yield a substantial reduction on the
cost of goods (COGs)
for TIL product expanded by the Gen 3 process.
10019031 IL-2 consumption indicates a general trend of IL-2 consumption on
Gen 2 process,
and in Gen 3 process IL-2 was higher due to the non-removal of the old media.
10019041 The Gen 3 process showed a higher clonal diversity measured by
CDR3 TCRab
sequence analysis.
10019051 The addition of feeders and OKT-3 on day 0 of the pre-REP allowed
an early
activation of TIL and allowed for TIL growth using the Gen 3 process.
10019061 Table 52 describes various embodiments and outcomes for the Gen 3
process as
compared to the current Gen 2 process.
TABLE 52. Exemplary Gen 3 process features.
Step Process Gen 2 embodiment Process Gen 3 embodiment
<240 fragments
<50 fragments <60
fragments/flask
IX G-REX-100MCS <4 flasks
Pre REP-
1 L media <2L media (500
mL/flask)
day 0
IL-2 (6000 IU/mL) IL-2 (6000 IU/mL)
11 days 2.5x108 feeder
cells/flask
15ug OKT3/flask
Fresh TIL direct to REP
Day 11 Fresh TIL direct
to REP
REP
<200e 6 viable cells Day 7
Initiation
x 1 09 feeder cells Activate entire
culture
G-REX-500MCS 5 > 108 feeder
cells
5L CM2 media + IL-2 (3000 IU/mL) 30 lag OKT3/flask
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150 lag OKT3 G-REX-100MCS
500 mL media+ IL-2 (6000 IU/mL)
<5 G-REX-500MCS <4 G-REX-500MCS
TIL Sub- <1x10 viable cells/ flask Scale up entire
culture
culture or
Scale up 5 L/flask 4
L/flask
Day 16 Day 10-
11
Harvest Day 22, Harvest Day 16
Harvest LOVO-automated cell washer LOVO-automated cell
washer
2 wash cycles 5 wash cycles
C
Cryopreserved Product ryopreserved
product
Final
formulation 300 IU/mL IL2- CS10 in LN2, 300 IU/mL IL-2-CS10
in LN2,
multiple aliquots multiple
aliquots
Process
22 days 16
days
time
EXAMPLE 11: AN EXEMPLARY GEN 3 PROCESS (ALSO REFERRED TO AS GEN 3.1)
10019071 This example describes further studies regarding the
"Comparability between the Gen
2 and Gen 3 processes for TIL expansion". The Gen 3 process was modified to
include an activation
step early in the process with the goal of increasing the final total viable
cell (TVC) output, while
maintaining the phenotypic and functional profiles. As described below, a Gen
3 embodiment was
modified as a further embodiment and is referred to herein in this example as
Gen 3.1.
10019081 In some embodiments, the Gen 3.1 TIL manufacturing
process has four operator
interventions:
1. Tumor Fragment Isolation and Activation: On Day 0 of the process the tumor
was
dissected and the final fragments generated awe-3x3mm each (up to 240
fragments total) and
cultured in 1-4 G-REX100MCS flasks. Each flask contained up to 60 fragments,
500 mL of
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CM1 or DM1 media, and supplemented with 6,000 IU rhIL-2, 15 lig OKT3, and
2.5x108
irradiated allogeneic mononuclear cells. The culture was incubated at 37 C for
6-8 days.
2. TIL Culture Reactivation: On Day 7-8 the culture was supplemented through
slow addition
of CM2 or DM1 media supplemented with 6,000 IU rhIL-2, 30 lag OKT3, and 5x108
irradiated allogeneic mononuclear cells in both cases. Care was taken to not
disturb the
existing cells at the bottom of the flask. The culture was incubated at 37 C
for 3-4 days.
3. Culture Scale Up: Occurs on day 10-11. During the culture scale-up, the
entire contents of
the G-REX100MCS was transferred to a G-REX500MCS flask containing 4L of CM4 or
DM2 supplemented with 3,000 IU/mL of IL-2 in both cases. Flasks were incubated
at 37 C
for 5-6 days until harvest.
4. Harvest/Wash/Formulate: On day 16-17 the flasks are volume reduced and
pooled. Cells
were concentrated and washed with PlasmaLyte A pH 7.4 containing 1% HSA. The
washed
cell suspension was formulated at a 1:1 ratio with CryoStorl 0 and
supplemented with rhIL-2
to a final concentration of 300 IU/mL.
10019091 The DP was cryopreserved with a controlled rate freeze
and stored in vapor phase
liquid nitrogen. *Complete Standard TIL media 1, 2, or 4 (CM1, CM2, CM4) could
be substituted for
CTSTmOpTmizerrm T-Cell serum free expansion Medium, referred to as Defined
Medium (DM1 or
DM2), as noted above.
10019101 Process description. On day 0, the tumor was washed 3
times, then fragmented in
3x3x3 final fragments. Once the whole tumor was fragmented, then the final
fragments were
randomized equally and divided into three pools. One randomized fragment pool
was introduced to
each arm, adding the same number of fragments per the three experimental
matrices.
10019111 Tumor L4063 expansion was performed with Standard Media
and tumor L4064
expansion was performed with Defined Media (CTS OpTmizer) for the entire TIL
expansion process.
Components of the media are described herein.
10019121 CM1 Complete Media 1: RPMI+ Glutamine supplemented with
2mM GlutaMaxTm,
10% Human AB Serum, Gentamicin (50ug/mL), 2-Mercaptoethanol (55uM). Final
media formulation
supplemented with 6000IU/mL IL-2.
10019131 CM2 Complete Media 2: 50% CM1 medium + 50% AIM-V medium.
Final media
formulation supplemented with 6000IU/mL IL-2.
[001914] CM4 Complete Media 4: AIM-V supplemented with GlutaMax TM
(2mM). Final
media formulation supplemented with 3000IU/mL IL-2.
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10019151 CTS OpTmizer CTSTmOpTmizerTm T-Cell Expansion Basal
Medium supplemented
with CTSTm OpTrnizerTm T-Cell Expansion Supplement (26 mL/L).
10019161 DM1: CTSTmOpTmizerTm T-Cell Expansion Basal Medium
supplemented with
CTSTm OpTmizerTm T-Cell Expansion Supplement (26 mL/L), and CTSTm Immune Cell
SR (3%),
with GlutaMaxTm (2mM). Final formulation supplemented with 6,000 IU/mL of IL-
2.
10019171 DM2: CTSTmOpTmizerTm T-Cell Expansion Basal Medium
supplemented with
CTSTm OpTmizerTm T-Cell Expansion Supplement (26 mL/L), and CTSTm Immune Cell
SR (3%),
with GlutaMaxTm (2mM). Final formulation supplemented with 3,000 IU/mL of IL-
2.
10019181 All types of media used, i.e., Complete (CM) and Defined
(DM) media, were
prepared in advance, held at 4 C degree until the day before use, and warmed
at 37 C in an incubator
for up to 24 hours in advance prior to process day.
10019191 TIL culture reactivation occurred on Day 7 for both
tumors. Scale-up occurred on day
for L4063 and day 11 for L4064. Both cultures were harvested and cryopreserved
on Day 16.
10019201 Results Achieved. Cells counted and % viability for Gen
3.0 and Gen 3.1 processes
were determined. Expansion in all the conditions followed details described in
this example.
10019211 For each tumor, the fragments were divided into three
pools of equal numbers. Due to
the small size of the tumors, the maximum number of fragments per flask was
not achieved. For the
three different processes, the total viable cells and cell viability were
assessed for each condition. Cell
counts were determined as TVC on day 7 for reactivation, TVC on day 10 (L4064)
or day 11 (L4063)
for scale-up, and TVC at harvest on day 16/17.
10019221 Cell counts for Day 7 and Day 10/11 were taken F10. Fold
expansion was calculated
by dividing the harvest day 16/17 TVC by the day 7 reactivation day TVC. To
compare the three
anus, the TVC on harvest day was divided by the number of fragments added in
the culture on Day 0
in order to calculate an average of viable cells per fragment.
10019231 Cell counts and viability assays were performed for L4063
and L4064. The Gen 3.1-
Test process yielded more cells per fragment than the Gen 3.0 Process on both
tumors.
10019241 Total viable cell count and fold expansion; % Viability
during the process. On
reactivation, scale up and harvest the percent viability was performed on all
conditions. On day 16/17
harvest, the final TVC were compared against release criteria for % viability.
All of the conditions
assessed surpassed the 70% viability criterion and were comparable across
processes and tumors.
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[001925] Immunophenotyping - Phenotypic characterization on TIL
final product. The final
products were subjected to flow cytometry analysis to test purity,
differentiation, and memory
markers. Percent populations were consistent for TCRa/13, CD4+ and CD8+ cells
for all conditions.
[001926] Extended phenotypic analysis of REP TIL was performed.
TIL product showed a
higher percentage of CD4+ cells for Gen 3.1 conditions compared to Gen 3.0 on
both tumors, and
higher percentage of CD28+ cells from CD8+ population for Gen 3.0 compared to
Gen 3.1 conditions
on both conditions.
[001927] TIL harvested from the Gen 3.0 and Gen 3.1 processes
showed comparable
phenotypic markers as CD27 and CD56 expression on CD4+and CD8+ cells, and a
comparable CD28
expression on CD4+ gated cells population. Memory markers comparison on TIL
final product:
[001928] Frozen samples of TIL harvested on day 16 were stained
for analysis. TIL memory
status was comparable between Gen 3.0 and Gen 3.1 processes. Activation and
exhaustion markers
comparison on TIL final product:
[001929] Activation and exhaustion markers were comparable between
the Gen 3.0 and Gen
3.1 processes gated on CD4+ and CD8+ cells.
[001930] Interferon gamma secretion upon restimulation. Harvested
TIL underwent an
overnight restimulation with coated anti-CD3 plates for L4063 and L4064.
Higher production of IFNy
from the Gcn 3.1 process was observed in the two tumors analyzed compared to
Gen 3.0 process.
[001931] Measurement of IL-2 levels in culture media. To compare
the levels of IL-2
consumption between all of the conditions and processes, cell supernatants
were collected at initiation
of reactivation on Day 7, at scale-up Day 10 (L4064) / 11 (L4063), and at
harvest Day 16 / 17, and
frozen. The supernatants were subsequently thawed and then analyzed. The
quantity of IL-2 in cell
culture supernatant was measured by the manufacturer protocol.
[001932] Overall Gen 3 and Gen 3.1 processes were comparable in
terms of IL-2 consumption
during the complete process assessed across same media conditions. IL-2
concentration (pg/mL)
analysis on spent media collected for L4063 and L4064.
[001933] Metabolite analysis. Spent media supernatants was
collected from L4063 and L4064
at reactivation initiation on day 7, scale-up on day 10 (L4064) or day 11
(L4063), and at harvest on
days 16/17 for L4063 and L4064, for every condition. Supernatants were
analyzed on a CEDEX Bio-
analyzer for concentrations of glucose, lactate, glutamine, GlutaMaxTm, and
ammonia.
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10019341 Defined media has a higher glucose concentration of 4.5
g/L compared to complete
media (2g/L). Overall, the concentration and consumption of glucose were
comparable for Gen 3.0
and Gen 3.1 processes within each media type.
10019351 An increase in lactate was observed and increase in
lactate was comparable between
the Gen 3.0 and Gen 3.1 conditions and between the two media used for
reactivation expansion
(complete media and defined media).
10019361 In some instances, the standard basal media contained 2
mM L-glutamine and was
supplemented with 2mM GlutaMaxTm to compensate for the natural degradation of
L-glutamine in
culture conditions to L-glutamate and ammonia.
10019371 In some instances, defined (scrum free) media used did
not contain L-glutamine on
the basal media, and was supplemented only with GlutaMaxTm to a final
concentration of 2mM.
GlutaMaxTm is a dipeptide of L-alanine and L-glutamine, is more stable than L-
glutamine in aqueous
solutions and does not spontaneously degrade into glutamate and ammonia.
Instead, the dipeptide is
gradually dissociated into the individual amino acids, thereby maintaining a
lower but sufficient
concentration of L-glutamine to sustain robust cell growth.
10019381 In some instances, the concentration of glutamine and
GlutaMaxTm slightly decreased
on the scale-up day, but at harvest day showed an increase to similar or
closer levels compared to
reactivation day. For L4064, glutamine and GlutaMaxTm concentration showed a
slight degradation in
a similar rate between different conditions, during the whole process.
10019391 Ammonia concentrations were higher samples grown in
standard media containing 2
mM glutamine + 2 mM GlutaMaxIm) than those grown in defined media containing 2
mM
GlutaMaxTm). Further, as expected, there was a gradual increase or
accumulation of ammonia over the
course of the culture. There were no differences in ammonia concentrations
across the three different
test conditions.
10019401 Telomere repeats by Flow ¨ FISH. Flow-FISH technology was
used to measure the
average length of the telomere repeat on L4063 and L4064 under Gen 3 and Gen
3.1 processes. The
determination of a relative telomere length (RTL) was calculated using
Telomere PNA kit/FITC for
flow cytometry analysis from DAKO. Telomere assay was performed. Telomere
length in samples
were compared to a control cell line (1301 leukemia). The control cell line is
a tetraploid cell line
having long stable telomeres that allows calculation of a relative telomere
length. Gen 3 and Gen 3.1
processes assessed in both tumors showed comparable telomere length.
TCR Vfl repertoire Analysis
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[001941] To determine the clonal diversity of the cell products
generated in each process, TIL
final products were assayed for clonal diversity analysis through sequencing
of the CDR3 portion of
the T-cell receptors.
[001942] Three parameters were compared between the three conditions:
= Diversity index of Unique CDR3 (uCDR3)
= % shared uCDR3
= For the top 80% of uCDR3:
o Compare the % shared uCDR3 copies
o Compare the frequency of unique clonotypes
[001943] Control and Gen 3.1 Test, percentage shared unique CDR3 sequences on
TIL harvested
cell product for: 975 sequences are shared between Gen 3 and Gen 3.1 Test
final product, equivalent
to 88% of top 80% of unique CDR3 sequences from Gcn 3 shared with Gen 3.1.
[001944] Control and Gen 3.1 Test, percentage shared unique CDR3 sequences on
TIL harvested
cell product for: 2163 sequences are shared between Gen 3 and Gen 3.1 Test
final product, equivalent
to 87% of top 80% of unique CDR3 sequences from Gen 3 shared with Gen 3.1.
10019451 The number of unique CD3 sequences identified from 1x106 cells
collected on Harvest
day 16, for the different processes. Gen 3.1 Test condition showed a slightly
higher clonal diversity
compared to Gen 3.0 based on the number of unique peptide CDRs within the
sample.
[001946] The Shannon entropy diversity index is a reliable and
common metric for
comparison, because Gen 3.1 conditions on both tumors showed slightly higher
diversity than Gen 3
process, suggesting that TCR VP repertoire for Gen 3.1 Test condition was more
polyclonal than the
Gen 3.0 process.
[001947] Additionally, the TCR VP repertoire for Gen 3.1 Test
condition showed more than
87% overlap with the corresponding repertoire for Gen 3.0 process on both
tumor L4063 and L4064.
[001948] The value of IL-2 concentration on spent media for Gen
3.1 Test L4064 on
reactivation day was below to the expected value (similar to Gen 3.1 control
and Gen 3.0 condition).
[001949] The low value could be due to a pipetting error, but
because of the minimal sample
taken it was not possible to repeat the assay.
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10019501 Conclusions. Gen 3.1 test condition including feeders and
OKT-3 on Day 0 showed a
higher TVC of cell doses at Harvest day 16 compared to Gen 3.0 and Gen 3.1
control. TVC on the
final product for Gen 3.1 test condition was around 2.5 times higher than Gen
3Ø
10019511 Gen 3.1 test condition with the addition of OKT-3 and
feeders on day 0, for both
tumor samples tested, reached a maximum capacity of the flask at harvest.
Under these conditions, if a
maximum of 4 flasks on day 0 is initiated, the final cell dose could be
between 80 - 100>< 109 TILs.
10019521 All the quality attributes such as phenotypic
characterization including purity,
exhaustion, activation and memory markers on final TIL product were maintained
between Gen 3.1
Test and Gen 3.0 process.
10019531 IFN-y production on final TIL product was 3 times higher
on Gen 3.1 with feeder and
OKT-3 addition on day 0, compared to Gen 3.0 in the two tumors analyzed,
suggesting Gen 3.1
process generated a potent TIL product.
10019541 No differences observed in glucose or lactate levels
across test conditions. No
differences observed on glutamine and ammonia between Gen 3.0 and Gen 3.1
processes across
media conditions. The low levels of glutamine on the media are not limiting
cell growth and suggest
the addition of GlutaMaxTm only in media is sufficient to give the nutrients
needed to make cells
proliferate.
10019551 The scale up on day 11 and day 10 respectively and did
not show major differences in
terms of cell number reached on the harvest day of the process and metabolite
consumption was
comparable in both cases during the whole process. This observation suggests
of Gen 3.0 optimized
process can have flexibility on processing days, thereby facilitating
flexibility in the manufacturing
schedule.
10019561 Gen 3.1 process with feeder and OKT-3 addition on day 0
showed a higher clonal
diversity measured by CDR3 TCRab sequence analysis compared to Gen 3Ø
10019571 Figure 32 describes an embodiment of the Gen 3 process
(Gen 3 Optimized process).
Standard media and CTS Optimizer serum free media can be used for Gen 3
Optimized process TIL
expansion. In case of CTS Optimizer serum free media is recommended to
increase the GlutaMaxTm
on the media to final concentration 4mM.
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EXAMPLE 12: AN EXEMPLARY EMBODIMENT OF SELECTING AND EXPANDING
PBLS FROM PBMCS IN CLL PATIENTS.
10019581 PBMCs are collected from patients and either frozen for later use, or
used fresh. Enough
volume of peripheral blood is collected to yield at least about 400,000,000
(400 x 106) PBMCs for
starting material in the method of the present invention. On Day 0 of the
method, IL-2 at 6x106IU/mL
is either prepared fresh or thawed, and stored at 4 C or on ice until ready to
use. 200 mL of CM2
medium is prepared by combining 100 mL of CM1 medium (containing GlutaMAX0),
then diluting
it with 100 mT, (1:1) with AIM-V to make CM2. The CM2 is protected from light,
and sealed tightly
when not in use.
10019591 All of the following steps are performed under sterile cell culture
conditions. An aliquot of
CM2 is warmed in a 50mL conical tube in a 37 C water bath for use in thawing
and/or washing a
frozen PBMC sample. If a frozen PBMC sample is used, the sample is removed
from freezer storage
and kept on dry ice until ready to thaw. When ready to thaw the PBMC cryovial,
5 mL of CM2
medium is placed in a sterile 50 mL conical tube. The PBMC sample cryovial is
placed in a 37 C
water bath until only a few ice crystals remain. Warmed CM2 medium is added,
dropwise, to the
sample vial in a 1:1 volume ratio of sample:medium (about 1 mL). The entire
contents is removed
from the cryovial and transferred to the remaining CM2 medium in the 50 mL
conical tube. An
additional 1-2 mL of CM2 medium is used to rinse the cryovial and the entire
contents of the cryovial
is removed and transferred to the 50 mL conical tube. The volume in the
conical tube is then adjusted
with additional CM2 medium to 15 mL, and swirled gently to rinse the cells.
The conical tube is then
centrifuged at 400g for 5 minutes at room temperature in order to collect the
cell pellet.
10019601 The supernatant is removed from the pellet, the conical tube is
capped, and then the cell
pellet is disrupted by, for example, scraping the tube along a rough surface.
About lmL of CM2
medium is added to the cell pellet, and the pellet and medium are aspirated up
and down 5-10 times
with a pipette to break up the cell pellet. An additional 3-5 mL of CM2 medium
is added to the tube
and mixed via pipette to suspend the cells. At this point, the volume of the
cell suspension is recorded.
Remove 100 uL of the cell suspension from the tube for cell counting with an
automatic cell counter,
for example, a Nexcelom Cellometer K2. Determine the number of live cells in
the sample and record.
10019611 Reserve a minimum of 5 x 106 cells for phenotyping and other
characterization
experiments. Spin the reserved cells at 400g for 5 minutes at room temperature
to collect the cell
pellet. Resuspend the cell pellet in freezing medium (sterile, heat-
inactivated FBS containing 20%
DMS0). Freeze one or two aliquots of the reserved cells in freezing medium,
and slow-freeze the
aliquots in a cell freezer (Mr. Frosty) in a -80 C freezer_ Transfer to liquid
nitrogen storage after a
minimum of 24 hours at -80 C.
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10019621 For the following steps, use pre-cooled solutions, work quickly, and
keep the cells cold. The
next step is to purify the T-cell fraction of the PBMC sample. This is
completed using a Pan T-cell
Isolation Kit (Miltenyi, catalog # 130-096-535). Prepare the cells for
purification by washing the cells
with a sterile-filtered wash buffer containing PBS, 0.5% BSA, and 2mM EDTA at
pH 7.2. The PBMC
sample is centrifuged at 400g for 5 minutes to collect the cell pellet. The
supernatant is aspirated off
and the cell pellet is resuspended in 40 uL of wash buffer for every 10'
cells. Add 10 uL of Pan T Cell
Biotin-Antibody Cocktail for every 107 cells. Mix well and incubate for 5
minutes in refrigerator or on
ice. Add 30 uL of wash buffer for every 107 cells. Add 20 uL of Pan T-cell
MicroBead Cocktail for
every 107 cells. Mix well and incubate for 10 minutes in refrigerator or on
ice. Prepare an LS column
and magnetically separate cells from the microbeads. The LS column is placed
in the QuadroMACS
magnetic field. The LS column is washed with 3 mL of cold wash buffer, and the
wash is collected
and discarded. The cell suspension is applied to the column and the flow-
through (unlabeled cells) is
collected. This flow-through is the enriched T-cell fraction (PBLs). Wash the
column with 3 mL of
wash buffer and collect the flow-through in the same tube as the initial flow-
through. Cap the tube
and place on ice. This is the T-cell fraction, or PBLs. Remove the LS column
from the magnetic field,
wash the column with 5 mL of wash buffer, and collect the non-T-cell fraction
(magnetically labeled
cells) into another tube. Centrifuge both fractions at 400g for 5 minutes to
collect the cell pellets.
Supernatants arc aspirated from both samples, disrupt the pellet, and
rcsuspcnd the cells in 1 mL of
CM2 medium supplemented with 3000 IU/mL IL-2 to each pellet, and pipette up
and down 5-10
times to break up the pellets. Add 1-2 mL of CM2 to each sample, and mix each
sample well, and
store in tissue culture incubator for next steps. Remove about a 50 uL aliquot
from each sample, count
cells, and record count and viability.
10019631 The T-cells (PBLs) are then cultured with DunabeadsTM Human T-
Expander CD3/CD28. A
stock vial of Dynabeads is vortexed for 30 seconds at medium spead. A required
aliquot of beads is
removed from the stock vial into a sterile 1.5 mL microtube. The beads are
washed with bead wash
solution by adding 1 mL of bead wash to the 1.5 mL microtube containing the
beads. Mix gently.
Place the tube onto the DynaMagTm-2 magnet and let sit for 30 minutes while
beads draw toward the
magnet. Aspirate the wash solution off the beads and remove tube from the
magnet. lmL of CM2
medium supplemented with 3000 IU/mL IL-2 is added to the beads. The entire
contents of the
microtube is transferred to a 15 or 50 mL conical tube. Bring the beads to a
final concentration of
about 500,000/mL using CM2 medium with IL-2.
10019641 The T-cells (PBLs) and beads are cultured together as follows. On day
0: In a G-Rex-REX
24 well plate, in a total of 7mL per well, add 500,000 T-cells, 500,000
CD3/CD28 Dynabeads, and
CM2 supplemented with IL-2. The G-Rex-REX plate is placed into a humidified 37
C, 5% CO2
incubator until the next step in the process (on Day 4). Remaining cells are
frozen in CS10
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cryopreservation medium using a Mr. Frosty cell freezer. The non-T-cell
fraction of cells are frozen in
CS10 cryopresenntion medium using a Mr. Frosty cell freezer. On day 4, medium
is exchanged. Half
of the medium (about 3.5mL) is removed from each well of the G-rex plate. A
sufficient volume
(about 3.5mL) of CM4 medium supplemented with 3000 IU/mL IL-2 warmed to 37 C
is added to
replace the medium removed from each sample well. The G-rex plate is returned
to the incubator.
10019651 On day 7, cells are prepared for expansion by REP. The G-rex plate is
removed from the
incubator and half of medium is removed from each well and discarded. The
cells are resuspended in
the remaining medium and transferred to a 15 mI, conical tube. The wells are
washed with 1 mI, each
of CM4 supplemented with 3000 IU/mL IL-2 warmed to 37 C and the wash medium is
transferred to
the same 15 mL tube with the cells. A representative sample of cells is
removed and counted using an
automated cell counter. If there are less than 1x106 live cells, the Dynabead
expansion process at Day
0 is repeated. The remainder of the cells are frozen for back-up expansion or
for phenotyping and
other characterization studies. If there are lx106 live cells or more, the REP
expansion is set up in
replicate according to the protocol from Day 0. Alternatively, with enough
cells, the expansion may
be set up in a G-rex 10M culture flask using 10-15x106 PBLs per flask and a
1:1 ratio of
Dynabeads:PBLs in a final volume of 100mL/well of CM4 medium supplemented with
3000 1U/mL
IL-2. The plate and/or flask is returned to the incubator. Excess PBLs may be
aliquotted and slow-
frozen in a Mr. Frosty cell freezer in a -80 C freezer, and the transferred to
liquid nitrogen storage
after a minimum of 24 hours at -80 C. These PBLs may be used as back-up
samples for expansion or
for phenotyping or other characterization studies.
10019661 On Day 11, the medium is exchanged. Half of the medium is removed
from either each well
of the G-rex plate or the flask and replaced with the same amount of fresh CM4
medium
supplemented with 3000 IU/mL IL-2 at 37 C.
10019671 On Day 14, the PBLs are harvested. If the G-rex plate is used, about
half of the medium is
removed from each well of the plate and discarded. The PBLs and beads are
suspended in the
remaining medium and transferred to a sterile 15 mL conical tube (Tube 1). The
wells are washed
with 1-2 mL of fresh AIM-V medium warmed to 37 C, and the wash is transferred
to Tube 1. Tube 1
is capped and placed in the DynaMagTm-15 Magnet for 1 minute to allow the
beads to be drawn to the
magnet. The cell suspension is transferred into a new 15 mL tube (Tube 2), and
the beads are washed
with 2mL of fresh AIM-V at 37 C. Tube 1 is placed back in the magnet for an
additional 1 minute,
and the wash medium is then transferred to Tube 2. The wells may be combined
if desired, after the
final washing step. Remove a representative sample of cells and count, record
count and viability.
Tubes may be placed in the incubator while counting. Additional AIM-V medium
may be added to
the Tube 2 if cells appear very dense. If a flask is used, the volume in the
flask should be reduced to
about 10 mL. The contents of the flask is mixed and transferred to a 15 mL
conical tube (Tube A).
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The flask is washed with 2mL of the AIM-V medium as described above and the
wash medium is also
transferred to Tube A. Tube A is capped and placed in the DynaMagTm-15 Magnet
for 1 minute to
allow the beads to be drawn to the magnet. The cell suspension is transfen-ed
into a new 15 mL tube
(Tube B), and the beads are washed with 2mL of fresh AIM-V at 37 C. Tube A is
placed back in the
magnet for an additional 1 minute, and the wash medium is then transferred to
Tube B. The wells may
be combined if desired, after the final washing step. Remove a representative
sample of cells and
count, record count and viability. Tubes may be placed in the incubator while
counting. Additional
AIM-V medium may be added to the Tube B if cells appear very dense. Cells may
be used fresh or
frozen in CS10 preservation medium at desired concentrations.
EXAMPLE 13: A PHASE 2, MULTICENTER STUDY OF AUTOLOGOUS TUMOR
INFILTRATING LYMPHOCYTES IN PATIENTS WITH SOLID TUMORS
10019681STUDY DESIGN
100196910verview
10019701This example describes a prospective, open-label, multi-cohort, non-
randomized,
multicenter Phase 2 study evaluating ACT using TIL in combination with
pembrolizumab or TIL as a
single therapy, using TILs prepared as described in the present application as
well as in this example.
10019711Objectives:
[0019721Primary:
10019731To evaluate the efficacy of autologous TIL in combination with
pembrolizumab in MM,
HNSCC, or NSCLC patients or TIL as a single therapy in relapsed or refractory
(r/r) NSCLC patients,
who had previously progressed on or after treatment with CPIs, as determined
by objective response
rate (ORR), using the Response Evaluation Criteria in Solid Tumors (RECIST
1.1), as assessed by
Investigator.
10019741To characterize the safety profile of TIL in combination with
pembrolizumab in MM,
HNSCC, and NSCLC patients or TIL as a single therapy in r/r NSCLC patients as
measured by the
incidence of Grade > 3 treatment-emergent adverse events (TEAEs).
10019751Secondary:
10019761To further evaluate the efficacy of autologous TIL in combination with
pembrolizumab in
MM, HNSCC, and NSCLC patients or TIL as a single therapy in r/r NSCLC patients
using complete
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response (CR) rate, duration of response (DOR), disease control rate (DCR),
progression-free survival
(PFS) using RECIST 1.1, as assessed by Investigator, and overall survival
(OS).
10019771 Cohorts:
10019781 Cohort 1A: TIL therapy in combination with pembrolizumab in patients
with Stage IIIC or
Stage IV unresectable or MM with < 3 prior lines of systemic therapy excluding
immunotherapy. If
previously treated, patients must have had radiographically documented
progression on or after most
recent therapy.
10019791 Cohort 2A: TIL therapy in combination with pembrolizumab in patients
with advanced,
recurrent or metastatic HNSCC (e.g., Stages T1N1-N2B, T2-4N0-N2b) with < 3
prior lines of
systemic therapy, excluding immunotherapy. If previously treated, patients
must have had
radiographically documented progression on or after most recent therapy.
10019801 Cohort 3A: TIL therapy in combination with pembrolizumab in patients
with locally
advanced or metastatic (Stage III¨ IV) NSCLC with <3 prior lines of systemic
therapy, excluding
immunotherapy. if previously treated, patients must have had radiographically
documented
progression on or after most recent therapy.
10019811 Cohort 3B: TIL therapy as a single agent in patients Stage HI or
Stage TV NSCLC who have
previously received systemic therapy with CPIs (e.g., anti-PD-1/anti-PD-L1) as
part of < 3 prior lines
of systemic therapy. If previously treated, patients must have had
radiographically documented
progression on or after most recent therapy.
10019821 Patients in Cohorts 3A and 3B (NSCLC) with oncogene-driven tumors
with available
effective targeted therapy must have received at least one line of targeted
therapy.
10019831 All patients received autologous cryopreserved TIL therapy (with or
without
pembrolizumab, depending on cohort assignment), preceded by a nonmycloablative
lymphodeplction
(NMA-LD) preconditioning regimen consisting of cyclophosphamide and
fludarabine. Following TIL
infusion, up to 6 IV interleukin-2 (IL-2) doses maximum were administered.
10019841 The following general study periods took place in all 4 cohorts,
unless specified otherwise.
10019851 Screening and Tumor Resection: Up to 4 weeks (28 days) from study
entry; manufacturing
of the TIL Product: approximately <22 days from tumor resection; and treatment
period, as discussed
below.
10019861 Treatment Period (Cohorts 1A, 2A, and 3A): up to 2 years, including
NMA-LD (7 days),
TIL infusion (1 day) followed by 1L-2 administrations (1 to 4 days).Patients
receive a single infusion
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of pembrolizumab after the completion of their tumor resection for TIL
production and baseline scans
but before the initiation of the NMA-LD regimen. The next dose of
pembrolizumab will be no earlier
than following the completion of 1L-2 and continue Q3W 3 days thereafter for
< 2 years (24
months) or until disease progression or unacceptable toxicity, whichever
occurs first. The end-of-
treatment (EOT) visit occurred within 30 days after the last dose of
pembrolizumab. The visit could
be combined with end-of-assessment (EOA) visit if applicable (e.g.,
pembrolizumab discontinuation
occurred at disease progression or at the start of new anticancer therapy).
10019871Treatment Period (Cohort 3B): up to 12 days, including NMA-LD (7
days), TIIõ infusion (I
day) followed by IL-2 administrations (Ito 4 days). The EOT visit occurred
once a patient received
the last dose of IL-2. The EOT visit was performed within 30 days after
treatment discontinuation and
it may be combined with any scheduled visit occurring within this interval
during the assessment
period.
10019881 Assessment Period: began after TIL infusion on Day 0 and ends upon
disease progression,
with the start of a new anticancer therapy, partial withdrawal of consent to
study assessments, or 5
years (Month 60), whichever occurred first. An end-of assessment (E0A) visit
occurred once a patient
reached disease progression or started a new anticancer therapy.
10019891 The TIL autologous therapy with the TILs prepared as described herein
was comprised of
the following steps:
100199011. Tumor resection to provide the autologous tissue that serves as the
source of the TIL
cellular product;
100199112. TIL product produced at a central Good Manufacturing Practice (GMP)
facility;
100199213. A 7-day NMA-LD preconditioning regimen;
100199314. Cohorts 1A, 2A, and 3A: Patients receive a single infusion of
pcmbrolizumab after the
completion of their tumor resection for TIL production and baseline scans but
before the initiation of
NMA-LD regimen. The next dose of pembrolizumab will be no earlier than
following the completion
of IL-2 and continue Q3W 3 days thereafter.
100199415. Infusion of the autologous TIL product (Day 0); and
100199516. IV IL-2 administrations for up to 6 doses maximum.
10019961111 Cohorts 1A, 2A, and 3A, the next dose of pembrolizumab was no
earlier than following
the completion of IL-2 and continue Q3W 3 days thereafter for < 2 years (24
months), or until
disease progression or unacceptable toxicity, whichever occurred first.
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10019971Flowcharts for Cohorts 1A, 2A, and 3A can be found in Figure 7. The
Flowchart for Cohort
3B can be found in Figure 8. Patients were assigned to the appropriate cohort
by tumor indication.
10019981 TIL Therapy + Pembrolizumab (Cohorts 1A. 2A, and 3A)
10019991Patients were screened and scheduled for surgery for tumor resection.
Patients then had one
or more tumor lesions resected, which were sent to a central manufacturing
facility for TIL
production.
10020001Next, the NMA-LD regimen was imitated and consisted of 2 days of IV
cycic-Thosphamide
(60 mg/kg) with mesna (per site standard of care or USPI/SmPC) on Days -7 and
Day -6 followed by
days of IV fludarabine (25 mg/m2: Day -5 through Day -1).
10020011Patients in Cohorts 1A, 2A, and 3A received a single infusion of
pembrolizumab after the
completion of their tumor resection for TIL production and baseline scans and
before the initiation of
NMA-LD regimen. IL-2 administrations at a dose of 600,000 IU/kg IV begun as
soon as 3 hours after,
but no later than 24 hours after, completion of the TIL infusion on Day 0.
Additional IL-2
administrations will be given approximately every 8 to 12 hours for up to 6
doses maximum. The
second dose of pembrolizumab was no earlier than following the completion of
IL-2. Patients should
have recovered from all IL-2-related toxicities (Grade <2), prior to the
second pembrolizumab
administration. Pembrolizumab will continue Q3W 3 days thereafter for <2
years (24 months) or
until disease progression or unacceptable toxicity, whichever occurred first.
10020021TIL Therapy as a Single Agent (Cohort 3B)
10020031Patients were screened and scheduled for surgery for tumor resection.
Patients then had one
or more tumor lesions resected, which were sent to a central manufacturing
facility for TIL
production.
10020041Next, the NMA-LD regimen consisted of 2 days of IV cyclophosphamide
(60 mg/kg) with
mesna (per site standard of care or USPI/SmPC) on Day -7 and Day -6 followed
by 5 days of IV
fludarabine (25 mg/m2: Day -5 through Day -1).
10020051Infusion of the tumor-derived autologous TIL product occurred no
sooner than 24 hours
after last dose of fludarabine. IL-2 administrations at a dose of 600,000
IU/kg IV may have begun as
soon as 3 hours after, but no later than 24 hours after, completion of the TIL
infusion.
10020061Additional 1L-2 administrations were given approximately every 8 to 12
hours for up to 6
doses maximum.
10020071Production and Expansion of Tumor Infiltrating Lymphocytes
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10020081The TIL autologous cellular product was composed of viable cytotoxic T
lymphocytes
derived from a patient's tumor/lesion, which are manufactured ex vivo at a
central GMP facility. An
exemplary flow diagram depicting the TIL production process is provided in
Figure 9, for example.
[002009] The TIL manufacturing process begun at the clinical site after
surgical excision of a primary
or secondary metastatic tumor lesion(s) of >1.5 cm in diameter in each
individual patient. Multiple
tumor lesions from various anatomical locations can be excised to compile a
total aggregate of tumor
tissue; however, the aggregate should not exceed 4.0 cm in diameter, or 10 g
in weight, due to the
limited quantity of the biopreservation media present in the transport bottle.
[002010] Once the tumor lesion(s) was placed in the biopreservation transport
bottle, it is shipped at
2 C to 8 C using an express courier to a central GMP manufacturing facility.
Upon arrival, the tumor
specimen(s) were dissected into fragments, which were then cultured in a pre-
rapid expansion
protocol (Pre-REP) with human recombinant IL-2 for ¨11 days.
[002011] These pre-REP cells were then further expanded using a rapid
expansion protocol (REP) for
11 days in the presence of IL-2, OKT3 (a murine monoclonal antibody to human
CD3, also known as
[muromonab-CD31) and irradiated allogenic peripheral blood mononuclear cells
(PBMC) as feeder
cells.
[002012] The expanded cells were then harvested, washed, formulated,
cryopreserved, and shipped to
the clinical site via an express courier. The dosage form of the TIL cellular
product was a
cryopreserved autologous "live-cell suspension" that was ready for infusion
into the patient from
whom the TIL were derived. Patients were to receive the full dose of product
that was manufactured
and released, which contained between 1 x 109 and 150 x 109 viable cells per
the product
specification. Clinical experience indicated that objective tumor responses
were achieved across this
dose range, which has also been shown to be safe (Radvanyi L.G., etal., Clin
Cancer Res.
2012;18(24):6758-70). The full dose of product was provided in up to four
infusion bags.
[002013] Preparation of Patients to Receive the TIL Cellular Product
[002014] The NMA-LD preconditioning regimen used in this study (i.e., 2 days
of cyclophosphamide
plus mesna, followed by 5 days of fludarabine) was based on the method
developed and tested by the
National Cancer Institute ( Rosenberg S.A., et al., Clin Cancer Res.
2011;17(13):4550-7; Radvanyi
L.G., et al., Clin Cancer Res. 2012;18(246758-70; Dudley ME., et al., J Clin
Oncol.
2008;26(32):5233-9; Pilon-Thomas S. et al., J Immunother. 2012;35(8):615-20;
Dudley M.E., et al., J
Clin Oncol. 2005;23(10):2346-57; and Dudley ME., et al., Science.
2002;298(5594):850-4).
Following the 7-day preconditioning regimen, the patient was infused with the
TIL cellular product
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[002015] The TIL infusion was followed by the administration of IV IL-2
(600,000 IU/kg) every 8 to
12 hours, with the first dose administered between 3 and 24 hours after the
completion of the TIL
infusion and continuing for up to 6 doses maximum. Per institutional
standards, the doses of IL-2 can
be calculated on the basis of actual weight.
[002016] SELECTION OF PATIENT POPULATIONS
[002017] Cohort IA:
[002018] Patients had a confirmed diagnosis of unresectable MM (Stage IITC or
Stage IV,
histologically confirmed as per American Joint Committee on Cancer [MCC]
staging system). Ocular
melanoma patients were excluded. Patients must not have received prior immuno-
oncology targeted
agents. If BRAF-mutation positive, patient could have received prior
BRAF/MEKtargetcd therapy.
[002019] Cohort 2A:
[002020] Patients had advanced, recurrent and/or metastatic HNSCC and can be
treatment naive;
histologic diagnosis of the primary tumor is required via the pathology
report. Patients must not have
received prior immunotherapy regimens.
10020211 Cohort 3A:
10020221 Patients had a confirmed diagnosis of Stage III or Stage IV NSCLC
(squamous,
adenocarcinoma, large cell carcinoma). Patients with oncogene-driven tumors
with available effective
targeted therapy had received at least one line of targeted therapy.
[002023] Cohort 3B:
10020241 Patients had a confirmed diagnosis of Stage III or Stage IV NSCLC
(squamous,
adenocarcinoma, large cell carcinoma) and had previously received systemic
therapy with CPIs (e.g.,
anti-PD-1/anti-PD-L1). Patients with oncogene-driven tumors with available
effective targeted
therapy had received at least one line of targeted therapy.
[002025] All patients had received up to 3 prior systemic anticancer therapies
(see, inclusion criteria
below), excluding immunotherapy for Cohorts 1A, 2A, and 3A. If previously
treated, patients had
radiographically confirmed progression on or after most recent therapy.
[002026] Inclusion Criteria
[002027] Patients must have met ALL of the following inclusion criteria for
participation in the
study:
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100202811. All patients had a histologically or pathologically confirmed
diagnosis of malignancy of
their respective histologies:
o Unresectable or metastatic melanoma (Cohort 1A)
o Advanced, recurrent or metastatic squamous cell carcinoma of the head and
neck
(Cohort 2A)
o Stage 111 or Stage IV NSCLC (squamous, nonsquamous, adenocarcinoma, large
cell
carcinoma) (Cohorts 3A and 3B).
100202912. Cohorts 1A, 2A, and 3A only: Patients were immunotherapy naive. If
previously treated,
patients had progressed on or after most recent therapy. Cohorts 1A, 2A, and
3A may have received
up to 3 prior systemic anticancer therapies, specifically:
o In Cohort 1A: Patients with unreseetable or metastatic melanoma (Stage
111C or Stage
IV); if BRAF mutation-positive, patients could have received a BRAF inhibitor.
o In Cohort 2A: Patients with unresectable or metastatic FINS CC. Those who
had
received initial chemo-radiotherapy were allowed.
o In Cohort 3A: Patients with Stage III or Stage IV NSCLC (squamous,
nonsquamous,
adenocarcinoma, or large cell carcinoma) and who were immunotherapy naive and
progressed after <3 lines of prior systemic therapy in the locally advanced or
metastatic setting. Patients who received systemic therapy in the adjuvant or
neoadjuvant setting, or as part of definitive chemoradiotherapy, were eligible
and
were considered to have had one line of therapy if the disease has progressed
within
12 months of completion of prior systemic therapy. Patients with known
oncogene
drivers (e.g., EGFR, ALK, ROS) who had mutations that were sensitive to
targeted
therapies must had progressed after at least 1 line of targeted therapy.
100203013. Cohort 3B only: Patients with Stage III or Stage IV NSCLC
(squamous, nonsquamous,
adenocarcinoma, or large cell carcinoma) who had previously received systemic
therapy with CPIs
(e.g., anti-PD-1/anti-PD-L1) as part of < 3 prior lines of systemic therapy.
o Patients had radiographically confirmed progression on or after most
recent therapy.
o Patients who received systemic therapy in the adjuvant or neoadjuvant
setting, or as
part of definitive chemoradiotherapy, were eligible and were considered to
have had 1
line of therapy if the disease had progressed within 12 months of completion
of prior
systemic therapy.
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o Patients with known oncogene drivers (e.g., EGFR, ALK, ROS) who had
mutations
that are sensitive to targeted therapies must have progressed after at least 1
line of
targeted therapy.
100203114. Patients had at least 1 resectable lesion (or aggregate lesions) of
a minimum 1.5 cm in
diameter post-resection for TIL investigational product production. It was
encouraged that tumor
tissue be obtained from multiple and diverse metastatic lesions, as long as
the surgical resection did
not pose additional risks to the patient.
o If the lesion considered for resection for TIL generation is within a
previously
irradiated field, the lesion must have demonstrated radiographic progression
prior to
resection.
o Patients must have an adequate histopathology specimen for protocol-
required
testing.
1002032] 5. Patients had remaining measurable disease as defined by the
standard and well known
RECIST 1.1 guidelines (see, for example, Eisenhauer, European Journal of
Cancer 45:228-247
(2009), also available on the World Wide Web at project.eortc.org/recist/wp-
content/uploads/sites/4/2015/03/RECISIGuidelines.pdf ) following tumor
resection for TIL
manufacturing:
o Lesions in previously irradiated areas were not be selected as target
lesions unless
there had been demonstrated progression of disease in those lesions;
o Lesions that were partially resected for TIL generation that were still
measurable per
RECIST may be selected as nontarget lesions but could not serve as a target
lesion for
response assessment.
100203316. Patients were > 18 years at the time of consent.
100203417. Patients had an Eastem Cooperative Oncology Group (ECOG)
performance status of 0 or
1, and an estimated life expectancy of >3 months.
100203518. Patients of childbearing potential or those with partners of
childbearing potential had to
be willing to practice an approved method of highly effective birth control
during treatment and
continue for 12 months after receiving all protocol-related therapy (Note:
Females of reproductive
potential were to use effective contraception during treatment and for 12
months after their last dose
of IL-2, or 4 months after their last dose of pembrolizumab whichever occurred
later). Males could
not donate sperm during the study or for 12 months after treatment
discontinuation, whichever
occurred later.
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100203619. Patients had the following hematologic parameters:
o Absolute ncutrophil count (ANC) >1000/mm3;
o Hemoglobin >9.0 g/dL;
o Platelet count >100,000/mm3.
1002037110. Patients had adequate organ function:
o Serum alanine aminotransferase (ALT)/serum glutamic-pyruvic transaminase
(SGPT)
and aspartate aminotransferase (AST)/SGOT <3 times the upper limit of normal
(ULN), patients with liver metastasis <5 times ULN.
o An estimated creatinine clearance >40 mL/min using the Cockcroft Gault
formula at
Screening.
o Total bilirubin <2 mg/dL.
o Patients with Gilbert's Syndrome must have a total bilirubin <3 mg/dL.
1002038111. Patients were seronegative for the human immunodeficiency virus
(HIV1 and HIV2).
Patients with positive serology for hepatitis B virus surface antigen (HBsAg),
hepatitis B core
antibody (anti HBc), or hepatitis C virus (anti-HCV) indicating acute or
chronic infection were
enrolled depending on the viral load based on polymerase chain reaction (PCR)
and the local
prevalence of certain viral exposures.
1002039112. Patients had a washout period from prior anticancer therapy(ies)
of a minimum
duration, as detailed below prior to the first study treatment (i.e., start of
NMA-LD or
pembrolizumab):
o Targeted therapy: prior targeted therapy with an epidermal growth factor
receptor
(EGFR), MEK, BRAF, ALK, ROS1 or other-targeted agents (e.g., erlotinib,
afatinib,
dacomitinib, osimertinib, crizotinib, ccritinib, lorlatinib) was allowed
provided the
washout is a minimum of 14 days prior to the start of treatment.
o Chemotherapy: adjuvant, neoadjuvant or definitive chemotherapy/
chemoradiation
was allowed provided the washout is a minimum of 21 days prior to the start of
treatment.
o Immunotherapy for Cohort 3B only, prior checkpoint-targeted therapy with
an anti-
PD-1, other mAbs, or vaccines were allowed with a washout period of > 21 days
before the start of NMA-LD.
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o Palliative radiation therapy: prior external beam radiation was allowed
provided all
radiation-related toxicities were resolved to Grade 1 or baseline, excluding
alopecia,
skin pigmentation change, or other clinically insignificant events, e.g.,
small area
radiation dermatitis or rectal or urinary urgency
o The tumor lesion(s) being assessed as target for response via RECIST 1.1
were
outside of the radiation portal; however, if within the portal, they must have
demonstrated progression (see Inclusion Criterion above).
o Surgery/pre-planned procedure: previous surgical procedure(s) was
permitted
provided that wound healing had occurred, all complications had resolved, and
at
least 14 days have elapsed (for major operative procedures) prior to the tumor
resection.
1002040113. Patients had recovered from all prior anticancer treatment-related
adverse events
( _______ IRAEs) to Grade <1 (per Common Terminology Criteria for Adverse
Events [CTCAE]), except for
alopecia or vitiligo, prior to cohort assignment.
1002041114. Patients with stable Grade >2 toxicity from prior anticancer
therapy were considered on
a case by case basis after consultation with the Medical Monitor.
1002042115. Cohorts 1A, 2A, and 3A patients with irreversible toxicity not
reasonably expected to be
exacerbated by treatment with pembrolizumab were included only after
consultation with the Medical
Monitor. For patients in Cohort 3B only, patients with documented Grade >2 or
higher diarrhea or
colitis as a result of a previous treatment with immune checkpoint inhibitor
CPI(s) must have been
asymptomatic for at least 6 months or had a normal by visual assessment
colonoscopy post-treatment
prior to tumor resection.
1002043116. Patients must have provided written authorization for use and
disclosure of protected
health information.
10020441Exclusion Criteria
10020451 Patients who meet ANY of the following criteria were excluded from
the study:
100204611. Patients with melanoma of uveal/ocular origin
100204712. Patients who had received an organ allograft or prior cell transfer
therapy that included a
nonmyeloablativc or mycloablativc chemotherapy regimen within the past 20
years. (Note: This
criterion was applicable for patients undergoing retreatment with TIL, with
the exception that they
had a prior NMA-LD regimen with their
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10020481 prior TIL treatment.)
100204913. Patients with symptomatic and/or untreated brain metastases.
o = Patients with definitively-treated brain metastases will be considered
for enrollment
10020501 after discussion with Medical Monitor; if, prior to the start of
treatment the patient is
10020511 clinically stable for >2 weeks, there are no new brain lesions via
magnetic resonance
10020521 imaging (MRI) post-treatment, and the patient does not require
ongoing
10020531 corticosteroid treatment.
100205414. Patients who are on a systemic steroid therapy within 21 days of
enrollment
100205515. Patients who are pregnant or breastfeeding.
100205616. Patients who had an active medical illness(es), which in the
opinion of the Investigator,
posed increased risks for study participation; such as systemic infections
(e.g., syphilis or any other
infection requiring antibiotics), coagulation disorders, or other active major
medical illnesses of the
cardiovascular, respiratory, or immune systems.
100205717. Patients may not have active or prior documented autoimmune or
inflammatory disorders
(including pneumonitis, inflammatory bowel disease [e.g., colitis or Crohn's
disease], diverticulitis
[with the exception of diverticulosisl, systemic lupus erythematosus,
sarcoidosis syndrome, or
Wegener syndrome [granulomatosis with polyangiitis, Graves. disease,
rheumatoid arthritis,
hypophysitis, uveitis, etc.1). The following were exceptions to this
criterion:
o Patients with vitiligo or alopecia.
o Patients with hypothyroidism (e.g., following Hashimoto syndrome) stable
on
o hormone replacement.
o Any chronic skin condition that did not require systemic therapy.
o Patients with celiac disease controlled by diet alone.
10020581 8. Patients who had received a live or attenuated vaccination within
28 days prior to the
start of treatment.
100205919. Patients who had any form of primary immunodeficiency (such as
severe combined
immunodeficiency disease I SCID I and acquired immune deficiency syndrome
'AIDS!).
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1002060110. Patients with a history of hypersensitivity to any component of
the study drugs. TILs
were not administered to patients with a known hypersensitivity to any
component of TIL product
formulation including, but not limited to any of the following:
o = NMA-LD (cyclophosphamide, mesna, and fludarabine)
o = ProleukinO, aldesleukin, IL-2
o = Antibiotics of the aminoglyco side group (i.e., streptomycin,
gcntamicin [excluding
those who are skin-test negative for gentamicin hypersensitivity])
o = Any component of the TIL product formulation including dimethyl
sulfoxide
o [DMS0], HSA, IL-2, and dextran-40
o = Pembrolizumab
1002061111. Patients who had a left ventricular ejection fraction (LVEF) <45%
or who are New
York Heart Association Class II or higher. A cardiac stress test demonstrating
any irreversible wall
movement abnormality in any patients >60 years of age or in patients who have
a history of ischemic
heart disease, chest pain, or clinically significant atrial and/or ventricular
arrhythmias.
o Patients with an abnormal cardiac stress test could be enrolled if they
had adequate
ejection fraction and cardiology clearance with approval of the Sponsor's
Medical
Monitor.
1002062112. Patients who had obstructive or restrictive pulmonary disease and
have a documented
FEV1 (forced expiratory volume in 1 second) of <60% of predicted normal.
o = If a patient was not able to perform reliable spirometry due to
abnormal upper
airway anatomy (i.e., tracheostomy), a 6-minute walk test was used to assess
pulmonary function. Patients who were unable to walk a distance of at least
80%
predicted for age and sex or demonstrates evidence of hypoxia at any point
during the
test (Sp02<90%) are excluded.
1002063113. Patients who had another primary malignancy within the previous 3
years (except for
those which did not require treatment or had been curatively treated greater
than 1 year ago, and in the
judgment of the Investigator, did not pose a significant risk of recurrence
including, but not limited to,
non-melanoma skin cancer, DCIS, LCIS, prostate cancer Gleason score <6 or
bladder cancer).
1002064114. Participation in another clinical study with an investigational
product within 21 days of
the initiation of treatment.
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10020651 Study Endpoints and Planned Analyses
10020661 The primary and secondary endpoints were analyzed separately by
cohort.
10020671Primary Endpoints:
10020681 The ORR was defined as the proportion of patients who achieved either
a confirmed PR or
CR as best response as assessed by Investigators per RECIST 1.1 among the
efficacy analysis set.
10020691 Objective response was evaluated per each disease assessment and the
ORR was expressed
as a binomial proportion with the corresponding 2-sided 90% CI. The primary
analysis for each
cohort occurred when all treated patients per cohort have an opportunity to be
followed for 12 months,
unless progressed/expired or discontinued early from the assessment period.
10020701 The safety primary endpoint was measured by any Grade 3 or higher
TEAE incidence rate
within each cohort expressed as binomial proportions with the corresponding 2-
sided 90% CI.
10020711 Secondary Endpoints:
10020721Efficacy:
10020731 The secondary efficacy endpoints were defined as follows:
[0020741 CR rate as based on responders who achieved confirmed CR as assessed
by Investigators.
DCR was derived as the sum of the number of patients who achieved confirmed
PR/CR or sustained
SD (at least 6 weeks) divided by the number of patients in the efficacy
analysis set >< 100%. The CR
rate and DCR was summarized using a point estimate and its 2-sided 90% CI.
10020751DOR was defined among patients who achieved objective response. It was
measured from
the first-time response (PR/CR) criteria are met until the first date that
recurrent or progressive disease
was objectively documented, or receipt of subsequent anticancer therapy or the
patient dies
(whichever is first recorded). Patients not experiencing PD or have not died
prior to the time of data
cut or the final database lock will have their event times censored on the
last date that an adequate
assessment of tumor status is made.
10020761 PFS was defined as the time (in months) from the time of
lymphodcpletion to PD, or death
due to any cause, whichever event is earlier. Patients not experiencing PD or
not having expired at the
time of the data cut or the final database lock had their event times censored
on the last date that an
adequate assessment of tumor status is made.
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[00207710S as defined as the time (in months) from the time of lymphodepletion
to death due to
any cause. Patients not having expired by the time of data cut or the final
database lock had their
event times censored on the last date of their known survival status.
10020781DOR, PFS, and OS was subjected to right censoring. The Kaplan-Meier
method will be
used to summarize the time-to-event efficacy endpoints. The baseline data for
the tumor assessment
was the last scan before the lymphodepletion for all cohorts.
10020791The above efficacy parameters will be estimated for applicable cohort
for subsets defined
by baseline disease characteristics; BRAF status (Cohort lA only), HPV status
(Cohort 2A only),
squamous or non-squamous lung disease (Cohorts 3A and 3B only), and anti-PD-Li
status.
EXAMPLE 14: A PHASE 2, MULTICENTER STUDY OF AUTOLOGOUS TUMOR
INFILTRATING LYMPHOCYTES IN PATIENTS WITH SOLID TUMORS
STUDY DESIGN
100208010verview
10020811This example describes a prospective, open-label, multi-cohort, non-
randomized,
multicenter Phase 2 study evaluating ACT using TIL in combination with
pembrolizumab or TIL as a
single therapy, using TILs prepared as described in the present application as
well as in this example.
10020821Objectives:
10020831Primary:
10020841To evaluate the efficacy of autologous TIL in combination with
pembrolizumab in MM,
HNSCC, or NSCLC patients or TIL as a single therapy in relapsed or refractory
(r/r) NSCLC patients,
who had previously progressed on or after treatment with CPIs, as determined
by objective response
rate (ORR), using the Response Evaluation Criteria in Solid Tumors (RECIST
1.1), as assessed by
Investigator.
10020851To characterize the safety profile of TIL in combination with
pembrolizumab in MM,
HNSCC, and NSCLC patients or TIL as a single therapy in r/r NSCLC patients as
measured by the
incidence of Grade > 3 treatment-emergent adverse events (TEAEs).
10020861Secondary:
10020871To further evaluate the efficacy of autologous TIL in combination with
pembrolizumab in
MM, HNSCC, and NSCLC patients or TIL as a single therapy in r/r NSCLC patients
using complete
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response (CR) rate, duration of response (DOR), disease control rate (DCR),
progression-free survival
(PFS) using RECIST 1.1, as assessed by Investigator, and overall survival
(OS).
10020881 Cohorts:
10020891 Cohort 1A: TIL therapy in combination with pembrolizumab in patients
with Stage IIIC or
Stage IV unresectable or MM with < 3 prior lines of systemic therapy excluding
immunotherapy. If
previously treated, patients must have had radiographically documented
progression on or after most
recent therapy.
10020901 Cohort 2A: TIL therapy in combination with pembrolizumab in patients
with advanced,
recurrent or metastatic HNSCC (e.g., Stages T1N1-N2B, T2-4N0-N2b) with < 3
prior lines of
systemic therapy, excluding immunotherapy. If previously treated, patients
must have had
radiographically documented progression on or after most recent therapy.
10020911 Cohort 3A: TIL therapy in combination with pembrolizumab in patients
with locally
advanced or metastatic (Stage III¨ IV) NSCLC with <3 prior lines of systemic
therapy, excluding
immunotherapy. if previously treated, patients must have had radiographically
documented
progression on or after most recent therapy.
10020921 Cohort 3B: TIL therapy as a single agent in patients Stage HI or
Stage TV NSCLC who have
previously received systemic therapy with CPIs (e.g., anti-PD-1/anti-PD-L1) as
part of < 3 prior lines
of systemic therapy. If previously treated, patients must have had
radiographically documented
progression on or after most recent therapy.
10020931 Patients in Cohorts 3A and 3B (NSCLC) with oncogene-driven tumors
with available
effective targeted therapy must have received at least one line of targeted
therapy.
10020941 All patients received autologous cryopreserved TIL therapy (with or
without
pembrolizumab, depending on cohort assignment), preceded by a nonmycloablative
lymphodeplction
(NMA-LD) preconditioning regimen consisting of cyclophosphamide and
fludarabine. Following TIL
infusion, up to 6 IV interleukin-2 (IL-2) doses maximum were administered.
10020951 The following general study periods took place in all 4 cohorts,
unless specified otherwise.
10020961 Screening and Tumor Resection: Up to 4 weeks (28 days) from study
entry; manufacturing
of the TIL Product: approximately <22 days from tumor resection; and treatment
period, as discussed
below.
10020971 Treatment Period (Cohorts 1A, 2A, and 3A): up to 2 years, including
NMA-LD (7 days),
TIL infusion (1 day) followed by 1L-2 administrations (1 to 4 days).Patients
receive a single infusion
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of pembrolizumab after the completion of their tumor resection for TIL
production and baseline scans
but before the initiation of the NMA-LD regimen. The next dose of
pembrolizumab will be no earlier
than following the completion of 1L-2 and continue Q3W 3 days thereafter for
< 2 years (24
months) or until disease progression or unacceptable toxicity, whichever
occurs first. The end-of-
treatment (EOT) visit occurred within 30 days after the last dose of
pembrolizumab. The visit could
be combined with end-of-assessment (EOA) visit if applicable (e.g.,
pembrolizumab discontinuation
occurred at disease progression or at the start of new anticancer therapy).
[0020981Treatment Period (Cohort 3B): up to 12 days, including NMA-LD (7
days), TII õ infusion (I
day) followed by IL-2 administrations (Ito 4 days). The EOT visit occurred
once a patient received
the last dose of IL-2. The EOT visit was performed within 30 days after
treatment discontinuation and
it may be combined with any scheduled visit occurring within this interval
during the assessment
period.
10020991Assessment Period: began after TIL infusion on Day 0 and ends upon
disease progression,
with the start of a new anticancer therapy, partial withdrawal of consent to
study assessments, or 5
years (Month 60), whichever occurred first. An end-of assessment (E0A) visit
occurred once a patient
reached disease progression or started a new anticancer therapy.
10021001The TIL autologous therapy with the TILs prepared as described herein
was comprised of
the following steps:
100210111. Tumor resection to provide the autologous tissue that serves as the
source of the TIL
cellular product;
100210212. TIL product produced at a central Good Manufacturing Practice (GMP)
facility;
10021031 3. A 7-day NMA-LD preconditioning regimen;
100210414. Cohorts 1A, 2A, and 3A: Patients receive a single infusion of
pembrolizumab after the
completion of their tumor resection for TIL production and baseline scans but
before the initiation of
NMA-LD regimen. The next dose of pembrolizumab will be no earlier than
following the completion
of IL-2 and continue Q3W 3 days thereafter.
100210515. Infusion of the autologous TIL product (Day 0); and
100210616. IV IL-2 administrations for up to 6 doses maximum.
10021071111 Cohorts 1A, 2A, and 3A, the next dose of pembrolizumab was no
earlier than following
the completion of IL-2 and continue Q3W 3 days thereafter for < 2 years (24
months), or until
disease progression or unacceptable toxicity, whichever occurred first.
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10021081Flowcharts for Cohorts 1A, 2A, and 3A can be found in Figure 7. The
Flowchart for Cohort
3B can be found in Figure 8. Patients were assigned to the appropriate cohort
by tumor indication.
10021091 TIL Therapy + Pembrolizumab (Cohorts 1A. 2A, and 3A)
10021101Patients were screened and scheduled for surgery for tumor resection.
Patients then had one
or more tumor lesions resected, which were sent to a central manufacturing
facility for TIL
production.
10021111Next the NMA-LD regimen was imitated and consisted of 2 days of IV
cycic-Thosphamide
(60 mg/kg) with mesna (per site standard of care or USPI/SmPC) on Days -7 and
Day -6 followed by
days of IV fludarabine (25 mg/m2: Day -5 through Day -1).
10021121Patients in Cohorts 1A, 2A, and 3A received a single infusion of
pembrolizumab after the
completion of their tumor resection for TIL production and baseline scans and
before the initiation of
NMA-LD regimen. IL-2 administrations at a dose of 600,000 IU/kg IV begun as
soon as 3 hours after,
but no later than 24 hours after, completion of the TIL infusion on Day 0.
Additional IL-2
administrations will be given approximately every 8 to 12 hours for up to 6
doses maximum. The
second dose of pembrolizumab was no earlier than following the completion of
IL-2. Patients should
have recovered from all IL-2-related toxicities (Grade <2), prior to the
second pembrolizumab
administration. Pembrolizumab will continue Q3W 3 days thereafter for <2
years (24 months) or
until disease progression or unacceptable toxicity, whichever occurred first.
10021131 TIL Therapy as a Single Agent (Cohort 3B)
10021141Patients were screened and scheduled for surgery for tumor resection.
Patients then had one
or more tumor lesions resected, which were sent to a central manufacturing
facility for TIL
production.
10021151Next, the NMA-LD regimen consisted of 2 days of IV cyclophosphamide
(60 mg/kg) with
mesna (per site standard of care or USPI/SmPC) on Day -7 and Day -6 followed
by 5 days of IV
fludarabine (25 mg/m2: Day -5 through Day -1).
10021161Infusion of the tumor-derived autologous TIL product occurred no
sooner than 24 hours
after last dose of fludarabine. IL-2 administrations at a dose of 600,000
IU/kg IV may have begun as
soon as 3 hours after, but no later than 24 hours after, completion of the TIL
infusion.
10021171Additional 1L-2 administrations were given approximately every 8 to 12
hours for up to 6
doses maximum.
10021181Production and Expansion of Tumor Infiltrating Lymphocytes
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10021191The TIL autologous cellular product was composed of viable cytotoxic T
lymphocytes
derived from a patient's tumor/lesion, which are manufactured ex vivo at a
central GMP facility. An
exemplary flow diagram depicting the TIL production process is provided in
Figure 9, for example.
10021201The TIL manufacturing process begun at the clinical site after
surgical excision of a primary
or secondary metastatic tumor lesion(s) of >1.5 cm in diameter in each
individual patient. Multiple
tumor lesions from various anatomical locations can be excised to compile a
total aggregate of tumor
tissue; however, the aggregate should not exceed 4.0 cm in diameter, or 10 g
in weight, due to the
limited quantity of the biopreservation media present in the transport bottle.
100212110nce the tumor lesion(s) was placed in the biopreservation transport
bottle, it is shipped at
2 C to 8 C using an express courier to a central GMP manufacturing facility.
Upon arrival, the tumor
specimen(s) were dissected into fragments, which were then cultured in a pre-
rapid expansion
protocol (Pre-REP) with human recombinant IL-2 for ¨11 days.
10021221 These pre-REP cells were then further expanded using a rapid
expansion protocol (REP) for
11 days in the presence of IL-2, OKT3 (a murine monoclonal antibody to human
CD3, also known as
[muromonab-CD31) and irradiated allogenie peripheral blood mononuclear cells
(PBMC) as feeder
cells.
10021231The expanded cells were then harvested, washed, formulated,
cryopreserved, and shipped to
the clinical site via an express courier. The dosage form of the TIL cellular
product was a
cryopre served autologous "live-cell suspension" that was ready for infusion
into the patient from
whom the TIL were derived. Patients were to receive the full dose of product
that was manufactured
and released, which contained between 1 x 109 and 150 x 109 viable cells per
the product
specification. Clinical experience indicated that objective tumor responses
were achieved across this
dose range, which has also been shown to be safe (Radvanyi L.G., etal., Clin
Cancer Res.
2012;18(24):6758-70). The full dose of product was provided in up to four
infusion bags.
10021241Preparation of Patients to Receive the TIL Cellular Product
10021251The NMA-LD preconditioning regimen used in this study (i.e., 2 days of
cyclophosphamide
plus mesna, followed by 5 days of fludarabine) was based on the method
developed and tested by the
National Cancer Institute ( Rosenberg S.A., et al., Clin Cancer Res.
2011;17(13):4550-7; Radvanyi
L.G., et al., Clin Cancer Res. 2012;18(246758-70; Dudley M.E., et al., J Clin
Oncol.
2008;26(32):5233-9; Pilon-Thomas S. et al., J Immunother. 2012;35(8):615-20;
Dudley M.E., et al., J
Clin Oncol. 2005;23(10):2346-57; and Dudley M.E., et al., Science.
2002;298(5594):850-4).
Following the 7-day preconditioning regimen, the patient was infused with the
TIL cellular product
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10021261 The TIL infusion was followed by the administration of IV IL-2
(600,000 IU/kg) every 8 to
12 hours, with the first dose administered between 3 and 24 hours after the
completion of the TIL
infusion and continuing for up to 6 doses maximum. Per institutional
standards, the doses of IL-2 can
be calculated on the basis of actual weight.
10021271 SELECTION OF PATIENT POPULATIONS
10021281 Cohort 1A:
10021291 Patients had a confirmed diagnosis of unresectable MM (Stage TITC or
Stage IV,
histologically confirmed as per American Joint Committee on Cancer [AJCC]
staging system). Ocular
melanoma patients were excluded. Patients must not have received prior immuno-
oncology targeted
agents. If BRAF-mutation positive, patient could have received prior
BRAF/MEKtargetcd therapy.
10021301 Cohort 2A:
10021311 Patients had advanced, recurrent and/or metastatic HNSCC and can be
treatment naive;
histologic diagnosis of the primary tumor is required via the pathology
report. Patients must not have
received prior immunotherapy regimens.
10021321 Cohort 3A:
10021331 Patients had a confirmed diagnosis of Stage III or Stage IV NSCLC
(squamous,
adenocarcinoma, large cell carcinoma). Patients with oncogene-driven tumors
with available effective
targeted therapy had received at least one line of targeted therapy.
10021341 Cohort 3B:
10021351 Patients had a confirmed diagnosis of Stage III or Stage IV NSCLC
(squamous,
adenocarcinoma, large cell carcinoma) and had previously received systemic
therapy with CPIs (e.g.,
anti-PD-1/anti-PD-L1). Patients with oncogene-driven tumors with available
effective targeted
therapy had received at least one line of targeted therapy.
10021361 All patients had received up to 3 prior systemic anticancer therapies
(see, inclusion criteria
below), excluding immunotherapy for Cohorts 1A, 2A, and 3A. If previously
treated, patients had
radiographically confirmed progression on or after most recent therapy.
[002137] Inclusion Criteria
10021381 Patients must have met ALL of the following inclusion criteria for
participation in the
study:
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100213911. All patients had a histologically or pathologically confirmed
diagnosis of malignancy of
their respective histologies:
o Unresectable or metastatic melanoma (Cohort 1A)
o Advanced, recurrent or metastatic squamous cell carcinoma of the head and
neck
(Cohort 2A)
o Stage 111 or Stage IV NSCLC (squamous, nonsquamous, adenocarcinoma, large
cell
carcinoma) (Cohorts 3A and 3B).
100214012. Cohorts 1A, 2A, and 3A only: Patients were immunotherapy naive. If
previously treated,
patients had progressed on or after most recent therapy. Cohorts 1A, 2A, and
3A may have received
up to 3 prior systemic anticancer therapies, specifically:
o In Cohort 1A: Patients with unresectable or metastatic melanoma (Stage
111C or Stage
IV); if BRAF mutation-positive, patients could have received a BRAF inhibitor.
o In Cohort 2A: Patients with unresectable or metastatic FINS CC. Those who
had
received initial chemo-radiotherapy were allowed.
o In Cohort 3A: Patients with Stage III or Stage IV NSCLC (squamous,
nonsquamous,
adenocarcinoma, or large cell carcinoma) and who were immunotherapy naive and
progressed after <3 lines of prior systemic therapy in the locally advanced or
metastatic setting. Patients who received systemic therapy in the adjuvant or
neoadjuvant setting, or as part of definitive chemoradiotherapy, were eligible
and
were considered to have had one line of therapy if the disease has progressed
within
12 months of completion of prior systemic therapy. Patients with known
oncogene
drivers (e.g., EGFR, ALK, ROS) who had mutations that were sensitive to
targeted
therapies must had progressed after at least 1 line of targeted therapy.
100214113. Cohort 3B only: Patients with Stage III or Stage IV NSCLC
(squamous, nonsquamous,
adenocarcinoma, or large cell carcinoma) who had previously received systemic
therapy with CPIs
(e.g., anti-PD-1/anti-PD-L1) as part of < 3 prior lines of systemic therapy.
o Patients had radiographically confirmed progression on or after most
recent therapy.
o Patients who received systemic therapy in the adjuvant or neoadjuvant
setting, or as
part of definitive chemoradiotherapy, were eligible and were considered to
have had 1
line of therapy if the disease had progressed within 12 months of completion
of prior
systemic therapy.
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o Patients with known oncogene drivers (e.g., EGFR, ALK, ROS) who had
mutations
that are sensitive to targeted therapies must have progressed after at least 1
line of
targeted therapy.
100214214. Patients had at least 1 resectable lesion (or aggregate lesions) of
a minimum 1.5 cm in
diameter post-resection for TIL investigational product production. It was
encouraged that tumor
tissue be obtained from multiple and diverse metastatic lesions, as long as
the surgical resection did
not pose additional risks to the patient.
o If the lesion considered for resection for TIL generation is within a
previously
irradiated field, the lesion must have demonstrated radiographic progression
prior to
resection.
o Patients must have an adequate histopathology specimen for protocol-
required
testing.
1002143] 5. Patients had remaining measurable disease as defined by the
standard and well known
RECIST 1.1 guidelines (see, for example, Eisenhauer, European Journal of
Cancer 45:228-247
(2009), also available on the World Wide Web at project.eortc.org/recist/wp-
content/uploads/sites/4/2015/03/RECISIGuidelines.pdf ) following tumor
resection for TIL
manufacturing:
o Lesions in previously irradiated areas were not be selected as target
lesions unless
there had been demonstrated progression of disease in those lesions;
o Lesions that were partially resected for TIL generation that were still
measurable per
RECIST may be selected as nontarget lesions but could not serve as a target
lesion for
response assessment.
100214416. Patients were > 18 years at the time of consent.
100214517. Patients had an Eastem Cooperative Oncology Group (ECOG)
performance status of 0 or
1, and an estimated life expectancy of >3 months.
100214618. Patients of childbearing potential or those with partners of
childbearing potential had to
be willing to practice an approved method of highly effective birth control
during treatment and
continue for 12 months after receiving all protocol-related therapy (Note:
Females of reproductive
potential were to use effective contraception during treatment and for 12
months after their last dose
of IL-2, or 4 months after their last dose of pembrolizumab whichever occurred
later). Males could
not donate sperm during the study or for 12 months after treatment
discontinuation, whichever
occurred later.
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100214719. Patients had the following hematologic parameters:
o Absolute ncutrophil count (ANC) >1000/mm3;
o Hemoglobin >9.0 g/dL;
o Platelet count >100,000/mm3.
1002148110. Patients had adequate organ function:
o Serum alanine aminotransferase (ALT)/serum glutamic-pyruvic transaminase
(SGPT)
and aspartate aminotransferase (AST)/SGOT <3 times the upper limit of normal
(ULN), patients with liver metastasis <5 times ULN.
o An estimated creatinine clearance >40 mL/min using the Cockcroft Gault
formula at
Screening.
o Total bilirubin <2 mg/dL.
o Patients with Gilbert's Syndrome must have a total bilirubin <3 mg/dL.
1002149111. Patients were seronegative for the human immunodeficiency virus
(HIV1 and HIV2).
Patients with positive serology for hepatitis B virus surface antigen (HBsAg),
hepatitis B core
antibody (anti HBc), or hepatitis C virus (anti-HCV) indicating acute or
chronic infection were
enrolled depending on the viral load based on polymerase chain reaction (PCR)
and the local
prevalence of certain viral exposures.
1002150112. Patients had a washout period from prior anticancer therapy(ies)
of a minimum
duration, as detailed below prior to the first study treatment (i.e., start of
NMA-LD or
pembrolizumab):
o Targeted therapy: prior targeted therapy with an epidermal growth factor
receptor
(EGFR), MEK, BRAF, ALK, ROS1 or other-targeted agents (e.g., erlotinib,
afatinib,
dacomitinib, osimertinib, crizotinib, ccritinib, lorlatinib) was allowed
provided the
washout is a minimum of 14 days prior to the start of treatment.
o Chemotherapy: adjuvant, neoadjuvant or definitive chemotherapy/
chemoradiation
was allowed provided the washout is a minimum of 21 days prior to the start of
treatment.
o Immunotherapy for Cohort 3B only, prior checkpoint-targeted therapy with
an anti-
PD-1, other mAbs, or vaccines were allowed with a washout period of > 21 days
before the start of NMA-LD.
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o Palliative radiation therapy: prior external beam radiation was allowed
provided all
radiation-related toxicities were resolved to Grade 1 or baseline, excluding
alopecia,
skin pigmentation change, or other clinically insignificant events, e.g.,
small area
radiation dermatitis or rectal or urinary urgency
o The tumor lesion(s) being assessed as target for response via RECIST 1.1
were
outside of the radiation portal; however, if within the portal, they must have
demonstrated progression (see Inclusion Criterion above).
o Surgery/pre-planned procedure: previous surgical procedure(s) was
permitted
provided that wound healing had occurred, all complications had resolved, and
at
least 14 days have elapsed (for major operative procedures) prior to the tumor
resection.
10021511 13. Patients had recovered from all prior anticancer treatment-
related adverse events
( _______ IRAEs) to Grade <1 (per Common Terminology Criteria for Adverse
Events [CTCAE]), except for
alopecia or vitiligo, prior to cohort assignment.
1002152114. Patients with stable Grade >2 toxicity from prior anticancer
therapy were considered on
a case by case basis after consultation with the Medical Monitor.
1002153115. Cohorts 1A, 2A, and 3A patients with irreversible toxicity not
reasonably expected to be
exacerbated by treatment with pembrolizumab were included only after
consultation with the Medical
Monitor. For patients in Cohort 3B only, patients with documented Grade >2 or
higher diarrhea or
colitis as a result of a previous treatment with immune checkpoint inhibitor
CPI(s) must have been
asymptomatic for at least 6 months or had a normal by visual assessment
colonoscopy post-treatment
prior to tumor resection.
1002154116. Patients must have provided written authorization for use and
disclosure of protected
health information.
10021551Exclusion Criteria
10021561 Patients who meet ANY of the following criteria were excluded from
the study:
100215711. Patients with melanoma of uveal/ocular origin
100215812. Patients who had received an organ allograft or prior cell transfer
therapy that included a
nonmyeloablativc or mycloablativc chemotherapy regimen within the past 20
years. (Note: This
criterion was applicable for patients undergoing retreatment with TIL, with
the exception that they
had a prior NMA-LD regimen with their
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[002159] prior TIL treatment.)
100216013. Patients with symptomatic and/or untreated brain metastases.
o = Patients with definitively-treated brain metastases will be considered
for enrollment
[002161] after discussion with Medical Monitor; if, prior to the start of
treatment the patient is
[002162] clinically stable for >2 weeks, there are no new brain lesions via
magnetic resonance
[002163] imaging (MRI) post-treatment, and the patient does not require
ongoing
[002164] corticosteroid treatment.
100216514. Patients who are on a systemic steroid therapy within 21 days of
enrollment
100216615. Patients who are pregnant or breastfeeding.
100216716. Patients who had an active medical illness(es), which in the
opinion of the Investigator,
posed increased risks for study participation; such as systemic infections
(e.g., syphilis or any other
infection requiring antibiotics), coagulation disorders, or other active major
medical illnesses of the
cardiovascular, respiratory, or immune systems.
100216817. Patients may not have active or prior documented autoimmune or
inflammatory disorders
(including pneumonitis, inflammatory bowel disease [e.g., colitis or Crohn's
disease], diverticulitis
[with the exception of diverticulosisl, systemic lupus erythematosus,
sarcoidosis syndrome, or
Wegener syndrome [granulomatosis with polyangiitis, Graves' disease,
rheumatoid arthritis,
hypophysitis, uveitis, etc.1). The following were exceptions to this
criterion:
o Patients with vitiligo or alopecia.
o Patients with hypothyroidism (e.g., following Hashimoto syndrome) stable
on
o hormone replacement.
o Any chronic skin condition that did not require systemic therapy.
o Patients with celiac disease controlled by diet alone.
[002169] 8. Patients who had received a live or attenuated vaccination within
28 days prior to the
start of treatment.
100217019. Patients who had any form of primary immunodeficiency (such as
severe combined
immunodeficiency disease I SCID I and acquired immune deficiency syndrome
'AIDS!).
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1002171110. Patients with a history of hypersensitivity to any component of
the study drugs. TILs
were not administered to patients with a known hypersensitivity to any
component of TIL product
formulation including, but not limited to any of the following:
o = NMA-LD (cyclophosphamide, mesna, and fludarabine)
o = ProleukinO, aldesleukin, IL-2
o = Antibiotics of the aminoglyco side group (i.e., streptomycin,
gcntamicin [excluding
those who are skin-test negative for gentamicin hypersensitivity])
o = Any component of the TIL product formulation including dimethyl
sulfoxide
o [DMS0], HSA, IL-2, and dextran-40
o = Pembrolizumab
1002172111. Patients who had a left ventricular ejection fraction (LVEF) <45%
or who are New
York Heart Association Class II or higher. A cardiac stress test demonstrating
any irreversible wall
movement abnormality in any patients >60 years of age or in patients who have
a history of ischemic
heart disease, chest pain, or clinically significant atrial and/or ventricular
arrhythmias.
o Patients with an abnormal cardiac stress test could be enrolled if they
had adequate
ejection fraction and cardiology clearance with approval of the Sponsor's
Medical
Monitor.
1002173112. Patients who had obstructive or restrictive pulmonary disease and
have a documented
FEV1 (forced expiratory volume in 1 second) of <60% of predicted normal.
o = If a patient was not able to perform reliable spirometry due to
abnormal upper
airway anatomy (i.e., tracheostomy), a 6-minute walk test was used to assess
pulmonary function. Patients who were unable to walk a distance of at least
80%
predicted for age and sex or demonstrates evidence of hypoxia at any point
during the
test (Sp02<90%) are excluded.
1002174113. Patients who had another primary malignancy within the previous 3
years (except for
those which did not require treatment or had been curatively treated greater
than 1 year ago, and in the
judgment of the Investigator, did not pose a significant risk of recurrence
including, but not limited to,
non-melanoma skin cancer, DCIS, LCIS, prostate cancer Gleason score <6 or
bladder cancer).
1002175114. Participation in another clinical study with an investigational
product within 21 days of
the initiation of treatment.
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10021761 Study Endpoints and Planned Analyses
10021771The primary and secondary endpoints were analyzed separately by
cohort.
10021781Pr1mary Endpoints:
10021791The ORR was defined as the proportion of patients who achieved either
a confirmed PR or
CR as best response as assessed by Investigators per RECIST 1.1 among the
efficacy analysis set.
10021801Objective response was evaluated per each disease assessment and the
ORR was expressed
as a binomial proportion with the corresponding 2-sided 90% CI. The primary
analysis for each
cohort occurred when all treated patients per cohort have an opportunity to be
followed for 12 months,
unless progressed/expired or discontinued early from the assessment period.
10021811The safety primary endpoint was measured by any Grade 3 or higher TEAE
incidence rate
within each cohort expressed as binomial proportions with the corresponding 2-
sided 90% CI.
10021821Secondary Endpoints:
10021831Efficacy:
10021841The secondary efficacy endpoints were defined as follows:
10021851CR rate as based on responders who achieved confirmed CR as assessed
by Investigators.
DCR was derived as the sum of the number of patients who achieved confirmed
PR/CR or sustained
SD (at least 6 weeks) divided by the number of patients in the efficacy
analysis set >< 100%. The CR
rate and DCR was summarized using a point estimate and its 2-sided 90% CI.
10021861DOR was defined among patients who achieved objective response. It was
measured from
the first-time response (PR/CR) criteria are met until the first date that
recurrent or progressive disease
was objectively documented, or receipt of subsequent anticancer therapy or the
patient dies
(whichever is first recorded). Patients not experiencing PD or have not died
prior to the time of data
cut or the final database lock will have their event times censored on the
last date that an adequate
assessment of tumor status is made.
10021871PFS was defined as the time (in months) from the time of
lymphodcpletion to PD, or death
due to any cause, whichever event is earlier. Patients not experiencing PD or
not having expired at the
time of the data cut or the final database lock had their event times censored
on the last date that an
adequate assessment of tumor status is made.
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[00218810S was defined as the time (in months) from the time of
lymphodepletion to death due to
any cause. Patients not having expired by the time of data cut or the final
database lock had their
event times censored on the last date of their known survival status.
10021891 DOR, PFS, and OS was subjected to right censoring. The Kaplan-Meier
method will be
used to summarize the time-to-event efficacy endpoints. The baseline data for
the tumor assessment
was the last scan before the lymphodepletion for all cohorts.
10021901 The above efficacy parameters will be estimated for applicable cohort
for subsets defined
by baseline disease characteristics; BRAF status (Cohort lA only), HPV status
(Cohort 2A only),
squamous or non-squamous lung disease (Cohorts 3A and 3B only), and anti-PD-Li
status.
EXAMPLE 15: A PHASE 2, MULTICENTER STUDY OF AUTOLOGOUS TUMOR
INFILTRATING LYMPHOCYTES IN PATIENTS WITH SOLID TUMORS
10021911 The study in this example is a phase 2, multicenter, global, open
label study of autologous
tumor infiltrating lymphocytes (TIL) in patients with select solid tumors
(metastatic melanoma, head
and neck squamous cell cancer, and non-small cell lung cancer (NSCLC)). The
example uses the TIL
product manufactured according to the Examples herein, including Examples 10-
17, as well as
dresibed in throughout the present application, which is cryopreserved and has
a 22-day
manufacturing process. A single infusion of TIL produce was given after
patients had completed the
preparatory regimen of non-myeloablative lymphodepletion with cyclophosphamide
(60mg/kg x2
days) and fludarabine (25mg2/m x 5 days). There are 4 patient cohorts as
described below:
= Cohort lA (combination cohort): Stage IIIC or IV unresectable or
metastatic melanoma
patients who are immunotherapy naive with <3 prior lines of systemic therapy.
These patients
will be receiving TIL product in combination with pembrolizumab.
= Cohort 2A (combination cohort): Advanced, recurrent or metastatic head
and neck squamous
cell carcinoma patients who are immunotherapy naive with <3 prior lines of
systemic therapy.
These patients will be receiving TIL product in combination with
pembrolizumab.
= Cohort 3A (combination cohort): Locally advanced or metastatic (Stage 111-
1V) NSCLC
patients who are immunotherapy naive with <3 prior lines of systemic therapy.
These patients
will be receiving TIL product in combination with pembrolizumab.
= Cohort 3B (single agent cohort): Stage III- IV NSCLC patients who have
previously received
systemic therapy with checkpoint inhibitors (anti-PD- I /anti-PD-L I) as part
of <3 prior lines
of systemic therapy. These patients will be receiving TIL product as single
agent.
10021921 Two patients with NSCLC have been enrolled in Cohort 3B (TIL alone
post immune
checkpoint inhibitor) who had completed their first response assessment visit
at day 42) are described
below.
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[002193] Patient A is a 63-year old female diagnosed with Stage IVA lung
adenocarcinoma. She had
received 3 prior lines of systemic therapy which included pembrolizumab,
carboplatin/ bevacizumab
and vinorelbine. Her best response to prior pembrolizumab therapy was
progressive disease; to
carboplatin/bevacizumab was a partial response, and her vinorelbine response
was non-evaluable (she
discontinued prior to response assessment). She underwent left lower lobe lung
resection for TIL
generation and was infused with 3.75 x 10 cells of TIL product. The patient
had her first response
assessment (Day 42) visit, at which computerized tomography (CT) scans showed
a 4% reduction in
the tumor load compared to baseline scans, which is a stable disease response
per RECIST 1.1.
10021941 Patient B is a 70-year old female diagnosed with Stage IVB basaloid
squamous cell
carcinoma. She had received 3 prior lines of therapy which included
carboplatin/abraxane, nivolumab
and cisplatin/gemcitabine. Her best response to prior carbo/abraxane was not
evaluable (therapy
discontinued due to toxicity); to nivolumab was progressive disease and for
cisplatin/gemcitabine was
a partial response. She underwent splenie lesion resection for TIL generation
and was infused with 39
x 109 cells of TIL product. The patient had her first response assessment (Day
42) visit, at which CT
scans showed a 44% reduction in the tumor load compared to baseline scans,
which is a partial
response per RECIST 1.1.
EXAMPLE 16: A PHASE 2, MULTICENTER STUDY OF AUTOLOGOUS TUMOR
INFILTRATING LYMPHOCYTES IN PATIENTS WITH LOCALLY ADVANCED OR
METASTATIC NON-SMALL-CELL LUNG CANCER
[002195] This example relates to treatment of patients with locally advanced,
unresectable or
metastatic non-small-cell lung cancer (NSCLC) without any actionable driver
mutations who have
disease progression on or following a single line of approved systemic therapy
consisting of combined
checkpoint inhibitor (CPI) + chemotherapy bevacizumab (including bevacizumab
(AVASTIN), a
VEGFA inhibitor) and the cohorts for treatment are summarized below:
= Cohort 1: Patients whose tumors did not express programmed cell death-
ligand 1 (PD-L1)
(tumor proportion score [TPS] < 1%) prior to their CPI treatment.
= Cohort 2: Patients whose tumors expressed PD-Ll (TPS > 1%) prior to their
CPI treatment.
Cohort 3: Patients whose tumors did not express PD-Li (TPS < 1%) prior to
their CPI
treatment and who are unable to safely undergo a surgical harvest for TIL
generation due to at
least one of the followin:
o Unacceptable surgical risk, or
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o Surgically approachable lesion was required for Response
Evaluation Criteria in Solid
Tumors (RECIST) v1.1 assessment.
= Cohort 4: Retreatment cohort: Patients who had been previously treated
with TIL-based
immunotherapy in Cohort 1, 2 or 3 of this study.
10021961 Treatment will be given using autologous TIL-based immunotherapy
derived from an
individual patient's tumor for patient-directed therapy
Study Details
Autologous TIL Therapy Regimen
10021971The TIL-based immunotherapy treatment regimen involved a course of the
NMA-LD
preparative regimen using cyclophosphamide and fludarabine for a total of 5
days prior to TIL-based
immunotherapy infusion, and a limited course of IL-2 administration (up to six
doses) following the
TIL-based immunotherapy infusion. The NMA-LD preparative regimen and 1L-2 were
included in the
regimen to support the engraftment, expansion, and activation of the
transferred TIL
10021981 Several preparative regimens had been used in conjunction with TIL
therapies. NMA-LD
preparative regimens included cyclophosphamide/fludarabine, total body
irradiation (TBI), or the
combination of both. The present exemplary study utilized the cy-flu regimen.
The NMA-LD
preparative regimen used in the current study was based on the method
developed and tested by the
National Cancer Institute (NCI), which involves 2 days of cyclophosphamide
concomitant with 5 days
of fludarabine in an effort to shorten the duration of the hospital stay of
patients. Each patient would
undergo an NMA-LD preparative regimen prior to infusion of TIL-based
immunotherapy.
Brief description of the Treatment
19021991 The therapy was a ready-to-infuse, autologous TIL-based
immunotherapy. The TIL-based
immunotherapy was composed of autologous TIL, which were obtained from an
individual patient's
tumor and expanded ex vivo through cell culture in the presence of the
cytokine 1L-2 and a murine
monoclonal antibody (mAb) to human CD3 (OKT3).
10022001 The final drug product was a cryopreserved live-cell suspension that
was formulated for IV
infusion. The ex vivo expanded autologous TIL were formulated in CryoStor
CS10 cryopreservation
medium/PlasmaLyte (final dimethyl sulfoxide [DMS01 concentration: 5%), with
0.5% human serum
albumin (HSA) and 300 TIJ/mL (12 ng/mL) of 1L-2. The formulated product was
frozen at a
controlled rate to <-150 C in vapor phase liquid nitrogen, shipped in a
eryoshipper to the appropriate
clinical site, and thawed before use for infusion into the patient.
Production and Expansion of TIL
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10022011 The manufacturing process began at the clinical site with the
surgical resection or core
biopsy of a tumor lesion containing viable tumor material. An aggregate of
multiple separate lesion
biopsies could also be resected from the patient and was encouraged if patient
safety allows. The
tumor specimen was placed in transport media and shipped by express courier at
2-8 C to the Good
Manufacturing Practices (GMP) manufacturing facility. Upon arrival at the GMP
manufacturing
facility, the tumor specimen was dissected into fragments, which are then
activated (initial expansion
step) to generate the minimum number of viable cells required for the rapid
expansion protocol (REP)
stage. The tumors could also be enzymatically dissociated, and TIL could be
selected for expression
of biomarkers prior to proceeding to the REP. The REP stage (second expansion
step) further expands
the cells in the presence of IL-2, OKT3, and irradiated allogeneic peripheral
blood mononuclear cells
(PBMC). The REP-expanded cells are then harvested, washed, and formulated in a
blood
transport/infusion bag for shipment by courier to the clinical site. A diagram
of the manufacturing
process for TEL-based immunotherapy is provided in Figures 34 and 35.
10022021 Each cryopreservation bag of the TIL-based immunotherapy final
product was labeled with
a patient-specific label. TIL-based immunotherapy was shipped from the
manufacturing facility to
clinical sites for administration to patients.
10022031 This example related to a prospective, open-label, multi-cohort, non-
randomized,
multicenter phase 2 study evaluating TIL-based immunotherapy in patients with
locally advanced
unresectable or metastatic NSCLC.
The following cohorts were studied:
10022041 Cohort 1: TIL-based immunotherapy as single-agent therapy in patients
with Stage IA/
NSCLC whose tumors did not express PD-L1 (tumor proportion score [TPS] < 1%)
prior to their CPI
treatment without a known actionable driver mutation, who had disease
progression on or following a
single line of approved systemic therapy consisting of combined CPI +
chemotherapy bevacizumab,
who had at least one resectable lesion (or aggregate lesions) of a minimum 1.5
cm in diameter for TIL
production and, following the resection, had at least one remaining measurable
lesion, as defined by
RECIST 1.1..
10022051 Cohort 2: TIL-based immunotherapy as single-agent therapy in patients
with Stage IA/
NSCLC whose tumors expressed PD-Li (TPS >1%) prior to their CPI treatment,
without any known
actionable driver mutations, who had disease progression on or following a
single line of approved
systemic therapy consisting of combined CPI + chemotherapy bevacizumab, and
who had at least
one resectable lesion (or aggregate lesions) of a minimum 1.5 cm in diameter
for TIL production and,
following the resection, had at least one remaining measurable lesion, as
defined by RECIST 1.1.
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10022061 Cohort 3: TIL-based immunotherapy as single-agent therapy in patients
with Stage IV
NSCLC whose tumors did not express PD-L1 (TPS < 1%) prior to their CPI
treatment, without any
known actionable driver mutations, who had disease progression on or following
a single line of
approved systemic therapy consisting of combined CPI + chemotherapy
bevacizumab, and who
were unable to safely undergo a surgical harvest for TIL generation due to at
least one of the
following: 1) unacceptable surgical risk, or 2) surgically approachable lesion
is required for RECIST
assessment.
10022071 Cohort 4: TIL-based immunotherapy single agent therapy as retreatment
in patients who
previously received TIL-based immunotherapy as part of their participation in
Cohorts 1, 2 or 3.
10022081 For Cohorts 1, 2, 3, and 4, all patients received autologous
cryopreserved TIL-based
immunotherapy, preceded by a nonmyeloablative lymphodepletion (NMA-LD)
preconditioning
regimen consisting of cyclophosphamidc and fludarabinc. Following TIL-based
immunotherapy
infusion, up to 6 doses of IV IL-2 (such as aldesleukin or a biosimilar or
variant thereof) were
administered. Alternatively, descrescendo IL-2 or low-dose IL-2 may be used as
set forth herein.
10022091 The autologous TIL therapy with TIL-based immunotherapy included the
following general
steps:
= Tumor harvest to provide the autologous tissue that served as the source
of the autologous
TIL cellular product,
= Production of autologous TIL-based immunotherapy investigational product
(IP) at a
central Good Manufacturing Practice (GMP) facility,
= A 5-day nonmyeloablative lymphodepletion (NMA-LD) preconditioning
regimen,
= Infusion of the TIL-based immunotherapy product (Day 0) , and
= Administration of < 6 doses IV 1L-2.
Primary Objectives:
10022101 Evaluated the efficacy of TIL-based immunotherapy in patients with
locally advanced
unresectable or metastatic NSCLC without an actionable driver mutation who
have disease
progression on or following a single line of approved systemic therapy
consisting of combined
checkpoint inhibitor(s) (CPI[s]) + chemotherapy bevacizumab, as determined
by objective response
rate (ORR), using the Response Evaluation Criteria in Solid Tumors (RECIST
1.1), as assessed by the
Independent Review Committee (IRC) (Cohorts 1 and 2) or by the Investigator
Cohort 3 and Cohort
4).
Secondary Objectives:
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10022111 Evaluated the efficacy of TIL-based immunotherapy as determined by
ORR, using RECIST
1.1, and as assessed by the Investigator (Cohorts 1 and 2).
10022121 Further evaluated the efficacy of TIL-based immunotherapy using
complete response (CR)
rate; duration of response (DOR); disease control rate (DCR); progression-free
survival (PFS) using
RECIST 1.1, as assessed by the IRC (Cohorts 1 and 2) and Investigator (all
cohorts); and overall
survival (OS).
10022131 Characterized the safety profile of TIL-based immunotherapy in NSCLC
patients, as
measured by the incidence of Grade > 3 treatment-emergent adverse events
(TEAEs).
10022141 For Cohort 3 only: Evaluated the efficiency of generating TIL-based
immunotherapy from
core biopsies.
Exploratory Objectives:
10022151 Evaluated the persistence of TIL-based immunotherapy and to identify
immune correlates
that may affect response, outcome, and toxicity variables.
10022161 Assessed respective, indication-specific, health-related quality of
life (HROoL) parameters.
Endpoints - Primary Endpoint:
10022171ORR was assessed per RECIST 1.1 by the IRC (Cohorts 1 and 2) or by the
Investigator
(Cohorts 3 and 4).
Endpoints - Secondary Endpoints:
10022181 Incidence of severity, seriousness, relationship to study treatment,
and characteristics of
treatment-emergent adverse events (TEAEs), including serious AEs (SAEs),
therapy-related AEs, and
AEs leading to early discontinuation from treatment or withdrawal from the
Assessment Period or
death.
[0022191 CR (complete response) rate, DOR (duration of response), DCR (disease
control rate), and
PFS (progression-free survival) as assessed by IRC per RECIST 1.1 (Cohorts 1
and 2).
10022201ORR (objective response rate), CR rate, DOR, DCR, and PFS as assessed
by the
Investigator per RECIST 1.1 (all cohorts).
[00222110S (overall survival).
10022221 Percentage successful TIL products generated from core biopsies
(Cohort 3).
Endpoints - Exploratory Endpoints:
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100222311n vivo persistence of the T cells comprising the TIL product was
assessed by monitoring
the presence of TIL product-specific T-cell receptor-beta complementarity
determining region 3
(CDR3) sequences in the patient's blood overtime. The CDR3 sequences present
in the product and
peripheral blood samples were identified using deep sequencing.
10022241 Exploratory endpoints aimed at identifying predictive and
pharmacodynamic clinical
biomarkers of the activity of TIL-based immunotherapy:
= Phenotypic and functional characteristics of TIL-based immunotherapy;
= Immune profile of the tumor tissues;
= Gene expression profiles of the TIL product, tumor tissues, and/or PBMCs;
= Mutational landscape of the tumors;
= Circulating immune factors; and
= Immune composition of PBMC.
10022251HRQoL (health-related quality of life) as assessed per the European
Organization for
Research and Treatment of Cancer (EORTC) quality of life questionnaire (QLQ)
C30 and QLQ
LC13.
Study Design Details:
[0022261A prospective, open-label, multi-cohort, non-randomized, multicenter
phase 2 study
evaluated adoptive cell therapy (ACT) with TIL-based immunotherapy.
10022271 All patients received TIL-based immunotherapy, consisting of these
steps:
= Tumor harvest provided the autologous tissue that serves as the source of
the autologous
TIL cellular product,
= Production of autologous TIL-based immunotherapy investigational product
(IP) at a
central Good Manufacturing Practice (GMP) facility,
= A 5-day nonmyeloablative lymphodepletion (NMA-LD) preconditioning
regimen,
= Infusion of the TIL-based immunotherapy product (Day 0) and
= Administration of < 6 doses IV IL-2.
10022281The following general sequential periods will occur in all 4 cohorts,
unless otherwise
specified:
1. Screening Period: From informed consent form (ICF) signature to enrollment
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2. Pre-fret:Owen' Period: From enrollment to initiation of preparative NMA-LD
regimen.
3. Treatment Period: From initiation of preparative NMA-LD to End of Treatment
(EOT)
Visit. This consisted of 8 to 9 days of therapy, including NMA-LD (Days - 5 to
-1), TIL-
based immunotherapy infusion (Day 0), followed by IL-2 administrations (Days 0
or 1 to 3 or
4). The EOT occurred approximately 30 days after Day 0.
4. Posttreatment Follow-up period, which is composed of:
a. Posttreatment Efficacy Follow-up Period (TEFU): From EOT Visit to study
completion (at 5 years [Month 601 after treatment) or the End of Efficacy
Assessment
(EOEA) Visit, which would be prompted by disease progression or start of a new
anticancer therapy, whichever occurs first.
b. Long-Term Follow-up Period (LTFU): From EOEA, as described above, to study
completion (at 5 years [Month 601 after treatment).
10022291 Study participants (enrolled patients) will transition early to LTFU
(e.g., at partial
withdrawal of consent, or if is determined that they would not receive TIL-
based immunotherapy for
any reason). Early study withdrawal was prompted by either consent withdrawal,
death, lost to follow-
up, or study termination by Sponsor. A flowchart for the study design is
presented in Figure 36.
Detailed Doses and Treatment Schedule:
TIL-Based Immunotherapy
10022301 Patients will undergo a 5-day preconditioning NMA-LD regimen that was
initiated prior to
the planned TIL-based immunotherapy infusion on Day 0 (i.e., Days -5 through -
1). The NMA LD
regimen consisted of 2 days of intravenous (IV) cyclophosphamide (60 mg/kg)
with mesna (per site
standard of care or USPI/SmPC) on Days -5 and -4, and 5 days of fludarabine IV
(25 mg/m2, Days -5
through -1).
10022311 IL-2 IV administrations at a dose of 600,000 IU/kg began as soon as 3
hours after, but no
later than 24 hours after, completion of the TIL-based immunotherapy infusion
on Day 0. Additional
IL-2 doses were given approximately every 8 to 12 hours for up to 6 total
doses.
Table 53: Treatment administration regimen
Treatment Administration
-5 -4 -3 -2 -1 0 1 2 3 4
Cyclophosphamide 60 mg/kg X X
Mesna X X
Fludarabine 25 mg/m2/day X X X X X
TIL-based immunotherapy
X
infusion
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Treatment Administration
-5 -4 -3 -2 -1 0 1 2 3 4
IL-2 600,000 IU/kg (X)a X X X (X)a
a 0 = If applicable.
Mesna Preparation
10022321 Mcsna was administered to reduce the risk of hemorrhagic cystitis
related to
cyclophosphamide administration. Mesna was administered as a continuous or
intermittent infusion as
per local standards.
10022331 The total dose of mesna was not adjusted if the amount of
cyclophosphamide is reduced.
Dilute the volume of mesna injection or infusion per institutional standard.
Infusion of Cyclophosphamide and Mesna
10022341Cyclophosphamide (60 mg/kg) in a total volume of 250 mL or 500 mL
(e.g., 5% dextrose in
water 1D5W1 or 0.9 % sodium chloride [NaCl]).
10022351 Mesna (15 mg/kg), if infused continuously, was infused over
approximately 2 hours with
cyclophosphamide (on Days -5 and -4), then at a rate of 3 mg/kg/hour for the
remaining 22 hours in a
suitable diluent over 24 'hours starting concomitantly with each
cyclophosphamide dose.
10022361 The total dose administered was at least 1.3 times that of the dose
of cyclophosphamide.
Higher or continued doses of mesna could be administered for prevention of
hemorrhagic cystitis.
Infusion of Fludarabine
10022371 Fludarabine (25 mg/m2) was to be given IV over approximately 30
minutes once daily for 5
consecutive days during Day -5 to Day -1.
Duration of Participation:
10022381 Overall, the study participation time will be up to 5 years from
treatment to completion.
Selected Inclusion Criteria:
10022391Had histologically or pathologically confirmed diagnosis of NSCLC
(squamous,
nonsquamous, adenocarcinoma, large cell, or mixed histologies), and must have
documented PD-Li
expression status, as determined by the tumor proportion score (TPS) prior to
the CPI treatment that
they received (ie, the historic TPS that informed the initial treatment
choice) (TPS < 1% for Cohorts 1
and 3, and TPS > 1% for Cohort 2).
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10022401 Have received a single line of systemic therapy that included CPI and
chemotherapy
concurrently, with documented radiographic disease progression on or following
this single line of
systemic therapy.
10022411 Prior systemic therapy in the adjuvant or neoadjuvant setting, or as
part of definitive
chemoradiotherapy was not counted as a line of therapy if the disease had not
progressed during or
within 12 months of the completion of such therapy. Prior TIL treatment on
this protocol did not
count as a line of therapy for Cohort 4 (retreatment) patients.
10022421 Had documented exercise tolerance no less than 85% of their age-
expected normal range
and no signs or symptoms of ischemia or clinically significant arrhythmias.
10022431 Had Eastern Cooperative Oncology Group (ECOG) performance status of 0
or 1 and an
estimated life expectancy of > 6 months, in the Investigator's opinion.
10022441 Cohorts 1 and 2: Must have had at least one resectable lesion (or
aggregate lesions) of a
minimum 1.5 cm in diameter for TIL production.
10022451 Cohort 3 only: Patients must have had a single RECIST 1.1 measurable
lesion and no
additional lesion available for surgical harvest, or be unable to safely
undergo a surgical harvest for
TIL generation, but able to safely have tumor harvest via radiology guided
core biopsy sufficient for
TIL generation.
10022461 Cohort 4: Followed either paradigm.
10022471 All Cohorts: If the lesion considered for harvest was within a
previously irradiated field, the
lesion must have demonstrated radiographic progression prior to harvest and
the irradiation must have
been completed at least 3 months prior to enrollment. Patients must have an
adequate histopathology
specimen for protocol-required testing.
10022481 Following tumor harvest for TIL manufacturing, all patients must have
had at least one
remaining measurable lesions, as defined by RECIST 1.1, with the following
considerations:
= Lesions in previously irradiated areas were not selected as target
lesions unless there had
been demonstrated progression in those lesions and the irradiation has been
completed at least
3 months prior to enrollment.
= Cohorts 1 and 2 only: Lesions that were surgically partially resected for
TIL generation
that were still measurable per RECIST v1.1 could be selected as nontarget
lesions but could
not serve as a target lesion for response assessment.
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= Cohort 3 only: If no other lesion was available for core biopsy for TIL
generation, the
single RECIST v1.1 measurable lesion may have served as both, the harvest site
for the core
biopsies, and the lesion for response monitoring.
= Cohort 4: May follow either paradigm but must have had at least 1 RECIST
v1.1
measurable lesion to follow for response.
Efficacy Assessment:
10022491 The following efficacy parameters for TIL-based immunotherapy as a
single therapy in
patients with NSCLC were investigated in each cohort: ORR, CR rate, DOR, DCR,
PFS, and OS.
Statistical Considerations:
10022501 The statistical analyses were based on the estimation of efficacy and
safety parameters and
will be performed by cohort. No formal statistical comparisons were applied
between cohorts.
10022511 The primary efficacy endpoint was ORR as assessed per RECIST v1.1 by
the IRC (Cohorts
1 and 2) or by the Investigator (Cohorts 3 and 4).
10022521 The ORR, CR rates, and the DCRs were summarized using point estimates
and 2-sided 95%
confidence limits based on the Clopper-Pearson exact method. Kaplan-Meier
methods were used to
summarize time-to-event efficacy endpoints, such as DOR, PFS, and OS. DOR
analyses were
performed for patients who achieve objective responses.
10022531 The safety analyses were descriptive and based on the summarization
of TEAEs, SAEs, and
AEs leading to discontinuation from the study, vital signs, and clinical
laboratory tests.
Sample Size Determination:
1002254] The total number of planned patients infused with TIL-based
immunotherapy in Cohorts 1,
2 and 3 was approximately 95.
10022551 Cohort 1 and 2: Approximately 40 patients in each cohort. For each
cohort, Simon's two-
stage design (Simon, 1989) with minimax was used to test the null hypothesis
of <10% ORR against
the alternative hypothesis of ORR >10%. In the first stage, twenty-five
patients were accrued. If there
are 2 or fewer patients responding to the therapy in these 25 patients, the
cohort could be terminated.
Otherwise, expansion into Stage 2 to a total of 40 patients occurred
concurrently with the analysis of
Stage 1. At the end of the second stage, if at least 7 patients respond to
therapy among the total of 40
patients, the null hypothesis was rejected. This 2-stage design provided 70%
power to reject the null
hypothesis of 10% ORR based on an assumption of 20% ORR for TIL-based
immunotherapy at a
one-sided alpha level of 0.1.
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10022561 Cohort 3: Approximately 15 patients were planned, which provided an
estimated ORR with
a half-width 90% confidence interval (CI) of <0.23 by the Clopper-Pearson
exact method.
10022571 Cohort 4: Retreatment cohort: Patients who had been previously
treated with TIL-based
immunotherapy in Cohort 1, 2 or 3 of this study.
EXAMPLE 17: COMPLETE RESPONSE (CR) TO IOVANCE TUMOR INFILTRATING
LYMPHOCYTES (TIL) ALONE ADMINISTERED TO A PATIENT WITH RELAPSED
NON-SMALL CELL LUNG CANCER (NSCLC): CASE REPORT
Introduction
10022581 NSCLC is the most common and lethal cancer with a world-wide
prevalence of over 2
million and 1.7 million deaths annually. Treatment options are limited, and
prognosis remains poor
for patients with relapsed metastatic NSCLC (mNSCLC) after failing standard of
care therapies
including platinum-doublet chemotherapy and checkpoint inhibitors (CPI). There
remains a
significant unmet medical need in NSCLC for patients who progress after CPI.
10022591 Adoptive cell therapy with tumor infiltrating lymphocytes (TIL) has
demonstrated
responses in various malignancies including, melanoma, cervical cancer, NSCLC
and HNSCC alone
or in combination with checkpoint inhibitors (CPI).
10022601 The safety and efficacy of TIL therapy, in a case study of a mNSCLC
heavily pre-treated
patient with PD-Li expression level less than< 1%, is presented.
Methods
10022611We report on a 72-year-old female, never-smoker with metastatic NSCLC
diagnosed after
presenting with cough and cold symptoms. The workup included a chest computer
tomography (CT)
showing a 135 mm lung mass. Staging identified pulmonary, splenic and nodular
lymphatic lesions
(T4N1M lb, Stage IVB), PD-Li <1%). Biopsy confirmed a basaloid squamous cell
carcinoma.
Therapy was initiated with carboplatin and +Nab paclitaxel but was
discontinued after 3 cycles due to
an allergic reaction and poor tolerability. The second-line of therapy
consisted of was nivolumab
which was, discontinued after 4 cycles due to disease progression. The patient
had a PD-L I level of
less than 1%. A third-line regimen of gemcitabine and cisplatin doublet
therapy was then
administered. Cisplatin was discontinued after 4 cycles and gemcitabine was
completed after 6 cycles.
After an initial response, disease progression was observed.
10022621 Approximately 4 months after completing gemcitabine, with disease
progressing rapidly
below the diaphragm, the patient enrolled in (NCT03645928), a prospective,
open-label, multi-cohort,
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non-randomized, multicenter phase 2 study evaluating ACT with TIL. The patient
had an ECOG
performance status of 1, baseline target lesion sum-of-diameters SOD of 50 mm
and had an increase
of 127% in her SOD from screening to baseline on study prior to receiving TIL.
The patient
underwent tumor resection for TIL generation and received TIL as a single
therapy and a one-time
treatment. Preconditioning chemotherapy consisted of
cyclophosphamide/fludarabine, then TIL
followed by 3 doses of 600,000 1U/kg 1L-2 (i.e., aldesleukin). Treatment
tolerability and safety were
assessed on an ongoing basis along with efficacy evaluation by the
Investigator using RECIST v1.1.
Results
10022631Adverse events were consistent with the known toxicities of the
lymphodepletion and IL-2.
Treatment emergent adverse events were acute, self-limiting, manageable, and
short in duration.
Adverse events > G3 were limited to G4 pancytopcnia, and G3 hypotension and
bactcremia. The
patient experienced no serious adverse events.
10022641At week 6, a partial response with PR (44% decrease) in target lesions
was observed at the
patient's first assessment. At 7 months after administration of TIL, when
reduction of sum of
diameters (SODs) was at a 48% decrease, a positron-emission tomography (PET)
CT was conducted
and showed no metabolic activity within the residual CT lesion. The patient is
considered a complete
response (CR) by PET-CT. The examination was ongoing at 15 months post TIL
administration and
total SOD reduction reached 60%. The patient has required no other anti-cancer
therapy to be
administered since the TIL administration.
Conclusions
10022651 This case presentation demonstrates that treatment with TILs as
described in the present
application can offer a therapeutic option for patients with metastatic NSCLC
who have disease
progression on mNSCLC after multiple lines of standard of care therapies,
including CPI. Enrollment
is ongoing and studies to continue to monitor and evaluate impact of TIL in
NSCLC patients.
EXAMPLE 18: EXEMPLARY PRODUCTION OF A CRYOPRESERVED TIL CELL
THERAPY
10022661 This example describes an exemplarty cGMP manufacture of
TIL Cell Therapy
Process in G-REX Flasks according to current Good Tissue Practices and current
Good
Manufacturing Practices.
Table 54 - Process Expansion Examplary Plan
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Estimated Day Estimated
Total
(post-seed) Activity Target Criteria Anticipated
Vessels
Volume (mL)
50 desirable tumor fragments
0 Tumor Dissection per G-REX100MCS
G-REX100MCS 1 flask 1000
¨ 200 X 106 viable cells per
11 REP Seed G-REX500MCS
G-REX500MCS 1 flasks 5000
1 x 109 viable cells per
16 REP Split G-REX500MCS
G-REX500MCS flasks 25000
22 Harvest Total available cells 3-4 CS-750 bags
530
Table 55 - Flask Volumes
Working
Flask Type
Volume/Flask
G-REX100MCS 1000
G-REX500MCS 5000
PROCESS INFORMATION - PRIMARY
Day 0 CM1 Mcdia Preparation
10022671 In the BSC added reagents to RPMI 1640 Media bottle.
Added the following reagents
t Added per bottle: Heat Inactivated Human AB Serum (100.0 mL); GlutaMax (10.0
mL); Gentamicin
sulfate, 50 mg/mL (1.0 mL); 2-mercaptoethanol (1.0 mL)
10022681 Removed unnecessary materials from BSC. Passed out media
reagents from BSC, left
Gentamicin Sulfate and HBSS in BSC for Formulated Wash Media preparation.
10022691 Thawed IL-2 aliquot. Thawed one 1.1 mL IL-2 aliquot
(6x106 IU/mL) (BR71424)
until all ice had melted. Recorded IL-2: Lot # and Expiry
10022701 Transferred IL-2 stock solution to media. In the BSC,
transferred 1.0 mL of IL-2
stock solution to the CM1 Day 0 Media Bottle prepared. Added CM1 Day 0 Media 1
bottle and IL-2
(6x106 IU/mL) 1.0 mL.
10022711 Passed G-REXIOOMCS into BSC. Aseptically passed G-
REX100MCS (W3013130)
into the BSC.
10022721 Pumped all Complete CMI Day 0 Media into G-REXIOOMCS
flask. Tissue
Fragments Conical or GRex100MCS .
Day 0 Tumor Wash Media Preparation
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10022731 In the BSC, added 5.0 mL Gentamicin (W3009832 or
W3012735) to 1 500 mL
HBSS Media (W3013128) bottle. Added per bottle: HBSS (500.0 mL); Gentamicin
sulfate, 50
mg/mL (5.0 mL). Filtered HBSS containing gentamicin prepared through a 11_,
0.22-micron filter unit
(W1218810).
Day 0 Tumor Processing
10022741 Obtained Tumor. Obtained tumor specimen from QAR and
transferred into suite at 2-
8 C immediately for processing.
10022751 Aliquoted Tumor Wash Media.
10022761 Tumor Wash 1 Using 8" forceps (W3009771), removed the
tumor from the specimen
bottle and transferred to the "Wash 1- dish prepared. Followed by Tumor Wash 2
and Tumor Wash 3.
10022771 Measured Tumor. Assessed Tumor. Assessed whether > 30% of
entire tumor area
observed to be necrotic and/or fatty tissue. If applicable: Clean-Up
Dissection. If tumor was large and
>30% of tissue exterior was observed to be necrotic/fatty, performed -clean up
dissection" by
removing necrotic/fatty tissue while preserving tumor inner structure using a
combination of scalpel
and/or forceps.
10022781 Dissect Tumor Using a combination of scalpel and/or
forceps, cut the tumor
specimen into even, appropriately sized fragments (up to 6 intermediate
fragments). Transferred
intermediate tumor fragments. Dissected Tumor Fragmentsinto pieces
approximately 3x3x3mm in
size. Stored Intermediate Fragments to Prevent Drying.
10022791 Repeated Intermediate Fragment Dissection. Determined
number of pieces collected.
If desirable tissue remains, selected additional Favorable Tumor Pieces from
the -favorable
intermediate fragments" 6-well plate to fill the drops for a maximum of 50
pieces.
10022801 Prepared Conical Tube. Transferred Tumor Pieces to 50mL
Conical Tube. Prepared
BSC for G- REX100MCS. Removed G-REX100MCS from Incubator. Aseptically passed G-
REX100MCS flask into the BSC. Added tumor fragments to G-REXIOOMCS flask.
Evenly
distributed pieces.
10022811 Incubated G-REX100MCS at the following parameters:
Incubated G-REX flask:
Temperature LED Display: 37.0 2.0 C; CO2 Percentage: 5.0 1.5 %CO2.
Calculations: Time of
incubation; lower limite = time of incubation + 252 hours; upper limit = time
of incubation + 276
hours.
10022821 After process was complete, discarded any remaining
warmed media and thawed
aliquots of IL-2.
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Day 11 ¨ Media Preparation
[002283] Monitored Incubator. Monitored Incubator. Incubator
parameters: Temperature LED
Display: 37.0 2.0 C; CO2 Percentage: 5.0+1.5 %CO2.
[002284] Warmed 3x 1000 mL RPMI 1640 Media (W3013112) bottles and
3x 1000 mL AIM-
V (W3009501) bottles in an incubator for?: 30 minutes. Removed RPM! 1640 Media
from incubator.
Prepared RPM! 1640 Media. Filter Media. Thawed 3 x 1.1mL aliquots of IL-2
(6x106 IU/mL)
(BR71424). Removed AIM-V Media from the incubator. Add IL-2 to AIM-V.
Aseptically transferred
a IOL Labtainer Bag and a repeater pump transferr set into the BSC.
[002285] Prepared 10L Labtainer media bag. Prepared Baxa pump.
Prepared 10L Labtainer
media bag. Pumped media into 10L Labtainer. Removed pumpmatic from Labtainer
bag.
[002286] Mixed media. Gently massaged the bag to mix. Sample media
per sample plan.
Removed 20.0mL of media and place in a 50mL conical tube.
[002287] Prepared Cell Count Dilution Tubes
In the BSC, added 4.5mL of AIM-V Media
that had been labelled with "For Cell Count Dilutions" and lot number to four
15mL conical tubes.
Transferred reagents from the BSC to 2-8 C. Prepared 1L Transfer Pack. Outside
of the BSC weld
(per Process Note 5.11) a 1L Transfer Pack to the transfer set attached to the
"Complete CM2 Day 11
Media" bag prepared. Prepared feeder cell transfer pack. Incubated Complete
CM2 Day 11 Media.
Day 11 - TII, Harvest
10022881 Preprocessing table. Incubator parameters: Temperature
LED Display: 37.02.0 C;
CO2 Percentage: 5.0+1.5 %CO2. Removed G-REX100MCS from incubator. Prepared
300mL
Transfer Pack. Welded transfer packs to G-REX100MCS.
[002289] Prepare flask for TIL Harvest and nitiation of TIL
Harvest. TIL Harvested. Using the
GatheRex, transferred the cell suspension through the blood filter into the
300mL transfer pack.
Inspect membrane for adherent cells.
[002290] Rinsed flask membrane. Closed clamps on G-REX100MCS.
Ensured all clamps are
closed. Heat sealed the TIL and the "Supernatant" transfer pack. Calculated
volume of TIL
suspension. Prepared Supernatant Transfer Pack for Sampling.
[002291] Pulled Bac-T Sample. In the BSC, draw up approximately
20.0 mL of supernatant
from the 1L "Supernatant" transfer pack and dispense into a sterile 50mL
conical tube.
[002292] Inoculated BacT per Sample Plan. Removed a 1.0 mL sample
from the 50mL conical
labeled BacT prepared using an appropriately sized syringe and inoculated the
anaerobic bottle.
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10022931 Incubated TIL. Placed TIL Transfer Pack in incubator
until needed. Performed cell
counts and calculations. Determined the Average of Viable Cell Concentration
and Viability of the
cell counts performed. Viability 2. Viable Cell Concentration 2. Determined
Upper and Lower
Limit for counts. Lower Limit: Average of Viable Cell Concentration >< 0.9.
Upper Limit: Average of
Viable Cell Concentration >< 1.1. Confirmed both counts within acceptable
limits. Determined an
average Viable Cell Concentration from all four counts performed.
10022941 Adjusted Volume of T1L Suspension. Calculate the adjusted
volume of TIL
suspension after removal of cell count samples. Total TIL Cell Volume (A).
Volume of Cell Count
Sample Removed (4.0 ml) (B) Adjusted Total TIL Cell Volume C=A-B.
10022951 Calculated Total Viable TIL Cells. Average Viable Cell
Concentraion*: Total
Volume; Total Viable Cells: C = A x B.
10022961 Calculation for flow cytometry: if the Total Viable TIL
Cell count from was >
4.0x107, calculated the volume to obtain 1.0x10' cells for the flow cytometry
sample.
10022971 Total viable cells required for flow cytometry: 1.0x107
cells. Volume of cells required
for flow cytometry: Viable cell concentration divived by 1.0x107 cells A.
10022981 Calculated the volume of TIL suspension equal to 2.0x108
viable cells. As needed,
calculated the excess volume of TIL cells to remove and removed excess TIL and
placed TIL in
incubator as needed. Calculated total excess TIL removed, as needed.
10022991 Calculated amount of CS-10 media to add to excess TIL
cells with the target cell
concentration for freezing is 1.0 x 108 cells/mL. Centrifuged excess TILs, as
needed. Observed
conical tube and added CS-10.
10023001 Filled Vials. Aliquoted 1.0mL cell suspension, into
appropriately sized
cryovials.Aliquoted residual volume into appropriately sized cryovial per SOP-
00242. If volume is
<0.5mL, add CS10 to vial until volume is 0.5mL.
10023011 TIL Cryopreservation of Sample
10023021 Calculated the volume of cells required to obtain 1x107
cells for cryopreservation.
Removed sample for Cryopreservation. Placed TIL in Incubator.
Cryopreservation of sample.
10023031 Observed conical tube and added CS-10 slowly and record
volume of 0.5mL of CS 10
added.
Day 11 - Feeder Cells
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10023041 Obtained feeder cells. Obtained 3 bags of feeder cells
with at least two different lot
numbers from LN2 freezer. Kept cells on dry ice until ready to thaw. Prepared
waterbath or
Cryotherrn. Thawed Feeder Cells at 37.0 2.0 C water bath or cytotherm for ¨3-
5 minutes or until ice
has just disappeared. Removed media from incubator. Pooled thawed feeder
cells. Added feeder cells
to transfer pack. Dispensed the feeder cells from the syringe into the
transfer pack. Mixed pooled
feeder cells and labeled transfer pack.
10023051 Calculated total volume of feeder cell suspension in
Transfer Pack
10023061 Removed cell count samples. Using a separate 3mL syringe
for each sample, pulled
4x1.0mL cell count samples from Feeder Cell Suspension Transfer Pack using the
needless injection
port. Aliquoted each sample into the cryovials labeled. Performed Cell Counts
and Determine
Multiplication FactorSelected protocols and entered multiplication factors.
Determined the Average of
Viable Cell Concentration and Viability of the cell counts performed.
Determined Upper and Lower
Limit for counts and confirm within limits.
10023071 Adjusted Volume of Feeder Cell Suspension. Calculated the
adjusted volume of
Feeder Cell suspension after removal of cell count samples. Calculated Total
Viable Feeder Cells.
Obtained additional Feeder Cells as needed. Thawed Additional Feeder Cells as
needed. Placed the
4th Feeder Cell bag into a zip top bag and thaw in a 37.0 2.0 C water bath
or cytotherm for ¨3-5
minutes and pooled additional feeder cells. Measured Volume. Measured the
volume of the feeder
cells in the syringe and recorded below (B). Calculated the new total volume
of feeder cells. Added
Feeder Cells to Transfer Pack.
10023081 Prepared dilutions as needed, adding 4.5mL of AIM-V Media
to four 15mL conical
tubes. Prepared cell counts. Using a separate 3mLsyringe for each sample,
removed 4 x 1.0mL cell
count samples from Feeder Cell Suspension transfer pack, using the needless
injection port.
Performed cell counts and calculations. Determined an average Viable Cell
Concentration from all
four counts performed. Adjusted Volume of Feeder Cell suspension and
calculated the adjusted
volume of Feeder Cell suspension after removal of cell count samples. Total
Feeder Cell Volume
minues 4.0 mL removed. Calculated the volume of Feeder Cell Suspension that
was required to obtain
5x109 viable feeder cells. Calculated excess feeder cell volume. Calculated
the volume of excess
feeder cells to remove. Removed excess feeder cells.
10023091 Using a 1.0mL syringe and 16G needle, drew up 0.15mL of
OKT3 and added OKT3.
Heat sealed the Feeder Cell Suspension transfer pack.
Day 11 G-REX Fill and Seed
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10023101 Set up G-REX500MCS. Removed "Complete CM2 Day 11 Media",
from incubator
and pumped media into G-REX500MCS. Pumped 4.5L of media into the G-REX500MCS,
filling to
the line marked on the flask. Heat sealed and incubated flask as needed.
Welded the Feeder Cell
suspension transfer pack to the G-REX500MCS. Added Feeder Cells to G-
REX500MCS. Heat sealed.
Welded the TIL Suspension transfer pack to the flask. Added TIL to G-
REX500MCS. Heat sealed.
Incubated G-REX500MCS at 37.0+2.0 C, CO2 Percentage: 5.0 1.5 %CO2.
10023111 Calculated incubation window. Performed calculations to
determine the proper time
to remove G-REX500MCS from incubator on Day 16. Lower limit: Time of
incubation + 108 hours.
Upper limit: Time of incubation + 132 hours.
Day 11 Excess TIL Cryopreservation
10023121 Applicable: Froze Excess TIL Vials. Verified the CRF has
been set up prior to freeze.
Perform Cryopreservation. Transferred vials from Controlled Rate Freezer to
the appropriate storage.
Upon completion of freeze, transfer vials from CRF to the appropriate storage
container. Transferred
vials to appropriate storage. Recorded storage location in LN2.
Day 16 Media Preparation
10023131 Pre-warmed AIM-V Media. Calculated time Media was warmed
for media bags 1, 2,
and 3. Ensured all bags have been warmed for a duration between 12 and 24
hours. Setup 10L
Labtainer for Supernatant. Attached the larger diameter end of a fluid pump
transfer set to one of the
female ports of a 10L Labtainer bag using the Luer connectors. Setup 10L
Labtainer for Supernatant
and label. Setup 10L Labtainer for Supernatant. Ensure all clamps were closed
prior to removing from
the BSC. NOTE: Supernatant bag was used during TIL Harvest, which may be
performed
concurrently with media preparation.
10023141 Thawed IL-2. Thawed 5x1.1mL aliquots of IL-2 (6x106
IU/mL) (BR71424) per bag
of CTS AIM V media until all ice had melted. Aliquoted 100.0mL GlutaMax. Added
IL-2 to
GlutaMax. Prepared CTS AIM V media bag for formulation. Prepared CTS AIM V
media bag for
formulation. Stage Baxa Pump. Prepared to formulate media. Pumped GlutaMax +IL-
2 into bag.
Monitored parameters: Temperature LED Display: 37.0+2.0 C, CO2 Percentage:
5.0+1.5 %CO2.
Warmed Complete CM4 Day 16 Media. Prepared Dilutions.
Day 16 REP Spilt
10023151 Monitored Incubator parameters: Temperature LED Display:
37.0+2.0 C, CO2
Percentage: 5.0+1.5 %CO2. Removed G-REX500MCS from the incubator. Prepared and
labeled IL
Transfer Pack as TIL Suspension and weighed 1L.
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10023161 Volume Reduction of G-REX500MCS. Transferred ¨4.5L of
culture supernatant
from the G-REX500MCS to the IOL Labtainer per SOP-01777.
10023171 Prepared flask for TIL Harvest. After removal of the
supernatant, closed all clamps to
the red line.
10023181 Initiation of TIL Harvest. Vigorously tap flask and swirl
media to release cellsensure
all cells have detached.
10023191 TIL Harvest. Released all clamps leading to the TIL
suspension transfer pack. Using
the GatheRex transferred the cell suspension into the TIL Suspension transfer
pack. NOTE: Be sure to
maintain the tilted edge until all cells and media arc collected. Inspected
membrane for adherent cells.
Rinsed flask membrane. Closed clamps on G-REX500MCS. Heat sealed the Transfer
Pack containing
the TIL. Heat sealed the 10L Labtainer containing the supernatant. Recorded
weight of Transfer Pack
with cell suspension and calculate the volume suspension. Prepared transfer
pack for sample removal.
Removed testing samples from cell supernatant.
10023201 Sterility & BacT Testing Sampling: removed a 1.0mL sample
from the 15 mL conical
labeled BacT prepared. Removed Cell Count Samples. In the BSC, using separate
3mL syringes for
each sample, removed 4x1.0 mL cell count samples from "TIL Suspension"
transfer pack.
10023211 Removed Mycoplasma Samples. Using a 3mL syringe, removed
1.0 mL from TIL
Suspension transfer pack and place into 15 mL conical labeled "Mycoplasma
diluent" prepared.
10023221 Prepared Transfer Pack for Seeding. Placed TIL in
Incubator. Removed cell
suspension from the BSC and place in incubator until needed. Performed cell
counts and calculations.
Diluted cell count samples initially by adding 0.5mL of cell suspension into
4.5mL of AIM-V media
prepared which gave a 1:10 dilution. Determined the Average of Viable Cell
Concentration and
Viability of the cell counts performed. Determined Upper and Lower Limit for
counts. NOTE:
Dilution may be adjusted according based off the expected concentration of
cells. Determined an
average Viable Cell Concentration from all four counts performed. Adjusted
Volume of TIL
Suspension. Calculated the adjusted volume of TIL suspension after removal of
cell count samples.
Total TIL Cell Volume minus 5.0 mL removed for testing.
10023231 Calculated Total Viable TIL Cells. Calculated the total
number of flasks to seed.
NOTE: The maximum number of G-REX500MCS flasks to seed was five. if the
calculated number of
flasks to seed exceeded five, only five were seeded USING THE ENTIRE VOLUME OF
CELL
SUSPENSION AVAILABLE.
10023241 Calculate number of flasks for subculture. Calculated the
number of media bags
required in addition to the bag prepared. Prepared one 10L bag of "CM4 Day 16
Media" for every two
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G-REX-500M flask needed as calculated. Proceeded to seed the first GREX-500M
flask(s) while
additional media is prepared and warmed. Prepared and warmed the calculated
number of additional
media bags determined. Filled G-REX500MCS. Prepared to pump media and pumped
4.5L of media
into G-REX500MCS. Heat Sealed. Repeated Fill. Incubated flask. Calculated the
target volume of
TIL suspension to add to the new G-REX500MCS flasks. If the calculated number
of flasks exceeds
five only five will be seeded, USING THE ENTIRE VOLUME OF CELL SUSPENSION.
Prepared
Flasks for Seeding. Removed G-REX500MCS from the incubator. Prepared G-
REX500MCS for
pumping. Closed all clamps on except large filter line. Removed TIL from
incubator. Prepared cell
suspension for seeding. Sterile welded (per Process Note 5.11) "TIL
Suspension" transfer pack to
pump inlet line. Placed TIL suspension bag on a scale.
10023251 Seeded flask with TIL Suspension. Pump the volume of TIL
suspension calculated
into flask. Heat sealed. Filled remaining flasks.
10023261 Monitored Incubator. Incubator parameters: Temperature
LED Display: 37.0+2.0 C,
CO2 Percentage: 5.0+1.5 %CO2. Incubated Flasks.
10023271 Determined the time range to remove G-REX500MCS from
incubator on Day 22.
Day 22 Wash Buffer Preparation
10023281 Prepared 10 L Labtainer Bag. In BSC, attach a 4- plasma
transfer set to a 10L
Labtainer Bag via luer connection. Prepared 10 L Labtainer Bag. Closed all
clamps before transferring
out of the BSC. NOTE: Prepared one 10L Labtainer Bag for every two G-REX500MCS
flasks to be
harvested. Pumped Plasmalyte into 3000mL bag and removed air from 3000mL
Origen bag by
reversing the pump and manipulating the position of the bag. Added Human
Albumin 25% to
3000mL Bag. Obtain a final volumeof 120.0 mL of Human Albumin 25%.
10023291 Prepared IL-2 Diluent. Using a 10mL syringe, removed 5.0
mL of LOVO Wash
Buffer using the needleless injection port on the LOVO Wash Buffer bag.
Dispensed LOVO wash
buffer into a 50mL conical tube.
10023301 CRF Blank Bag LOVO Wash Buffer Aliquotted. Using a 100mL
syringe, drew up
70.0 mL of LOVO Wash Buffer from the needleless injection port.
10023311 Thawed 1L-2. Thawed one 1.1mL of 1L-2 (6x106 1U/mL) ),
until all ice has melted.
IL-2 Preparation. Added 501.11_, IL-2 stock (6x106 IU/mL) to the 50mL conical
tube labeled "IL-2
Diluent."
10023321 Cryopreservation Prep. Placed 5 cryo-cassettes at 2-8 C
to precondition them for
final product cryopreservation.
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10023331 Prepared Cell Count Dilutions. In the BSC, added 4.5mL of
AIM-V Media that has
been labelled with lot number and For Cell Count Dilutions" to 4 separate 15mL
conical tubes.
Prepared Cell Counts. Labeled 4 cryovials with vial number (1-4). Kept vials
under BSC to be used.
Day 22 TIL Harvest
10023341 Monitored Incubator. Incubator Parameters Temperature LED
display: 37 2.0 C,
CO2 Percentage: 5% 1.5%. Removed G-REX500MCS Flasks from Incubator. Prepared
TIL
collection bag and labeled. Sealed off extra connections. Volume Reduction:
Transfered ¨4.5L of
supernatant from the G-REX500MCS to the Supernatant bag.
10023351 Prepared flask for TIL Harvest. Initiated collection of
TIL. Vigorously tap flask and
swirl media to release cells. Ensure all cells have detached. Initiated
collection of TIL. Released all
clamps leading to the TIL suspension collection bag. TIL Harvest. Using the
GatheRex, transferred
the TIL suspension into the 3000mL collection bag. Inspect membrane for
adherent cells. Rinsed flask
membrane. Closed clamps on G- Rex500MCS and ensured all clamps are closed.
Transferred cell
suspension into LOVO source bag. Closed all clamps. Heat Sealed. Removed
4x1.0mL Cell Counts
Samples
10023361 Performed Cell Counts. Peifonned cell counts and
calculations utilizing NC-200 and
Process Note 5.14. Diluted cell count samples initially by adding 0.5mL of
cell suspension into 4.5mL
of AIM-V media prepared. This gave a 1:10 dilution. Determined the Average
Viability, Viable Cell
Concentration, and Total Nucleated Cell concentration of the cell counts
performed. Determined
Upper and Lower Limit for counts. Determined the Average Viability, Viable
Cell Concentration, and
Total Nucleated Cell concentration of the cell counts performed. Weighed LOVO
Source Bag.
Calculated Total Viable TIL Cells. Calculated Total Nucleated Cells.
10023371 Prepared Mycoplasma Diluent. Removed 10.0 mL from one
supernatant bag via luer
sample port and placed in a 15mL conical.
LOVO
10023381 Performed "TIL G-REX Harvest" protocoland determined the
final product target
volume. Loaded disposable kit. Removed filtrate bag. Entered Filtrate
capacity. Placed Filtrate
container on bcnchtop. Attached PlasmaLyte. Verified that the PlasmaLyte was
attached and observed
that the PlasmaLyte is moving. Attached Source container to tubing and
verified Source container was
attached. Confirmed PlasmaLyte was moving.
Final Formulation and Fill
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10023391 Target volume/bag calculation. Calculated volume of CS-10
and LOVO wash buffer
to formulate blank bag. Prepared CRF Blank.
10023401 Calculated the volume of IL-2 to add to the Final
Product. Final IL-2 Concentration
desired (IU/mL) ¨ 300IU/mL. IL-2 working stock: 6 x 104 IU/mL. Assembled
Connect apparatus.
Sterile welded a 4S-4M60 to a CC2 Cell Connection. Sterile welded (per Process
Note 5.11) the
CS750 Cryobags to the harness prepared. Welded CS-10 bags to spikes of the 4S-
4M60.Prepared TIL
with 1L-2. Using an appropriately sized syringe, removed amount of 1L-2
determined from the "1L-2
6x104" aliquot. Labeled Forumlated TIL Bag. Added the Formulated TIL bag to
the apparatus. Added
CS10. Switched Syringes. Drew ¨10mL of air into a 100mL syringe and replaced
the 60mL syringe
on the apparatus. Added CS10. Prepared CS-750 bags. Dispensed cells.
10023411 Removed air from final product bags and take retain. Once
the last final product bag
was filled, closed all clamps. Drew 10mL of air into a new 100mL syringe and
replace the syringe on
the apparatus. Dispensed retain into a 50mL conical tube and label tube as
"Retain" and lot number.
Repeat air removal step for each bag.
10023421 Prepared final product for cryopreservation, incuding
visual inspection. Held the
cryobags on cold pack or at 2-8 C until cryopreservation.
10023431 Removed Cell Count Sample. Using an appropriately sized
pipette, remove 2.0 mL of
retain and place in a 15mL conical tube to be used for cell counts. Performed
cell counts and
calculations. NOTE: Diluted only one sample to appropriate dilution to verify
dilution is sufficient.
Diluted additional samples to appropriate dilution factor and proceed with
counts. Determined the
Average of Viable Cell Concentration and Viability of the cell counts
performed. Determined Upper
and Lower Limit for counts. NOTE: Dilution may be adjusted according based off
the expected
concentration of cells. Determined the Average of Viable Cell Concentration
and Viability.
Determined Upper and Lower Limit for counts. Calculated IFN-y. Heat Sealed
Final Product Bags.
10023441 Labeled and Collected Samples per exemplary Sample Plan
below.
Table 56: Sample Plan
Sample
Number of Volume to Container
Sample
Containers Add to Type
Each
15 mL
*Mycoplasma 1 1.0 mL
Conical
Endotoxin 2 1.0 mL 2 mL
Cryovial
Gram Stain 1 1.0 mL 2 mL
Cryovial
IFN-g 1 1.0 mL 2 mL
Cryovial
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Flow
Cytometry 1 1.0 mL 2 mL
Cryovial
**BacT
2 1.0 mL Bac-T
Bottle
Sterility
QC Retain 4 1.0 mL 2 mL
Cryovial
Satellite Vials 10 0.5 mL 2 mL
Cryovial
[002345] Sterility & BacT. Testing Sampling. In the BSC, remove a
1.0mL sample from the
retained cell suspension collected using an appropriately sized syringe and
inoculate the anaerobic
bottle. Repeat the above for the aerobic bottle
Final Product Cryopresei-vation
[002346] Prepared Controlled Rate Freezer. Verified the CRF had
been set up. Set up CRF
probes. Placed final product and samples in CRF. Determined the time needed to
reach 4 C 1.5 C
and proceed with the CRF run. CRF Completed and Stored. Stopped the CRF after
the completion
of the run. Remove cassettes and vials from CRF. Transferred cassettes and
vials to vapor phase LN2
for storage. Recorded storage location
POST PROCESSING SUMMARY
Post-Processing: Final Drug Product
[002347] (Day 22) Determination of CD3+ Cells on Day 22 REP by
Flow Cytometry
[002348] (Day 22) Gram Staining Method (GMP)
[002349] (Day 22) Bacterial Endotoxin Test by Gel Clot LAL Assay
(GMP)
[002350] (Day 16) BacT Sterility Assay (GMP)
[002351] (Day 16) Mycoplasma DNA Detection by TD-PCR (GMP)
[002352] Acceptable Appearance Attributes
[002353] (Day 22) BacT Sterility Assay (GMP)(Day 22)
[002354] (Day 22) IFN-gamma Assay
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EXAMPLE 19: FIRST PHASE 2 RESULTS OF AUTOLOGOUS TUMOR-INFILTRATING
LYMPHOCYTE MONOTHERAPY IN PATIENTS WITH ADVANCED, IMMUNE
CHECKPOINT INHIBITOR-TREATED, NON-SMALL CELL LUNG CANCER (NSCLC)
Introduction
Background
10023551A majority of patients with advanced NSCLC develop disease progression
with first-line
ICI chemotherapy'.
100235611n the setting of ICI resistance, effective strategies to provide deep
and durable responses
are urgently needed.
10023571Gen 2 process TILs are centrally manufactured autologous TIL cell
products that have
demonstrated activity in advanced melanoma, cervical cancer, and head and neck
carcin0ma2-5.
[0023581TM nivolumab has demonstrated safety and efficacy in ICI-naive
patients with advanced
NSCLC in a phase 1 trial'.
10023591This example demonstrates first safety and efficacy data for single-
agent Gen 2 process TIL
cell therapy in patients with advanced NSCLC from a multicenter phase 2 study.
Methods
Study Design
10023601 Reported is a prospective, phase 2, multicenter, multicohort, open-
label study evaluating
autologous TIL cell therapy in multiple settings and indications.
10023611 Provided here is data from Cohort 3B, investigating TIL monotherapy
in patients with
advanced or metastatic NSCLC.
Cohort 3B Patients
10023621 Eligibility required age >18 years, 1-3 prior lines of systemic
therapy including either ICI
or oncogene-directed therapy, ECOG performance status 0-1, >lresectable lesion
(-1.5 cm in
diameter) for TIL manufacturing, and >1 measurable lesion post-resection for
response assessment.
Endpoints
10023631 Primary
10023641Efficacy, defined as investigator-assessed ORR per RECIST v1.1.
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10023651 Safety, as measured by incidence of Grade >3 TEAEs (defined as AEs
that occur from the
time of TIL infusion, up to 30 days after TIL infusion or start of a new
anticancer therapy).
10023661 Exploratory
10023671 Biomarker analyses, including TCR repertoire of the TIL product using
RNA sequencing
(HTBlvc assay, iRepertoire, Inc., Huntsville, AL); clones present above the
limits of detection in each
individual patient TIL product lot were counted and their proportion estimated
to assess TIL clonality
and diversity.
10023681 The patient journey and central Gen 2 GMP manufacturing of TIL
product is depicted in
Fig. 37.
10023691 The cohort 3B patient treatment schema is depicted in Fig. 38.
Results
10023701 Patient disposition is presented in Fig. 39.
10023711 Full analysis set includes all patients who received TIL therapy
infusion within
specifications.
10023721 Efficacy-evaluable set includes all patients who received TIL therapy
within specifications
and had >1 efficacy evaluation.
10023731 Translational set includes all patients who received TIL therapy
infusion and had TIL
available from the final drug-product for translational analysis.
10023741 TABLE 57. Baseline Patient Characteristics (FAS)
COM-202 Cohort 3B
Characteristic (N=28)
Sex, n (%)
Male 14 (50.0)
Female 14 (50.0)
Median (min, max) age, y 61.0 (40, 74)
Smoker (current or former), n (%) 24 (85.7)
Histologic cell type, n (%)
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Adenocarcinoma 22 (78.6)
Squamous 5 (17.9)
Other 1(3.6)
Tumor PD-Li expression, n (%)*
TPS <1% 4 (14.3)
TPS 1%-49% 10 (35.7)
TPS >50% 8(28.6)
Median (min, max) number of target and
4.5 (2, 11)
non-target lesions
Median (min, max) target
79.0 (22, 179)
lesion sum of diameters, mm
Prior brain metastases, n (%) 10 (35.7)
Median (min, max) number of prior
2.0 (1, 6)
systemic therapies
Prior systemic therapies, n (%)
Anti¨PD-1 and/or anti¨PD-Li 28 (100)
Chemotherapy 27 (96.4)
Anti¨PD-1 23 (82.1)
Anti¨PD-Li 7 (25.0)
Anti¨VEGF 6 (21.4)
Anti¨CTLA-4 6 (21.4)
EGFR inhibitor 1 (3.6)
Tyrosine kinase inhibitor 1 (3.6)
Other 3 (10.7)
10023751 Per central laboratory from tumor harvest specimen; tumor PD-Li
expression data were
missing for 6 patients.
10023761 All patients received prior ICI.
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10023771 TILs were most commonly harvested from lung metastases (60.7%).
10023781 TABLE 58. Treatment-Emergent Adverse Events* (>30%, FAS)
COM-202 Cohort 3B (N=28)
TEAE, n CYO Any Grade Grade 3/4
Grade 5
Any event 28 (100) 27 (96.4) 2 (7.1)*
Thrombocytopenia 20 (71.4) 19 (67.9) 0
Anemia 19 (67.9) 14 (50.0) 0
Hypotension 17 (60.7) 7 (25.0) 0
Chills 16 (57.1) 1(3.6) 0
Pyrexia 16 (57.1) 1(3.6) 0
Hypoxia 13 (46.4) 5 (17.9) 0
Diarrhea 10 (35.7) 3(10.7) 0
Neutropeniat 10 (35.7) 6(21.4) 0
Peripheral edema 10 (35.7) 0 0
Alopecia 9(32.1) 0 0
Decreased appetite 9 (32.1) 3 (10.7) 0
Dyspnea 9 (32.1) 3 (10.7) 0
Fatigue 9(32.1) 4(14.3) 0
10023791 TEAEs include AEs that occurred from the time of TIL infusion, up to
30 days after TIL
infusion or start of a new anticancer therapy.
10023801 Only laboratory abnormalities considered clinically significant were
reported as AEs.
10023811 One Grade 5 event each was reported for chronic cardiac failure and
multiple organ
dysfunction syndrome.
10023821 Safety was consistent with the underlying advanced disease and known
safety profiles of
NMA-LD and IL-2.
10023831Any-grade tumor harvest-related AEs were reported for 16 (41.0%)
patients, most
commonly procedural pain, n=7 (17.9%) and hypoxia, n=4 (10.3%).
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10023841Majority of tumor harvest-related AEs were Grade 1 or 2.
10023851 Adverse Events Over Time (FAS) are depicted in Fig. 40.
10023861Most AEs occurred prior to or within the first 2 weeks after TIL
infusion.
10023871Median number of IL-2 doses: 5.5.
10023881 TABLE 59a. Efficacy
COM-202 Cohort 3B (N=28)
Response n/N % (95% CI)
Full-Analysis Set (FAS)
ORR 6/28 21.4 (8.3, 41.0)
CR 1/28 3.6
PR 5/28 17.9
SD 12/28 42.9
PD 6/28 21.4
DCR 18/28 64.3 (44.1, 81.4)
NE* 4/28 14.3
Efficacy-Evaluable Set
ORR 6/24 25.0 (9.8, 46.7)
DCR 18/24 75.0 (53.3, 90.2)
10023891*Excluded from efficacy-eyaluable set due to death prior to first
assessment.
10023901 TABLE 59b. Efficacy
Median
Duration, months (95% CI) MM, Max
Study follow-up
9.8 (5.8, 14.5) 0.1+, 22.1
(FAS)
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10023911 ORR: 21.4% in the FAS; 25.0% in the efficacy-evaluable set.
10023921All responders received >2 prior lines of systemic therapy.
10023931Median number of TIL infused was 20.9 x 109. Median time from
resection to infusion was
35.0 days. Median time from infusion to BOR was 2.2 months.
10023941 Best percentage change from baseline in target lesion sum of
diameters (efficacy-evaluable
set) data are depicted in Fig. 41. For patient 2, the overall response of CR
was based on investigator
assessment of a complete metabolic response via negative FDG-PET scan.
10023951Time to first response, duration of response, and time on efficacy
assessment for confirmed
responders who achieved PR or better are depicted in Fig. 42. Per central
laboratory from tumor
harvest specimen, except for patient 2, who had TPS assessed locally using
archival tumor sample.
Patient 25 had PD due to new lesion; patients 26 and 22 had unequivocal PD of
non-target disease.
10023961 One patient had a complete metabolic response, ongoing at 20.7
months.
100239712 responses, including the CR, occurred in patients with TPS <1%.
10023981 Percentage change from baseline in target lesion sum of diameters
(FAS) data are depicted
in Fig. 43.
10023991The overall response of CR is based on a negative FDG-PET scan.
10024001 TABLE 60. TIL TCR Repertoire Analyses
COM-202 Cohort 3B, Translational Set
(N=27)
TIL Product Parameter Median*
Min, Max
Unique TCR clones 4396
865, 17,317
Shannon Entropy Index (TCR clone diversity) 7.18
3.55, 11.66
Simpson Clonality Index (TCR clonality): 0.20
0.02, 0.60
10024011 Comparison with prior published datasets7,8 using the limit of
detection applied to this
dataset. Unique TCR clones: 5596 for melanoma and 6874 for cervical. Shannon
Entropy Index: 7.60
for melanoma and 7.11 for cervical. Simpson Clonality Index: 0.18 for melanoma
and 0.20 for
cervical.
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[0024021A larger Shannon Entropy Index indicates a more diverse CDR3
population. Values can
range from 0 (monoclonal sample) to 10g2(R) (evenly distributed, polyclonal
sample with R unique
clones).
10024031 Simpson Clonality Index reflects mono- or poly-clonality of a sample
and is inversely
related to diversity (Shannon Entropy Index). Values can range from 0 (evenly
distributed, polyclonal
sample) to 1 (monoclonal sample).
1002404127 patients had TIL available from the final drug-product for TCR
repertoire analysis;
analyses of correlation with clinical outcome are ongoing.
Conclusions
10024051 This signal-finding study demonstrated the feasibility of tumor
harvest. TIL manufacturing,
and TIL treatment in patients with advanced NSCLC.
10024061 Patients tolerated surgical resection, including pulmonary lesions.
TIL manufacturing was
feasible for most patients. One-time TIL treatment with conditioning regimen
was well-tolerated.
10024071 The TCR repertoire of TILs generated from NSCLC tumors demonstrated a
similar number
of unique TCR clones, as well as measures of diversity and clonality, as
previously published for
lifileucel for melanoma7 and TIL therapy for cervical cancer'.
10024081 Despite multiple prior lines of therapy, 6 patients experienced
responses, including 2 with
durable responses, consistent with published experience including durable CRs
extending beyond 1
vear6.
Abbreviations
AE, adverse event; BOR, best overall response; CR, complete response; CTLA-4,
cytotoxic T
lymphocyte antigen-4; CY, cyclophosphamide; ECOG, Eastern Cooperative Oncology
Group; EGFR,
epidermal growth factor receptor; EOA, end of assessment; EOS, end of study;
EOT, end of
treatment; FAS, full-analysis set; FDG-PET, fluorodeoxyglucose -positron
emission tomography;
FLU, fludarabine; GMP, good manufacturing practices; ICI, immune checkpoint
inhibitors; IL-2,
interleukin-2; NA, not assessed; ND, none detected; NMA-LD, nonmyeloablative
lymphodepletion;
NSCLC, non-small cell lung cancer; ORR, objective response rate; PD,
progressive disease; PR,
partial response; Pt, patient; PD-1, programmed cell death protein-1; PD-L1,
programmed death
ligand-1; RECIST, Response Evaluation Criteria in Solid Tumors; SD, stable
disease; TCR, T-cell
receptor; TEAEs, treatment-emergent adverse events; TIL, tumor-infiltrating
lymphocytes; TPS,
tumor proportion score.
References
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1. Horvath L, etal. Molecular Cancer (2020) 19:141.
2. Samaik AA, et al. J Clin Oncol (2021); doi: 10.1200/JC0.21.00612.
3. Thomas SS, et al. J Clin Oncol (2021); 3 9 (suppl; abstract 9537).
4. Jazaeri A, etal. J Clin Oncol (2019);37 (suppl; abstract 2538).
5. Jimeno A, et al. J Immunother Cancer (2020);8 (suppl; abstract A378).
6. Creelan BC, etal. Nature Med (2021): doi: 10.1038/s41591-021-01462-y.
7. Gontcharova V. et al. Cancer Research (2019); 79:13 (suppl; abstract 14).
8. Jazaeri A, etal. Annals Oncol (2020);31:S642 (suppl; abstract 3688).
10024091 The examples set forth above are provided to give those of ordinary
skill in the art a
complete disclosure and description of how to make and use the embodiments of
the compositions,
systems and methods of the invention, and are not intended to limit the scope
of what the inventors
regard as their invention. Modifications of the above-described modes for
carrying out the invention
that are obvious to persons of skill in the art are intended to be within the
scope of the following
claims. All patents and publications mentioned in the specification are
indicative of the levels of skill
of those skilled in the art to which the invention pertains.
10024101 All headings and section designations are used for clarity and
reference purposes only and
are not to be considered limiting in any way. For example, those of skill in
the art will appreciate the
usefulness of combining various aspects from different headings and sections
as appropriate
according to the spirit and scope of the invention described herein.
[0024111AR references cited herein are hereby incorporated by reference herein
in their entireties
and for all purposes to the same extent as if each individual publication or
patent or patent application
was specifically and individually indicated to be incorporated by reference in
its entirety for all
purposes.
002412J Many modifications and variations of this application can be made
without departing from
its spirit and scope, as will be apparent to those skilled in the art. The
specific embodiments and
examples described herein are offered by way of example only, and the
application is to be limited
only by the terms of the appended claims, along with the full scope of
equivalents to which the claims
are entitled.
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