Note: Descriptions are shown in the official language in which they were submitted.
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EXPANSION OF PERIPHERAL BLOOD LYMPHOCYTES (PBLS) FROM PERIPHERAL BLOOD
CROSS REFERENCE TO RELATED APPLICATIONS
[0001] This application is a continuation-in-part of International Patent
Application No.
PCT/U518/032109 filed May 10, 2018, which claims priority to U.S. Provisional
Application
No. 62/504,337, filed May 10, 2017; U.S. Provisional Application No.
62/530,681, filed July 10,
2017; U.S. Provisional Application No. 62/550,398, filed August 25, 2017; U.S.
Provisional
Application No. 62/590,034, filed November 22, 2017; U.S. Provisional
Application No.
62/621,462, filed January 24, 2018; U.S. Provisional Application No.
62/621,798, filed January
25, 2018; and U.S. Provisional Application No. 62/647,367, filed March 23,
2018, all of which
are herein incorporated by reference in their entireties.
FIELD OF THE INVENTION
[0002] Methods of expanding tumor infiltrating lymphocytes (TILs) derived
from blood
and/or bone marrow of a patient with a hematological malignancy, such as a
liquid tumor,
including lymphomas and leukemias, and compositions comprising populations of
TILs obtained
therefrom, are disclosed herein. In addition, therapeutic uses of TILs
expanded from blood or
bone marrow of a patient with a hematological malignancy, such as a liquid
tumor, including in
the treatment of such hematological malignancies, are disclosed herein.
BACKGROUND OF THE INVENTION
[0003] Treatment of bulky, refractory cancers using adoptive autologous
transfer of tumor
infiltrating lymphocytes (TILs) represents a powerful approach to therapy for
patients with poor
prognoses. Gattinoni, et al., Nat. Rev. Immunol. 2006, 6, 383-393. TILs are
dominated by T
cells, and IL-2-based TIL expansion followed by a "rapid expansion process"
(REP) has become
a preferred method for TIL expansion because of its speed and efficiency.
Dudley, et at.,
Science 2002, 298, 850-54; Dudley, et at., I Cl/n. Oncol. 2005, 23, 2346-57;
Dudley, et at.,
Cl/n. Oncol. 2008, 26, 5233-39; Riddell, et al., Science 1992, 257, 238-41;
Dudley, et al.,
Immunother. 2003, 26, 332-42. A number of approaches to improve responses to
TIL therapy in
melanoma and to expand TIL therapy to other tumor types have been explored
with limited
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success, and the field remains challenging. Goff, et al., I Cl/n. Oncol. 2016,
34, 2389-97;
Dudley, et al., I Cl/n. Oncol. 2008, 26, 5233-39; Rosenberg, et al., Cl/n.
Cancer Res. 2011, /7,
4550-57. Earlier approaches to expansions of TILs from B cell lymphomas
yielded poor results,
with only 2 of 12 attempts at TIL growth providing for potential activity
against tumors.
Schwartzentruber, et al., Blood 1993, 82, 1204-1211. There is an urgent need
to provide for
more efficacious therapies in many hematological malignancies, including acute
myeloid
leukemia (AML) and chronic lymphocytic leukemia (CLL).
[0004] The present invention provides the surprising finding that TIL
expansion processes
can result in efficacious TIL populations obtained from hematological
malignancies, such as
liquid tumors, including lymphomas or leukemias.
SUMMARY OF THE INVENTION
[0005] In an embodiment, the invention provides a method of treating a
cancer in a patient
with a population of tumor infiltrating lymphocytes (TILs) comprising the
steps of:
(a) optionally pre-treating the patient with a regimen comprising at least one
kinase inhibitor;
(b) obtaining a tumor from the patient by resection, biopsy, needle
aspiration, or apheresis,
the tumor comprising a first population of TILs;
(c) optionally fragmenting or dissociating the tumor to obtain tumor fragments
and
contacting the tumor or tumor fragments with a first cell culture medium;
(d) performing an initial 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, and wherein the initial expansion is performed
over a
period of 21 days or less;
(e) performing a second expansion of the second population of TILs in a second
cell culture
medium to obtain a third population of TILs, wherein the third population of
TILs is at
least 50-fold greater in number than the second population of TILs after 7
days from the
start of the second expansion; wherein the second cell culture medium
comprises IL-2,
OKT-3 (anti-CD3 antibody), and irradiated allogeneic peripheral blood
mononuclear
cells (PBMCs), and wherein the second expansion is performed over a period of
14 days
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or less;
(f) harvesting the third population of TILs; and
(g) administering a therapeutically effective portion of the third population
of TILs to the
patient;
wherein the tumor is a liquid tumor, and wherein the cancer is a hematological
malignancy.
[0006] In an embodiment, the invention provides a method of treating a
cancer in a patient
with a population of tumor infiltrating lymphocytes (TILs) comprising the
steps of:
(a) optionally pre-treating the patient with a regimen comprising at least one
kinase inhibitor;
(b) obtaining a tumor from the patient by resection, biopsy, needle
aspiration, or apheresis,
the tumor comprising a first population of TILs;
(c) optionally fragmenting or dissociating the tumor to obtain tumor fragments
and
contacting the tumor or tumor fragments with a first cell culture medium;
(d) performing an initial 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, and wherein the initial expansion is performed
over a
period of 21 days or less;
(e) performing a second expansion of the second population of TILs in a second
cell culture
medium to obtain a third population of TILs, wherein the third population of
TILs is at
least 50-fold greater in number than the second population of TILs after 7
days from the
start of the second expansion; wherein the second cell culture medium
comprises IL-2,
OKT-3 (anti-CD3 antibody), and irradiated allogeneic peripheral blood
mononuclear
cells (PBMCs), and wherein the second expansion is performed over a period of
14 days
or less;
(f) harvesting the third population of TILs; and
(g) administering a therapeutically effective portion of the third population
of TILs to the
patient;
wherein the tumor is a liquid tumor, and wherein the cancer is a hematological
malignancy
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selected from the group consisting of acute myeloid leukemia (AML), mantle
cell lymphoma
(MCL), follicular lymphoma (FL), diffuse large B cell lymphoma (DLBCL),
activated B cell
(ABC) DLBCL, germinal center B cell (GCB) DLBCL, chronic lymphocytic leukemia
(CLL), small lymphocytic leukemia (SLL), non-Hodgkin's lymphoma (NHL),
Hodgkin's
lymphoma, relapsed and/or refractory Hodgkin's lymphoma, B cell acute
lymphoblastic
leukemia (B-ALL), mature B-ALL, Burkitt's lymphoma, Waldenstrom's
macroglobulinemia
(WM), multiple myeloma, myelodysplatic syndromes, myelofibrosis, chronic
myelocytic
leukemia, follicle center lymphoma, indolent NHL, human immunodeficiency virus
(HIV)
associated B cell lymphoma, and Epstein¨Barr virus (EBV) associated B cell
lymphoma.
[0007] In an embodiment of the invention, the method further comprises
addition of an ITK
inhibitor. In an embodiment, the ITK inhibitor is added to the cell culture
medium during at
least one of steps (d) and (e). In an embodiment of the invention, the ITK
inhibitor is a covalent
ITK inhibitor that covalently and irreversibly binds to ITK. In an embodiment
of the invention,
the ITK inhibitor is an allosteric ITK inhibitor that binds to ITK. In another
embodiment, the
ITK inhibitor is selected from the group consisting of aminothiazole-based ITK
inhibitors,
benzimidazole-based ITK inhibitors, aminopyrimidine-based ITK inhibitors, 3-
aminopyride-2-
ones-based ITK inhibitors, indolylndazole-based ITK inhibitors, pyrazolyl-
indole-based
inhibitors, thienopyrazole inhibitors, and ITK inhibitors targeting cysteine-
442 in the ATP
pocket. In another embodiment, ITK inhibitor is the ITK inhibitor is
ibrutinib, dasatinib,
bosutinib, nilotinib, erlotinib BMS509744, CTA056, GSK2250665A, PF06465469
((R)-3-(1-(1-
acryloylpiperidin-3-y1)-4-amino-1H-pyrazolo[3,4-d]pyrimidin-3-y1)-N-(3-methyl-
4-(1-
methylethyl))benzamide), and combinations thereof In another embodiment, the
ITK inhibitor
is ibrutinib, also known as 1-[(3R)-344-amino-3-(4-phenoxypheny1)-1H-
pyrazolo[3,4-
d]pyrimidin-1-y1]-1-piperidiny1]-2-propen-1-one. In another embodiment, the
ITK inhibitor is
(R)-3-(1-(1-acryloylpiperidin-3-y1)-4-amino-1H-pyrazolo[3,4-d]pyrimidin-3-y1)-
N-(3-methy1-4-
(1-methylethyl))benzamide. The foregoing ITK inhibitors are available
commercially from
various sources, including Tocris Bioscience, Inc. (Minneapolis, MN, USA),
Selleckchem, Inc.
(Houston, TX, USA), and AK Scientific, Inc. (Union City, CA, USA). In another
embodiment,
the ITK inhibitor is added at a concentration of from about 0.1 nM to about 5
M. In another
embodiment, the ITK inhibitor is added at a concentration of from about 0.1 nM
to about 5 M.
In another embodiment, the ITK inhibitor is added at a concentration of from
about 0.1 nM to
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about 100 nM. In another embodiment, the ITK inhibitor is added at a
concentration of from
about 0.5 nM to about 50 nM. In another embodiment, the ITK inhibitor is added
at a
concentration of from about 1 nM to about 10 nM. In an embodiment, the ITK
inhibitor is added
at a concentration of about 0.01 nM, 0.05 nM, 0.1 nM, 0.5 nM, 1 nM, 2 nM, 5
nM, 10 nM, 20
nM, 30 nM, 40 nM, 50 nM, 60 nM, 70 nM, 80 nM, 90 nM, 100 nM, 150 nM, 200 nM,
300 nM,
400 nM, 500 nM, 600 nM, 700 nM, 800 nM, 900 nM, 1 [tM, 2 [tM, 3 [tM, 4 [tM, 5
[tM, 10 [tM,
20 M, 30 [tM, 40 [tM, and 50 M.
[0008] In an embodiment of the invention, a method for expanding peripheral
blood
lymphocytes (PBLs) from peripheral blood comprises:
a. Obtaining a sample of peripheral blood mononuclear cells (PBMCs) from
peripheral blood, wherein said sample is optionally cryopreserved;
b. Isolating PBLs from said sample by selecting and removing CD19+ B cells;
c. Optionally co-culturing said PBLs with said CD19+ B cells;
d. Stimulating said PBLs in a first cell culture medium with IL-2 and anti-
CD3/anti-
CD28 antibodies for a period of from about 2 days to about 6 days in a gas
permeable container;
e. Culturing the PBLs from step (d) for a period of from about 2 days to
about 6
days with IL-2 and anti-CD3/anti-CD28 antibodies;
f. Isolating the antibody-bound PBLs from the culture in step (e);
g. Removing the antibodies from the PBLs isolated in step (e); and
h. Harvesting the PBLs.
[0009] In an embodiment of the invention, the method further comprises
addition of IL-2
after step (d), and exchanging the first culture medium to a second cell
culture medium. In
another embodiment, the method further comprises addition of IL-2 after step
(e), and
exchanging the second culture medium to a third culture medium. In an
embodiment, the first
cell culture medium, second cell culture medium, or third culture medium is
selected from the
group consisting of CM-2, CM-4, and AIM-V. In another embodiment, the first
and second cell
culture media are the same. In another embodiment, the first and second cell
culture media are
different. In an embodiment of the invention, one or more of the first, second
and third cell
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culture media are the same. In another embodiment, the first, second, and
third cell culture
media are all different.
[0010] In an embodiment, the optional co-culturing of said PBLs with said
CD19+ B cells is
performed for a period of 1 hour to 3 days.
[0011] In an embodiment of the invention, the ratio of T-cells to B-cells
in step (c) is from
about 0.1:1 to about 10:1 (B-cells:T-cells). In another embodiment, the ratio
of B-cells to T-cells
in step (c) is selected from the group consisting of 0.1:1, 1:1, and 10:1 (B-
cells:T-cells).
[0012] In an embodiment of the invention, the starting cell number of PBLs
at the beginning
of step (d) is at least from about lx105 to about 10x105 PBLs. In another
embodiment, the
starting cell number of PBLs at the beginning of step (d) is at least from
about 2.5x105 to 10x105
PBLs. In another embodiment, the starting cell number of PBLs at the beginning
of step (d) is at
least 5x105 PBLs.
[0013] In an embodiment of the invention, the IL-2 in each of steps (c) and
(d) is used at a
concentration of from about 1000 IU/mL to about 6000 IU/mL. In another
embodiment, the IL-2
in each of steps (c) and (d) is used at a concentration of about 3000 IU/mL.
[0014] In an embodiment of the invention, the anti-CD3/anti-CD28 antibodies
are coated
onto beads. In an embodiment of the invention, the anti-CD3/anti-CD28
antibodies-coated beads
are DynaBeads . In an embodiment, the method includes co-culturing the anti-
CD3/anti-CD28
antibody beads with the PBLs in about a 1:1 bead:PBL ratio in each of steps
(c) and (d).
[0015] In an embodiment of the invention, the method comprises adding an
ITK inhibitor. In
an embodiment, the ITK inhibitor is added during at least one of steps (c),
(d), and (e). In an
embodiment of the invention, the ITK inhibitor is selected from the group
consisting of
aminothiazole-based ITK inhibitors, benzimidazole-based ITK inhibitors,
aminopyrimidine-
based ITK inhibitors, 3-aminopyride-2-ones-based ITK inhibitors,
indolylndazole-based ITK
inhibitors, pyrazolyl-indole-based inhibitors, thienopyrazole inhibitors, and
ITK inhibitors
targeting cysteine-442 in the ATP pocket. In another embodiment, the ITK
inhibitor is ibrutinib,
dasatinib, bosutinib, nilotinib, erlotinib BMS509744, CTA056, GSK2250665A,
PF06465469,
and combinations thereof. In another embodiment, the ITK inhibitor is
ibrutinib.
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[0016] In an embodiment of the invention, any of the foregoing methods for
preparing PBLs
is performed in a closed, sterile system.
[0017] In an embodiment of the present invention, a method for expanding
peripheral blood
lymphocytes (PBLs) from peripheral blood comprises:
a. Obtaining a sample of peripheral blood mononuclear cells (PBMCs) from
peripheral blood, wherein said sample is optionally cryopreserved;
b. Isolating PBLs from said sample by selecting and removing CD19+ B cells;
c. Co-culturing said PBLs with said CD19+ B cells for a period of 4 days;
d. Adding from about 2.5x105 to about 5x105 PBLs to a gas-permeable container
in
CM-2 cell culture medium and stimulating said PBLs with 3000 IU/ml IL-2 and
anti-
CD3/anti-CD28 antibodies immobilized on beads for a period of about 4 days;
e. Exchanging the CM-2 cell culture medium with AIM-V cell culture medium and
additional IL-2 at about 3000 IU/ml;
f. Culturing the PBLs in the AIM-V cell culture medium from step (e) for an
additional period of about 3 days with IL-2 and anti-CD3/anti-CD28 antibodies
immobilized on beads;
g. Isolating the antibody-bound PBLs from the culture in step (f);
h. Removing the antibodies from the PBLs isolated in step (g); and
i. Harvesting the PBLs.
[0018] In an embodiment of the invention, a method for treating a
hematological malignancy
comprises:
a. Obtaining a sample of peripheral blood mononuclear cells (PBMCs) from
peripheral blood of a patient suffering from a hematological malignancy;
b. Isolating PBLs from said sample by selecting and removing CD19+ B cells;
c. Optionally co-culturing said PBLs with said CD19+ B cells;
d. Stimulating said PBLs in a first cell culture medium with IL-2 and anti-
CD3/anti-
CD28 antibodies for a period of at least about 4 days in a gas permeable
container;
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e. Culturing the PBLs from step (d) for a period of 3 days with IL-2 and
anti-
CD3/anti-CD28 antibodies;
f. Isolating the antibody-bound PBLs from the culture in step (e);
g. Removing the antibodies from the PBLs isolated in step (e); and
h. Harvesting the PBLs; and
i. Administering the PBLs to the patient in a therapeutically effective
amount to
treat said hematological malignancy.
[0019] In an embodiment of the invention, the method further comprises
obtaining a PBMC
sample from a patient that is pre-treated with an ITK inhibitor. In an
embodiment of the
invention, the ITK inhibitor is selected from the group consisting of
aminothiazole-based ITK
inhibitors, benzimidazole-based ITK inhibitors, aminopyrimidine-based ITK
inhibitors, 3-
aminopyride-2-ones-based ITK inhibitors, indolylndazole-based ITK inhibitors,
pyrazolyl-
indole-based inhibitors, thienopyrazole inhibitors, and ITK inhibitors
targeting cysteine-442 in
the ATP pocket. In an embodiment of the invention, the ITK inhibitor is
ibrutinib, BMS509744,
CTA056, GSK2250665A, PF06465469, and combinations thereof In another
embodiment, the
ITK inhibitor is ibrutinib. In another embodiment, the patient is pre-treated
with at least three
rounds of an ibrutinib regimen.
[0020] In an embodiment of the invention, the hematological malignancy is
selected from the
group consisting of acute myeloid leukemia (AML), mantle cell lymphoma (MCL),
follicular
lymphoma (FL), diffuse large B cell lymphoma (DLBCL), activated B cell (ABC)
DLBCL,
germinal center B cell (GCB) DLBCL, chronic lymphocytic leukemia (CLL), CLL
with
Richter's transformation (Richter's syndrome), small lymphocytic leukemia
(SLL), non-
Hodgkin's lymphoma (NHL), Hodgkin's lymphoma, relapsed and/or refractory
Hodgkin's
lymphoma, B cell acute lymphoblastic leukemia (B-ALL), mature B-ALL, Burkitt's
lymphoma,
Waldenstrom's macroglobulinemia (WM), multiple myeloma, myelodysplatic
syndromes,
myelofibrosis, chronic myelocytic leukemia, follicle center lymphoma, indolent
NHL, human
immunodeficiency virus (HIV) associated B cell lymphoma, and Epstein¨Barr
virus (EBV)
associated B cell lymphoma. In another embodiment, the hematological
malignancy is chronic
lymphocytic leukemia (CLL). In an embodiment of the invention, the PBLs are
administered in
an amount of from about 0.1x109 to about 15x109 PBLs.
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[0021] In an embodiment of the invention, a method for expanding marrow-
infiltrating
lymphocytes (MILs) from bone marrow comprises:
a. Obtaining a sample of peripheral blood mononuclear cells (PBMCs) from
bone
marrow, wherein said sample is optionally cryopreserved;
b. Sorting a CD3+, CD33+, CD20+ and CD14+ cell fraction comprising MILs (MIL
cell fraction) and a non-CD3+, non-CD33+, non-CD20+, non-CD14+ cell fraction
(AML blast cell fraction) from the sample of PBMCs;
c. Optionally disrupting the AML blast cell fraction;
d. Adding the optionally disrupted AML blast cell fraction to the MIL cell
fraction
in a cell number ratio of from about 0.1:1 to about 10:1
e. Culturing the MIL cell fraction, with or without the AML blast cell
fraction, in a
gas permeable container in a first cell culture medium, comprising IL-2;
f. Stimulating the MILs with anti-CD3/anti-CD28 antibodies to obtain
expansion of
MILs;
g. Restimulating the MILs with IL-2 and anti-CD3/anti-CD28 antibodies for an
additional period of about 2 to about 6 days;
h. Culturing the MILs with additional IL-2 for an additional period of from
about 1
to about 3 days; and
i. Harvesting said MILs.
[0022] In an embodiment of the invention, the method further comprises
addition IL-2 after
step (e), and exchanging the first cell culture medium to a second cell
culture medium. In an
embodiment, the first cell culture medium and the second cell culture medium
are each
individually selected from the group consisting of CM-2, CM-4, and AIM-V. In
another
embodiment, the first and second cell culture media are the same. In another
embodiment, the
first and second cell culture media are different.
[0023] In an embodiment, there are at least from about 2x104 to about 5x105
MILs in the gas
permeable container at the beginning of step (e). In another embodiment, there
are at least from
about 2.8x104 to 3.4x105 MILs in the gas permeable container at the beginning
of step (e). In
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another embodiment, there are at least 5x105 MILs in the gas permeable
container at the
beginning of step (e).
[0024] In an embodiment of the invention, the IL-2 is present in a
concentration of between
1000 IU/ml and 6000 IL/ml in step (e). In another embodiment, the IL-2 is
present in a
concentration of about 6000 IU/ml. In another embodiment, the IL-2 is present
in a
concentration of about 3000 IU/ml in step (g). In another embodiment, the IL-2
is present in a
concentration of about 3000 IU/ml in step (h).
[0025] In an embodiment of the invention, the culturing in step (e) is
performed over a
period of about 3 days. In an embodiment, the stimulation in step (f) is
performed over a period
of about 4 days. In an embodiment, the stimulation in step (g) is performed
over a period of
about 7 days.
[0026] In an embodiment of the invention, the optionally disrupted AML
blast cell fraction is
disrupted using a method selected from the group consisting of sonication,
homogenization,
vortexing, vibration, and lysis. In an embodiment of the invention, the non-
CD3+, non-CD33+,
non-CD20+, non-CD14+ cell fraction (AML blast cell fraction) is lysed using a
suitable lysis
method, including high temperature lysis, chemical lysis (such as organic
alcohols), enzyme
lysis, and other cell lysis methods known in the art.
[0027] In an embodiment of the invention, the anti-CD3/anti-CD28 antibodies
are coated
onto beads and the MILs:bead ratio is about 1:1 in each of steps (f) and (g).
[0028] In an embodiment of the invention, the method is performed in a
closed, sterile
system.
[0029] In an embodiment of the invention, a method for expanding marrow
infiltrating
lymphocytes (MILs) from bone marrow comprises:
a. Obtaining a sample of peripheral blood mononuclear cells (PBMCs) from
bone
marrow, wherein said sample is optionally cryopreserved;
b. Sorting a CD3+, CD33+, CD20+ and CD14+ cell fraction comprising MILs (MIL
cell fraction) and a non-CD3+, non-CD33+, non-CD20+, non-CD14+ cell fraction
(AML blast cell fraction) from the sample of PBMCs;
c. Disrupting the AML blast cell fraction and adding the disrupted AML blast
cell
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fraction to a MIL cell fraction in a cell number ratio of about 1:1;
d. Culturing the MILs and AML blast cell fractions in a cell culture contained
in a gas
permeable container with a first cell culture medium comprising IL-2 at about
6000
IU/ml for a period of about 3 days;
e. Adding anti-CD3/anti-CD28 antibodies immobilized on beads to the cell
culture
at a ratio of about 1:1 (MILs:beads) and culturing the MILs and antibodies for
a
period of about 1 day;
f. Exchanging the first cell culture medium to a second cell culture medium
comprising additional IL-2 at about 3000 IU/ml;
g. Culturing the antibodies and MILs for an additional period of about 3
days;
h. Restimulating the MILs with IL-2 and anti-CD3/anti-CD28 antibodies
immobilized on beads for an additional period of at least about 4 days;
i. Exchanging the second cell culture medium to a third cell culture medium
comprising additional IL-2 at about 3000 IU/ml and culturing for an additional
period
of at least about 3 days;
j. Harvesting said MILs.
[0030] In an embodiment of the invention, a method for treating a
hematological malignancy
in a patient comprises:
a. Obtaining a sample of peripheral blood mononuclear cells (PBMCs) from
bone
marrow of the patient, wherein said sample is optionally cryopreserved;
b. Sorting a CD3+, CD33+, CD20+ and CD14+ cell fraction comprising MILs (MIL
cell fraction) and a non-CD3+, non-CD33+, non-CD20+, non-CD14+ cell fraction
(AML blast cell fraction) from the sample of PBMCs;
c. optionally disrupting the AML blast cell fraction;
d. Adding the optionally disrupted AML blast cell fraction to the MIL cell
fraction
in a cell number ratio of from about 0.1:1 to about 10:1;
e. Culturing the MIL cell fraction, with or without the AML blast cell
fraction, in a
gas permeable container in a first cell culture medium comprising IL-2;
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f. Stimulating the MILs with anti-CD3/anti-CD28 antibodies;
g. Restimulating the MILs with IL-2 and anti-CD3/anti-CD28 antibodies for an
additional period of at least about 4 days;
h. Culturing the MILs with additional IL-2 for an additional period of at
least about
3 days;
i. Harvesting said MILs; and
j. Administering said MILs to the patient in a therapeutically effective
amount to
treat the hematological malignancy.
[0031] In an embodiment of the invention, the hematological malignancy is
selected from the
group consisting of acute myeloid leukemia (AML), mantle cell lymphoma (MCL),
follicular
lymphoma (FL), diffuse large B cell lymphoma (DLBCL), activated B cell (ABC)
DLBCL,
germinal center B cell (GCB) DLBCL, chronic lymphocytic leukemia (CLL), CLL
with
Richter's transformation (Richter's syndrome), small lymphocytic leukemia
(SLL), non-
Hodgkin's lymphoma (NHL), Hodgkin's lymphoma, relapsed and/or refractory
Hodgkin's
lymphoma, B cell acute lymphoblastic leukemia (B-ALL), mature B-ALL, Burkitt's
lymphoma,
Waldenstrom's macroglobulinemia (WM), multiple myeloma, myelodysplatic
syndromes,
myelofibrosis, chronic myelocytic leukemia, follicle center lymphoma, indolent
NHL, human
immunodeficiency virus (HIV) associated B cell lymphoma, and Epstein¨Barr
virus (EBV)
associated B cell lymphoma. In another embodiment, the hematological
malignancy is acute
myeloid leukemia (AML). In an embodiment of the invention, the MILs are
administered in an
amount of from about 4x108 to about 2.5x109 MILs.
BRIEF DESCRIPTION OF THE DRAWINGS
[0032] The foregoing summary, as well as the following detailed description
of the
invention, will be better understood when read in conjunction with the
appended drawings.
[0033] FIG. 1 illustrates pathology information for lymphoma tumors.
[0034] FIG. 2 illustrates a comparison of different subsets of lymphoma and
melanoma TILs,
showing that effector memory (EM) subsets in lymphoma TILs are significantly
higher than EM
subsets in melanoma TILs.
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[0035] FIG. 3 illustrates a comparison of different subsets of lymphoma and
melanoma TILs,
showing that CD28+CD4+ subsets in lymphoma TIL are significantly higher than
these subsets in
melanoma TILs.
[0036] FIG. 4 illustrates a comparison of CD4+ T cell subsets of non-
Hodgkin's lymphoma
TILs and melanoma TILs, showing differentiation markers. Red lines in the
graphs represent
median values. CM refers to central memory T cells, EM refers to effector
memory T cells, and
TEMRA refers to effector memory CD45RA+ T cells.
[0037] FIG. 5 illustrates a comparison of CD8+ T cell subsets of non-
Hodgkin's lymphoma
TILs and melanoma TILs, showing differentiation markers. Red lines in the
graphs represent
median values. CM refers to central memory T cells, EM refers to effector
memory T cells, and
TEMRA refers to effector memory CD45RA+ T cells.
[0038] FIG. 6 illustrates a comparison of CD4+ T cell subsets of non-
Hodgkin's lymphoma
TILs and melanoma TILs, showing exhaustion markers. Red lines in the graphs
represent
median values. LAG3 refers to lymphocyte-activation gene 3, PD1 refers to
programmed death
1, and TIGIT refers to T cell immunoreceptor with Ig and ITIM domains.
[0039] FIG. 7 illustrates a comparison of CD8+ T cell subsets of non-
Hodgkin's lymphoma
TILs and melanoma TILs, showing exhaustion markers. Red lines in the graphs
represent
median values. LAG3 refers to lymphocyte-activation gene 3, PD1 refers to
programmed death
1, and TIGIT refers to T cell immunoreceptor with Ig and ITIM domains.
[0040] FIG. 8 illustrates a comparison of cell types between non-Hodgkin's
lymphoma TILs
and melanoma TILs. NK refers to natural killer cells, and TCRab refers to
cells expressing a T
cell receptor with alpha and beta chains.
[0041] FIG. 9 illustrates bioluminescent redirected lysis assay (BRLA)
results.
[0042] FIG. 10 illustrates interferon-y (IFN- y) enzyme-linked
immunosorbent assay
(ELISA) results for lymphoma TILs versus melanoma TILs.
[0043] FIG. 11 illustrates enzyme-linked immunospot (ELIspot) assay results
for lymphoma
TILs.
[0044] FIG. 12 illustrates ELIspot assay results for melanoma TILs.
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[0045] FIG. 13 illustrates the results of NANOSTRING NCOUNTER analysis,
showing that
lymphoma TILs express higher levels of RORC IL17A (TH17 phenotype) and GATA3
(Th2
phenotype) compared to melanoma TILs. Respective genes are highlighted in red
boxes in the
heat map.
[0046] FIG. 14 illustrates a TIL expansion and treatment process. Step 1
refers to the
addition of 4 tumor fragments into 10 G-Rex 10 flasks. At step 2,
approximately 40 x 106 TILs
or greater are obtained. At step 3, a split occurs into 36 G-Rex 100 flasks
for REP. TILs are
harvested by centrifugation at step 4. Fresh TIL product is obtained at step 5
after a total process
time of approximate 43 days, at which point TILs may be infused into a
patient.
[0047] FIG. 15 illustrates a treatment protocol for use with TILs obtained
from lymphomas
of the present disclosure. Surgery (and tumor resection) occurs at the start,
and lymphodepletion
chemo refers to non-myeloablative lymphodepletion with chemotherapy as
described elsewhere
herein.
[0048] FIG. 16 demonstrates the results of the flow cytometry analysis
using standard
phenotype panel DF2 as described in Example 4, below. Lymphoma and melanoma
TILs were
stained using standard phenotype panel DF2 as described in Example 4. Data
shown represents
different subpopulations of total CD4 and CD8 T cells in TIL. Figure 16A
demonstrates the
proportion of CD4 and CD8 cells for Naïve T-cell subsets; Figure 16B for
central memory T-cell
subsets (CM); Figure 16C for effector memory T-cell subsets (EM), and Figure
16D for
terminally differentiated effector memory (TEMRA) T-cell subsets. P-values
were calculated
using two-tailed Mann-Whitney Test (unpaired). The mean proportion of cell
subsets is
prepresnted by horizontal bars.
[0049] FIG. 17 demonstrates the results of the flow cytometry analysis
using the standard
phenotype panel DF1 as described in Example 4, below. Lymphoma and melanoma
TIL were
stained using standard phenotype panel DF1, as described in Example 4. Data
shown represents
different CD27+ (FIG. 17A) and CD28+ (FIG. 17B) subpopulations of total CD4
and CD9 T-
cells in TIL, which indicates a higher proportion of costimulatory molecule-
CD28 expressing
CD4 T-cells in lymphoma TIL. P values were calculated using the two-tailed
Mann-Whitney
Test (unpaired).
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[0050] FIG. 18 demonstrates the results of an interferon-gamma (IFN-y) test
conducted in
accordance with Example 4, below. FIG. 18A demonstrates the results using
ELIspot. ELIspot
data is expressed as IFN-y producing cells per 106 TIL. FIG. 18B demonstrates
the results using
ELISA. ELISA data is expressed as IFN-y levels in the supernatants from TIL
cultures at 5x105
TIL/well) as measured by ELISA (logarithmic scale. P values were calculated
using the two-
tailed Mann-Whitney Test (unpaired).
[0051] FIG. 19 demonstrates the lytic potential of TIL. FIG. 19A shows the
LU50 of target
cells normalized to 106 TIL at 4 hours (FIG. 19A) and 24 hours (FIG. 19B) in
co-culture (TIL
effector cells with GFP+P815 target cells).
[0052] FIG. 20 demonstrates the cytolytic activity of different TIL against
allogeneic and
autologous tumor types. FIG. 20A shows the cytolytic activity of melanoma TIL
against
allogeneic 526 target cells. FIG. 20B shows the cytolytic activity of lymphoma
TIL against
autologous tumor cells determined by 7-AAD uptake. The data in FIGS. 20A and
20B FIG. are
shown as percent dead cels in co-cultures with 50:1 effector cell:target cell
(E:T) ratio. FIG. 20C
represents percent killing of target cells induced by melanoma TIL. FIG. 20D
represents percent
killing of target cells induced by lymphoma TIL at different E:T ratios.
[0053] FIG. 21 is a heat map showing the gene expression profiles of
lymphoma and
melanoma TIL. The expression profiles were determined by 579 plex nCounter GX
Human
Immunology V2 CSO panel from NanoString. The heat map shows the fold change in
expression of a particular set of genes in lymphoma TIL compared to melanoma
TIL, and
suggests a higher expression of IL-17A and RORC from lymphoma-derived TIL. The
cancers
shown in this figure include follicular lymphoma (FL), diffuse large B cell
Lymphoma (DLBCL)
and mantle cell lymphoma (MCL).
[0054] FIG. 22 is a schematic demonstrating the 2A process for preparing
TIL, harvest, and
ship schedule.
[0055] FIG. 23 is a flow chart demonstrating the 2A process for preparing
TIL.
[0056] FIG. 24 is a flow chart demonstrating three different methods for
expanding
Peripheral Blood Lymphocytes (PBLs).
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[0057] FIGS. 25A-25C represent three different methods for expanding marrow
infiltrating
lymphocytes (MILs) from bone marrow.
[0058] FIG. 26 represents a graph of the fold expansion for PBLs isolated
from fresh
peripheral blood mononuclear cells (PBMCs) and from cryopreserved PBMCs. The
cryopreserved PBMCs are derived from patients with CLL who have not been
(PreRx PBL) or
who have been (PostRx PBL) treated with an ibrutinib regimen. For each of
FIGS. 26-34, each
dot is one patient. Shaded dots are patients whose PBLs were expanded using
PBL Method 1;
open dots are patients whose PBLs were expanded using PBL Method 2; black dots
are patients
whose PBLs were expanded using PBL Method 3.
[0059] FIG. 27 represents a graph of IFN-y producing cells for PBLs
isolated from fresh
PBMCs and cryopreserved PBMCs. Within cryopreserved PBMCs, PreRx PBLs and
PostRx
PBLs are also represented.
[0060] FIG. 28 represents the proportion of CD4+ and CD8+ T cell subsets in
PreRx PBL
and PostRx PBL, using melanoma TIL as a comparator.
[0061] FIGS. 29A-29D and FIGS. 30A-30D represent a comparison between CD4
(FIG. 29)
and CD8 (FIG. 30) memory subsets of PreRx PBLs and PostRx PBLs, using melanoma
TIL as a
comparator. FIGS. 29A and 30A show data for naïve (CCR7+/CD45RA+); FIGS. 29B
and 30B
show data for central memory t-cells (CM) (CCR7+/CD45RA-); FIGS. 29C and 30C
show data
for effector memory T-cells (EM) (CCR7-/CD45RA-); and FIGS. 29D and 30D show
data for
terminally differentiated effector memory cells (TEMRA) (CCR7-/CD45RA+).
[0062] FIGS. 31A and 31B represent a comparison of CD27 subsets of CD4
(FIG. 31A) and
CD8 (FIG. 31B) subsets for PreRx PBLs and PostRx PBLs, using melanoma TIL as a
comparator.
[0063] FIGS. 32A and 32B represent a comparison of CD28 subsets of CD4
(FIG. 32A) and
CD8 (FIG. 32B) subsets for PreRx PBLs and PostRx PBLs, using melanoma TIL as a
comparator.
[0064] FIGS. 33A and 33B represent a comparison of LAG3+ subsets within the
CD4 (FIG.
33A) and CD8 (FIG. 33B) populations for both PreRx PBLs and PostRx PBLs.
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[0065] FIGS. 34A and 34B represent a comparison of PD1+ subsets within the
CD4 (FIG.
34A) and CD8 (FIG. 34B) populations for both PreRx PBLs and PostRx PBLs.
[0066] FIGS. 35A and 35B show results of cytolytic activity of PreRx PBLs
(FIG. 35A) and
PostRx PBLs (FIG35B), measured using an autologous tumor killing assay. The
cytotoxicity is
measured as the LUso (the number of PBLs required to kill 50% of the target
cells).
[0067] FIGS. 36A and 36B represent graphs of the fold expansion for MILs
(FIG. 36A) and
PBLs (FIG. 36B) isolated from either bone marrow (MILs) or peripheral blood
(PBLs) of AML
patients. MIL 1.1 was expanded using MIL Method 1, MIL1.2 was expanded using
MIL
Method 2, and MIL1.3 was expanded using MIL Method 3. MIL2 and MIL3 were
expanded
using MIL Method 3. All PBLs were expanded using PBL Method 3. Starting cell
number for
MIL1.3 was 138,000 cells, for MIL2 was 62,000 and for MIL 3 was 28,000 cells.
Starting cell
number for PBL2 was 338,000 and for PBL3 was 336,000.
[0068] FIGS. 37A and 37B illustrate IFN-y producing cells for each of MILs
(FIG. 37A) and
PBLs (FIG. 37B).
[0069] FIGS. 38A-38F represent graphs illustrating T cell subsets in MILs
(FIGS. 38A-38C)
and PBLs (FIGS. 38D-38F) isolated from ANIL patients. FIGS. 38A and 38D
illustrate TCRal3+
subsets, FIGS. 38B and 38E illustrate CD4+ subsets, and FIGS. 38C and 38F
illustrate CD8
subsets. PBLs are shown at Day 0 and at Day 14.
[0070] FIGS. 39A-39D represent graphs illustrating CD4 memory subsets for
MILs isolated
from ANIL patients. FIG. 39A shows data for naïve (CCR7+/CD45RA+); FIG. 39B
shows data
for central memory t-cells (CM) (CCR7+/CD45RA-); FIG. 39C shows data for
effector memory
T-cells (EM) (CCR7-/CD45RA-); and FIG. 39D shows data for terminally
differentiated effector
memory cells (TEMRA) (CCR7-/CD45RA+).
[0071] FIGS.40A-40D represent graphs illustrating CD4 memory subsets for
PBLs isolated
from ANIL patients. FIG. 40A shows data for naïve (CCR7+/CD45RA+); FIG. 40B
shows data
for central memory t-cells (CM) (CCR7+/CD45RA-); FIG. 40C shows data for
effector memory
T-cells (EM) (CCR7-/CD45RA-); and FIG. 40D shows data for terminally
differentiated effector
memory cells (TEMRA) (CCR7-/CD45RA+).
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[0072] FIGS. 41A-41D represent graphs illustrating CD8 memory subsets for
MILs isolated
from ANIL patients. FIG. 41A shows data for naïve (CCR7+/CD45RA+); FIG. 41B
shows data
for central memory t-cells (CM) (CCR7+/CD45RA-); FIG. 41C shows data for
effector memory
T-cells (EM) (CCR7-/CD45RA-); and FIG. 41D shows data for terminally
differentiated effector
memory cells (TEMRA) (CCR7-/CD45RA+).
[0073] FIGS.42A-42D represent graphs illustrating CD8 memory subsets for
PBLs isolated
from ANIL patients. FIG. 42A shows data for naïve (CCR7+/CD45RA+); FIG. 42B
shows data
for central memory t-cells (CM) (CCR7+/CD45RA-); FIG. 42C shows data for
effector memory
T-cells (EM) (CCR7-/CD45RA-); and FIG. 42D shows data for terminally
differentiated effector
memory cells (TEMRA) (CCR7-/CD45RA+).
[0074] FIGS. 43A and 43B represent graphs illustrating CD27 subsets of CD4
and CD8 cell
populations for MILs (FIG. 43A) and PBLs (FIG 43B).
[0075] FIGS. 44A and 44B represent graphs illustrating CD28 subsets of CD4
and CD8 cell
populations for MILs (FIG. 44A) and PBLs (FIG. 44B).
[0076] FIGS. 45A and 45B represent graphs illustrating PD1+ subsets of CD4
and CD8 cell
populations for MILs (FIG. 45A) and PBLs (FIG. 45B).
[0077] FIGS. 46A and 46B represent graphs illustrating LAG3+ subsets of CD4
and CD8
cell populations for MILs (FIG. 46A) and PBLs (FIG. 46B).
[0078] FIG. 47 is a timeline illustrating exemplary embodiments of PBL
Method 1 and PBL
Method 3. In this figure, the addition of IL-2 can take place at any point in
time during the
process, and in an exemplary embodiment, over the bracketed area.
[0079] FIG. 48 is a timeline illustrating an exemplary embodiment of the
MIL Method 3. In
this figure, the addition of IL-2 can take place at any point in time during
the process, and in an
exemplary embodiment, over the bracketed area.
[0080] FIG. 49 is an exemplary embodiment of the present invention, showing
a method for
expanding PBLs (including PBLs from a patient pre-treated with ibrutinib)
useful for treatment
of hematological malignancies, such as CLL, as described herein.
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BRIEF DESCRIPTION OF THE SEQUENCE LISTING
[0081] SEQ ID NO:1 is the amino acid sequence of the heavy chain of
muromonab.
[0082] SEQ ID NO:2 is the amino acid sequence of the light chain of
muromonab.
[0083] SEQ ID NO:3 is the amino acid sequence of a recombinant human IL-2
protein.
[0084] SEQ ID NO:4 is the amino acid sequence of aldesleukin.
[0085] SEQ ID NO:5 is the amino acid sequence of a recombinant human IL-4
protein.
[0086] SEQ ID NO:6 is the amino acid sequence of a recombinant human IL-7
protein.
[0087] SEQ ID NO:7 is the amino acid sequence of a recombinant human IL-15
protein.
[0088] SEQ ID NO:8 is the amino acid sequence of a recombinant human IL-21
protein.
DETAILED DESCRIPTION OF THE INVENTION
[0089] 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.
Definitions
[0090] The terms "co-administration," "co-administering," "administered in
combination
with," "administering in combination with," "simultaneous," and "concurrent,"
as used herein,
encompass administration of two or more active pharmaceutical ingredients 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 are present. Simultaneous
administration
in separate compositions and administration in a composition in which both
agents are present
are preferred.
[0091] The term "in vivo" refers to an event that takes place in a
mammalian subject's body.
[0092] The term "ex vivo" refers to an event that takes place outside of a
mammalian
subject's body, in an artificial environment.
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[0093] The term "in vitro" refers to an event that takes places in a test
system. In vitro
assays encompass cell-based assays in which alive or dead cells may be are
employed and may
also encompass a cell-free assay in which no intact cells are employed.
[0094] 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.
[0095] 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.
[0096] 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. Optionally, the peripheral blood mononuclear cells are irradiated
allogeneic
peripheral blood mononuclear cells. PBMCs include antigen presenting cells.
The term "PBLs"
refers to peripheral blood lymphocytes and are T-cells expanded from
peripheral blood. The
terms PBL and TIL are used interchangeably herein.
[0097] 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, and UCHT-1. Other anti-CD3
antibodies
include, for example, otelixizumab, teplizurnab, and visilizumab.
[0098] 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
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hybridoma capable of producing OKT-3 is deposited 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.
Identifier Sequence (One-Letter Amino Acid Symbols)
SEQ ID NO:1 QVQLQQSGAE LARPGASVKM SOKASGYTFT RYTMHWVKQR PGQGLEWIGY
INPSRGYTNY 60
Muromonab heavy NQKFKDKATL TTDKSSSTAY MQLSSLTSED SAVYYOARYY DDHYCLDYWG
QGTTLTVSSA 120
chain KTTAPSVYPL APVOGGTTGS SVTLGOLVKG YFPEPVTLTW NSGSLSSGVH
TFPAVLQSDL 180
YTLSSSVTVT SSTWPSQSIT CNVAHPASST KVDKKIEPRP KSCDKTHTCP PCPAPELLGG
240
PSVFLFPPKP KDTLMISRTP EVTCVVVDVS HEDPEVKFNW YVDGVEVHNA KTKPREEQYN
300
STYRVVSVLT VLHQDWLNGK EYKCKVSNKA LPAPIEKTIS KAKGQPREPQ VYTLPPSRDE
360
LTKNQVSLTC LVKGFYPSDI AVEWESNGQP ENNYKTTPPV LDSDGSFFLY SKLTVDKSRW
420
QQGNVFSCSV MHEALHNHYT QKSLSLSPGK
450
SEQ ID NO:2 QIVLTQSPAI MSASPGEKVT MTCSASSSVS YMNWYQQKSG TSPKRWIYDT
SKLASGVPAH 60
Muromonab light FRGSGSGTSY SLTISGMEAE DAATYYCQQW SSNPFTFGSG TKLEINRADT
APTVSIFPPS 120
chain SEQLTSGGAS VVCFLNNFYP KDINVKWKID GSERQNGVLN SWTDQDSKDS
TYSMSSTLTL 180
TKDEYERHNS YTCEATHKTS TSPIVKSFNR NEC
213
[0099] 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, Annu.
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 IU 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 IL-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 IL2 prodrug NKTR-214, available from Nektar Therapeutics, South San
Francisco,
CA, USA. NKTR-214 and pegylated IL-2 suitable for use in the invention is
described in U.S.
Patent Application Publication No. US 2014/0328791 Al and International Patent
Application
Publication No. WO 2012/065086 Al, the disclosures of which are incorporated
by reference
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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.
TABLE 2. Amino acid sequences of interleukins.
Identifier Sequence (One-Letter Amino Acid Symbols)
SEQ ID NO:3 MAPTSSSTKK TQLQLEHLLL DLQMILNGIN NYKNPKLTRM LTFKFYMPKK
ATELKHLQCL 60
recombinant EEELKPLEEV LNLAQSKNFH LRPRDLISNI NVIVLELKGS ETTFMCEYAD
ETATIVEFLN 120
human IL-2 RWITFCQSII STLT
134
(rhIL-2)
SEQ ID NO:4 PTSSSTKKTQ LQLEHLLLDL QMILNGINNY KNPKLTRMLT FKFYMPKKAT
ELKHLQCLEE 60
Aldesleukin ELKPLEEVLN LAQSKNFHLR PRDLISNINV IVLELKGSET TFMCEYADET
ATIVEFLNRW 120
ITFSQSIIST LT
132
SEQ ID NO:5 MHKCDITLQE IIKTLNSLTE QKTLCTELTV TDIFAASKNT TEKETFCRAA
TVLRQFYSHH 60
recombinant EKDTRCLGAT AQQFHRHKQL IRFLKRLDRN LWGLAGLNSC PVKEANQSTL
ENFLERLKTI 120
human IL-4 MREKYSKCSS
130
(rhIL-4)
SEQ ID NO:6 MDCDIEGKDG KQYESVLMVS IDQLLDSMKE IGSNCLNNEF NFFKRHICDA
NKEGMFLFRA 60
recombinant ARKLRQFLKM NSTGDFDLHL LKVSEGTTIL LNCTGQVKGR KPAALGEAQP
TKSLEENKSL 120
human IL-7 KEQKKLNDLC FLKRLLQEIK TCWNKILMGT KEH
153
(rhIL-7)
SEQ ID NO:7 MNWVNVISDL KKIEDLIQSM HIDATLYTES DVHPSCKVTA MKCFLLELQV
ISLESGDASI 60
recombinant HDTVENLIIL ANNSLSSNGN VTESGCKECE ELEEKNIKEF LQSFVHIVQM FINTS
115
human IL-15
(rhIL-15)
SEQ ID NO:8 MQDRHMIRMR QLIDIVDQLK NYVNDLVPEF LPAPEDVETN CEWSAFSCFQ
KAQLKSANTG 60
recombinant NNERIINVSI KKLKRKPPST NAGRRQKHRL TCPSCDSYEK KPPKEFLERF
KSLLQKMIHQ 120
human IL-21 HLSSRTHGSE DS
132
(rhIL-21)
[00100] 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.
IL-4 regulates the differentiation of naive 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-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 IL-4 suitable for use in the invention is given in Table 2 (SEQ ID
NO:5).
[00101] 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. IL-7 can
22
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WO 2019/103857 PCT/US2018/060183
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-7 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:6).
[00102] The term "IL-15" (also referred to herein as "IL15") refers to the T
cell growth factor
known as interleukin-15, and includes all forms of IL-15 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 0 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:7).
[00103] 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 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 IL-21 recombinant
protein, Cat. No.
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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:8).
[00104] 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 the 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.
[00105] 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
VH) and a heavy chain constant region. The heavy chain constant region is
comprised of three
domains, CHL 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 VL regions of an antibody may be
further subdivided
into regions of hypervariability, which are referred to as complementarity
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.
[00106] 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
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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 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.
[00107] 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. coil 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.
[00108] 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 CHI
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domains; (iv) a Fv fragment consisting of the VL and VH 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 complementarity
determining region (CDR).
Furthermore, although the two domains of the Fv fragment, VL and VH, 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. Sci. USA 1988, 85, 5879-5883). Such scFv
antibodies are
also intended to be encompassed within the terms "antigen-binding portion" or
"antigen-binding
fragment" of an antibody. These antibody fragments are 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.
[00109] 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.
[00110] 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 an embodiment,
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 genome
comprising a human
heavy chain transgene and a light chain transgene fused to an immortalized
cell.
[00111] 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
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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 antibodies are sequences
that, while
derived from and related to human germline VH and VL sequences, may not
naturally exist within
the human antibody germline repertoire in vivo.
[00112] As used herein, "isotype" refers to the antibody class (e.g., IgM
or IgG1) that is
encoded by the heavy chain constant region genes.
[00113] 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."
[00114] 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.
[00115] 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 are human immunoglobulins (recipient antibody)
in which
residues from a hypervariable region of the recipient are replaced by residues
from a 15
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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 (Fc),
typically that of a human immunoglobulin. For further details, see Jones, et
at., Nature 1986,
321, 522-525; Riechmann, et al., 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 Fc
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 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.
[00116] 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.
[00117] A "diabody" is a small antibody fragment with two antigen-binding
sites. The
fragments comprises a heavy chain variable domain (VH) connected to a light
chain variable
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domain (VI) in the same polypeptide chain (VH-VL or VL-VH). 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.
[00118] 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 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
at., 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
reducing 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
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PCT/US2018/060183
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 at., 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, et al., 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, et al., Biochem. 1975, 14, 5516-5523.
[00119]
"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. Pegylation may, for example, increase the biological (e.g., serum)
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-
polyethylene 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.
[00120] The terms "fusion protein" or "fusion polypeptide" refer to proteins
that combine the
properties of two or more individual proteins. Such proteins have at least two
heterologous
polypeptides covalently linked either directly or via an amino acid linker.
The polypeptides
forming the fusion protein are typically linked C-terminus to N-terminus,
although they can also
be linked C-terminus to C-terminus, N-terminus to N-terminus, or N-terminus to
C-terminus.
The polypeptides of the fusion protein can be in any order and may include
more than one of
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either or both of the constituent polypeptides. The term encompasses
conservatively modified
variants, polymorphic variants, alleles, mutants, subsequences, interspecies
homologs, and
immunogenic fragments of the antigens that make up the fusion protein. Fusion
proteins of the
disclosure can also comprise additional copies of a component antigen or
immunogenic fragment
thereof The fusion protein may contain one or more binding domains linked
together and
further linked to an Fc domain, such as an IgG Fc domain. Fusion proteins may
be further linked
together to mimic a monoclonal antibody and provide six or more binding
domains. Fusion
proteins may be produced by recombinant methods as is known in the art.
Preparation of fusion
proteins are known in the art and are described, e.g., in International Patent
Application
Publication Nos. WO 1995/027735 Al, WO 2005/103077 Al, WO 2008/025516 Al, WO
2009/007120 Al, WO 2010/003766 Al, WO 2010/010051 Al, WO 2010/078966 Al, U.S.
Patent Application Publication Nos. US 2015/0125419 Al and US 2016/0272695 Al,
and U.S.
Patent No. 8,921,519, the disclosures of each of which are incorporated by
reference herein.
[00121] 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).
[00122] The term "conservative amino acid substitutions" in means amino acid
sequence
modifications which do not abrogate the binding of an antibody or fusion
protein to the antigen.
Conservative amino acid substitutions include the substitution of an amino
acid in one class by
an amino acid of the same class, where a class is defined by common
physicochemical amino
acid side chain properties and high substitution frequencies in homologous
proteins found in
nature, as determined, for example, by a standard Dayhoff frequency exchange
matrix or
BLOSUM matrix. Six general classes of amino acid side chains have been
categorized and
include: Class I (Cys); Class II (Ser, Thr, Pro, Ala, Gly); Class III (Asn,
Asp, Gln, Glu); Class IV
(His, Arg, Lys); Class V (Ile, Leu, Val, Met); and Class VI (Phe, Tyr, Trp).
For example,
substitution of an Asp for another class III residue such as Asn, Gln, or Glu,
is a conservative
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substitution. Thus, a predicted nonessential amino acid residue in an antibody
is preferably
replaced with another amino acid residue from the same class. Methods of
identifying amino
acid conservative substitutions which do not eliminate antigen binding are
well-known in the art
(see, e.g., Brummell, et al., Biochemistry 1993, 32, 1180-1187; Kobayashi, et
al., Protein Eng.
1999, 12, 879-884 (1999); and Burks, et al., Proc. Natl. Acad. Sci. USA 1997,
94, 412-417.
[00123] 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 are 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
particular alignment
software. In certain embodiments, the default parameters of the alignment
software are used.
[00124] 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.
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[00125] Nucleic acid sequences implicitly encompass conservatively modified
variants
thereof (e.g., degenerate codon substitutions) and complementary sequences, as
well as the
sequence explicitly indicated. Specifically, degenerate codon substitutions
may be achieved by
generating sequences in which the third position of one or more selected (or
all) codons is
substituted with mixed-base and/or deoxyinosine residues. Batzer, et at.,
Nucleic Acid Res.
1991, 19, 5081; Ohtsuka, et at., I Biol. Chem. 1985, 260, 2605-2608;
Rossolini, et at., Mol. Cell.
Probes 1994, 8, 91-98. The term nucleic acid is used interchangeably with
cDNA, mRNA,
oligonucleotide, and polynucleotide.
[00126] 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 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
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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 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 polypeptide. 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
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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 pharmacokinetic (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.
[00127] The term "hematological malignancy" refers 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 may
result in the
formation of a "liquid tumor." Hematological malignancies include, but are not
limited to, acute
lymphoblastic leukemia (ALL), chronic lymphocytic lymphoma (CLL), small
lymphocytic
lymphoma (SLL), acute myeloid leukemia (AML), chronic myelogenous leukemia
(CML), 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.
[00128] 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,
including liquid
tumors resident in bone marrow, 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.
[00129] The term "biopsy" refers to any medical procedure used to obtain
cancerous cells,
including bone marrow biopsy.
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[00130] The terms "acute myeloid leukemia" or "AML" refers to cancers of the
myeloid
blood cell lines, which are also known in the art as acute myelogenous
leukemia and acute
nonlymphocytic leukemia. Although AML is a liquid tumor, some manifestations
of AML,
including extramedullary manifestations such as chloroma, exhibit properties
of a solid tumor,
but are classified herein as a liquid tumor.
[00131] 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 at., 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.
[00132] 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.
[00133] A "therapeutic effect" as that term is used herein, encompasses a
therapeutic benefit
and/or a prophylactic benefit. A prophylactic effect includes delaying or
eliminating the
appearance of a disease or condition, delaying or eliminating the onset of
symptoms of a disease
or condition, slowing, halting, or reversing the progression of a disease or
condition, or any
combination thereof
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[00134] 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.
[00135] The terms "QD," "qd," or "q.d." mean quaque die, once a day, or once
daily. The
terms "BID," "bid," or "b.i.d." mean bis in die, twice a day, or twice daily.
The terms "TID,"
"tid," or "t.i.d." mean ter in die, three times a day, or three times daily.
The terms "QID," "qid,"
or "q.i.d." mean quater in die, four times a day, or four times daily.
[00136] 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.
[00137] 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 c43, 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
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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.
[00138] By "cryopreserved TILs" (or cryopreserved MILs or PBLs) 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.
[00139] By "thawed cryopreserved TILs" (or thawed MILs or PBLs) 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.
[00140] 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
x 108 cells. REP
expansion is generally done to provide populations of 1.5 x 109 to 1.5 x 1010
cells for infusion.
[00141] In general, TILs are initially obtained from a patient tumor sample
("primary TILs")
and then expanded into a larger population for further manipulation as
described herein,
optionally cyropreserved, restimulated as outlined herein and optionally
evaluated for phenotype
and metabolic parameters as an indication of TIL health.
[00142] In
general, the harvested cell suspension is called a "primary cell population"
or a
"freshly harvested" cell population.
[00143] In general, as discussed herein, the TILs are initially prepared by
obtaining a primary
population of TILs from a tumor resected from a patient as discussed herein
(the "primary cell
population" or "first cell population"). This is followed with an initial bulk
expansion utilizing a
culturing of the cells with IL-2, forming a second population of cells
(sometimes referred to
herein as the "bulk TIL population" or "second population").
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[00144] The term "cytotoxic lymphocyte" includes cytotoxic T (CTL) cells
(including CD8+
cytotoxic T lymphocytes and CD4+ T-helper lymphocytes), natural killer T (NKT)
cells and
natural killer (NK) cells. Cytotoxic lymphocytes can include, for example,
peripheral blood-
derived ccp TCR-positive or y6 TCR-positive T cells activated by tumor
associated antigens
and/or transduced with tumor specific chimeric antigen receptors or T-cell
receptors, and tumor-
infiltrating lymphocytes (TILs).
[00145] The term "central memory T cell" refers to a subset of T cells that in
the human are
CD45R0+ and constitutively express CCR7 (CCR7h i) and CD62L (CD62 hi). 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 BMII.
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.
[00146] 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 (CCR71o) and are heterogeneous or low for CD62L expression (CD62L1o). 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 BLIMP1. 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 proportionally enriched in the lung, liver, and
gut. CD8+ effector
memory T cells carry large amounts of perforin. 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 outsside environment until the TILs are ready
to be adminsitered
to the patient.
[00147] In some embodiments, methods of the present disclosure further include
a "pre-REP"
stage in which tumor tissue or cells from tumor tissue are grown in standard
lab media (including
without limitation RPMI) and treated the with reagents such as irradiated
feeder cells and anti-
CD3 antibodies to achieve a desired effect, such as increase in the number of
TILS and/or an
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enrichment of the population for cells containing desired cell surface markers
or other structural,
biochemical or functional features. The pre-REP stage may utilize lab grade
reagents (under the
assumption that the lab grade reagents get diluted out during a later REP
stage), making it easier
to incorporate alternative strategies for improving TIL production. Therefore,
in some
embodiments, the disclosed TLR agonist and/or peptide or peptidomimetics can
be included in
the culture medium during the pre-REP stage. The pre-REP culture can in some
embodiments,
include IL-2.The present invention is directed in preferred aspects to novel
methods of
augmenting REPs with one or more additional restimulation protocols, also
referred to herein as
a "restimulation Rapid Expansion Protocol" or "reREP", which leads
surprisingly to expanded
memory T cell subsets, including the memory effector T cell subset, and/or to
markes
enhancement in the glycolytic respiration as compared to freshly harvested
TILs or thawed
cryopreserved TILs for the restimulated TILs (sometimes referred to herein as
"reTILs"). That is,
by using a reREP procedure on cyropreserved TILs, patients can receive highly
metabolically
active, healthy TILs, leading to more favorable outcomes.
[00148] When "an anti-tumor effective amount", "an 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
genetically modified cytotoxic lymphocytes described herein may be
administered at a dosage of
104 to 1011 cells/kg body weight (e.g., i05 to 106, i05 to 1010, i05 to 1-11,
u 106 to 1010, 106 to
10",107 to 1011, io7 to 1010, 108 to 1011, 108 to 1-1 , u 109to
1011, or 109 to 1010 cells/kg body
weight), including all integer values within those ranges. Genetically
modified cytotoxic
lymphocytes compositions may also be administered multiple times at these
dosages. The
genetically modified cytotoxic lymphocytes can be administered by using
infusion techniques
that are commonly known in immunotherapy (see, e.g., Rosenberg et al., New
Eng. J. of Med.
319: 1676, 1988). 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.
[00149] For the avoidance of doubt, it is intended herein that particular
features (for example
integers, characteristics, values, uses, diseases, formulae, compounds or
groups) described in
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conjunction with a particular aspect, embodiment or example of the invention
are to be
understood as applicable to any other aspect, embodiment or example described
herein unless
incompatible therewith. Thus such features may be used where appropriate in
conjunction with
any of the definition, claims or embodiments defined herein. All of the
features disclosed in this
specification (including any accompanying claims, abstract and drawings),
and/or all of the steps
of any method or process so disclosed, may be combined in any combination,
except
combinations where at least some of the features and/or steps are mutually
exclusive. The
invention is not restricted to any details of any disclosed embodiments. The
invention extends to
any novel one, or novel combination, of the features disclosed in this
specification (including any
accompanying claims, abstract and drawings), or to any novel one, or any novel
combination, of
the steps of any method or process so disclosed.
[00150] 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 are 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.
[00151] 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 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
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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 of," and "consisting of."
Embodiments of Methods of Expanding Therapeutic T-Cells Including Peripheral
Blood (PBLs)
and/or Bone Marrow (MILs)
Methods of Expanding Peripheral Blood Lymphocytes (PBLs) from Peripheral Blood
[00152] PBL Method 1. In an embodiment of the invention, PBLs are expanded
using the
processes described herein. In an embodiment of the invention, the method
comprises obtaining
a PBMC sample from whole blood. In an embodiment, the method comprises
enriching T-cells
by isolating pure T-cells from PBMCs using negative selection of a non-CD19+
fraction. On
Day 0, the pure T-cells are cultured with antiCD3/antiCD28 antibodies
(DynaBeads ) in a 1:1
ratio (beads:cells) and IL-2 at 3000 IU/ml. On Day 4, additional IL-2 is added
to the culture at
3000 IU/ml. On Day 7, the culture is again stimulated with antiCD3/antiCD28
antibodies
(DynaBeads ) in a 1:1 ratio (beads:cells), and additional IL-2 at 3000 IU/ml
is added to the
culture. PBLs are harvested on Day 14, beads are removed, and PBLs are counted
and
phenotyped. In an embodiment, the method comprises enriching T-cells by
isolating pure T-
cells from PBMCs using magnetic bead-based negative selection of a non-CD19+
fraction.
[00153] In an embodiment 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). The isolated
T cells are
counted and seeded at 5x105 cells per well of a GRex 24-well plate and are co-
cultured with
DynaBeads (anti-CD3/anti-CD28) at a 1:1 ratio with IL-2 at 3000 IU/ml in a
total of 8m1 of
CM2 media per well. On Day 4, the media in each well is exchanged from CM2 to
AIM-V with
fresh IL-2 at 3000 IU/ml. On Day 7, the expanded cells are harvested, counted,
then cultured at
15x106 cells per flask in GRex TOM flasks with IL-2 at 3000 IU/ml and
DynaBeads at a 1:1
ratio (beads:cells) in a total of 100m1 AIM-V media. On Day 11, the media is
exchanged to CM-
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4 media supplemented with fresh IL-2 at 3000 IU/ml. On Day 14, the DynaBeads
are removed
using a DynaMag Magnet (DynaMagTm-15) and the cells are counted.
[00154] In an embodiment 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). The isolated
T cells are
counted and seeded at 5x105 cells per well of a GRex 24-well plate and are co-
cultured with
DynaBeads (anti-CD3/anti-CD28) at a 1:1 ratio with IL-2 at 3000 IU/ml in a
total of 8m1 of
CM2 media per well. On Day 4, the media in each well is exchanged from CM2 to
AIM-V with
fresh IL-2 at 3000 IU/ml. On Day 7, the PBLs are harvested, counted, then
reseeded at 1x106
cells per well of a new GRex-24 well plate with IL-2 at 3000 IU/ml and
DynaBeads at a 1:1
ratio (beads:cells) in a total of 8m1 AIM-V media. On Day 11, the media is
exchanged to CM-4
media supplemented with fresh IL-2 at 3000 IU/ml. On Day 14, the DynaBeads
are removed
using a DynaMag Magnet (DynaMagTm-15) and the cells are counted.
[00155] PBL Method 2. In an embodiment 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. The non-adherent cells are the expanded similarly as
PBL Method 1, that
is, on Day 0, the non-adherent cells are cultured with antiCD3/antiCD28
antibodies
(DynaBeads ) in a 1:1 ratio (beads:cells) and IL-2 at 3000 IU/ml. On Day 4,
additional IL-2 is
added to the culture at 3000 IU/ml. On Day 7, the culture is again stimulated
with
antiCD3/antiCD28 antibodies (DynaBeads ) in a 1:1 ratio (beads:cells), and
additional IL-2 at
3000 IU/ml is added to the culture. PBLs are harvested on Day 14, beads are
removed, and
PBLs are counted and phenotyped.
[00156] In an embodiment 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.
The PBLs are
cultured with anti-CD3/anti-CD28 DynaBeads in a 1:1 ratio of beads:cells, at
lx106 cells per
well and IL-2 at 3000 IU/ml in a total of 7m1 of CM-2 media in each well of a
GRex 24-well
plate. On Day 4, the media in each well is exchanged with AIM-V media and
fresh IL-2 at 3000
IU/ml. On Day 7, the expanded cells are harvested, counted, then cultured at
15x106 cells per
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flask in GRex TOM flasks with IL-2 at 3000 IU/m1 and DynaBeads at a 1:1 ratio
(T-cells:beads)
in a total of 100m1 AIM-V media. On Day 11, the media is changed to CM-4 media
and
supplemented with fresh IL-2 (3000 IU/m1). On Day 14, the DynaBeads are
removed using a
DynaMagTm Magnet (DynaMagTm-15) and the cells are counted.
[00157] In an embodiment 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.
The PBLs are
cultured with anti-CD3/anti-CD28 DynaBeads in a 1:1 ratio of beads:cells, at
lx106 cells per
well and IL-2 at 3000 IU/ml in a total of 7m1 of CM-2 media in each well of a
GRex 24-well
plate. On Day 4, the media in each well is exchanged with AIM-V media and
fresh IL-2 at 3000
IU/ml. On Day 7, the expanded cells are harvested, counted, then cultured at
1x106 cells per
well in a new GRex 24-well plate with IL-2 at 3000 IU/ml and DynaBeads at a
1:1 ratio (T-
cells:beads) in a total of 8m1 AIM-V media. On Day 11, the media is changed to
CM-4 media
and supplemented with fresh IL-2 (3000 IU/ml). On Day 14, the DynaBeads are
removed using
a DynaMagTm Magnet (DynaMagTm-15) and the cells are counted.
[00158] PBL Method 3. In an embodiment 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. On Day 0, the T-cells and B-cells are co-
cultured with
antiCD3/antiCD28 antibodies (DynaBeads ) in a 1:1 ratio (beads:cells) and IL-2
at 3000 IU/ml.
On Day 4, additional IL-2 is added to the culture at 3000 IU/ml. On Day 7, the
culture is again
stimulated with antiCD3/antiCD28 antibodies (DynaBeads ) in a 1:1 ratio
(beads:cells), and
additional IL-2 at 3000 IU/ml is added to the culture. PBLs are harvested on
Day 14, beads are
removed, and PBLs are counted and phenotyped.
[00159] In an embodiment 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 Biotec). Of the non-
CD19+ cell
fraction, T-cells are purified using the Human Pan T-cell Isolation Kit and LS
Columns
(Miltenyi Biotec). The T-cells (PBLs) and B-cells are co-cultured at different
ratios in a Grex
24-well plate in about 8m1 of CM2 media in the presence of IL-2 at about
30001U/ml. B-cell:T-
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cell ratios are 0.1:1; 1:1, and 10:1. The T-cell/B-cell co-culture is
stimulated with
antiCD3/antiCD28 antibodies (DynaBeadsg) in a 1:1 ratio (beads:cells). On Day
4, the media is
exchanged from CM2 to AIM-V media and additional IL-2 is added to the culture
at 3000 IU/ml.
On Day 7, the cells are harvested and counted and re-seeded on a new Grex 24-
well plate in
AIM-V media at a cell range of from about 1.5x105 to about 4x105 cells per
well and stimulated
with antiCD3/antiCD28 antibodies (DynaBeadsg) in a 1:1 ratio (beads:cells),
with additional IL-
2 at 3000 IU/ml. On Day 14, the DynaBeads are removed using a DynaMagTm Magnet
(DynaMagTm-15) and the cells are counted.
[00160] In an embodiment, PBMCs are isolated from a whole blood sample. In an
embodiment, the PBMC sample is used as the starting material to expand the
PBLs. In an
embodiment, the sample is cryopreserved prior to the expansion process. In
another
embodiment, a fresh sample is used as the starting material to expand the
PBLs. In an
embodiment of the invention, T-cells are isolated from PBMCs using methods
known in the art.
In an embodiment, the T-cells are isolated using a Human Pan T-cell isolation
kit and LS
columns. In an embodiment of the invention, T-cells are isolated from PBMCs
using antibody
selection methods known in the art, for example, CD19 negative selection.
[00161] In an embodiment of the invention, the process is performed over about
7 days, about
8 days, about 9 days, about 10 days, about 11 days, about 12 days, about 13
days, or about 14
days. In another embodiment, the process is performed over about 7 days. In
another
embodiment, the process is performed over about 14 days.
[00162] In an embodiment of the invention, the PBMCs are cultured with
antiCD3/antiCD28
antibodies. In an embodiment, any available antiCD3/antiCD28 product is useful
in the present
invention. In an embodiment of the invention, the commercially available
product used are
DynaBeads . In an embodiment, the DynaBeads are cultured with the PBMCs in a
ratio of 1:1
(beads:cells). In another embodiment, the antibodies are DynaBeads cultured
with the PBMCs
in a ratio of 1.5:1, 2:1, 2.5:1, 3:1, 3.5:1, 4:1, 4.5:1, or 5:1 (beads:cells).
In an embodiment of the
invention, the antibody culturing steps and/or the step of restimulating cells
with antibody is
performed over a period of from about 2 to about 6 days, from about 3 to about
5 days, or for
about 4 days. In an embodiment of the invention, the antibody culturing step
is performed over a
period of about 2 days, 3 days, 4 days, 5 days, or 6 days.
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[00163] In an embodiment, the PBMC sample is cultured with IL-2. In an
embodiment of the
invention, the cell culture medium used for expansion of the PBLs from PBMCs
comprises IL-2
at a concentration selected from the group consisting of about 100 IU/mL,
about 200 IU/mL,
about 300 IU/mL, about 400 IU/mL, about 100 IU/mL, about 100 IU/mL, about 100
IU/mL,
about 100 IU/mL, about 100 IU/mL, about 500 IU/mL, about 600 IU/mL, about 700
IU/mL,
about 800 IU/mL, about 900 IU/mL, about 1,000 IU/mL, about 1,100 IU/mL, about
1,200
IU/mL, about 1,300 IU/mL, about 1,400 IU/mL, about 1,500 IU/mL, about 1,600
IU/mL, about
1,700 IU/mL, about 1,800 IU/mL, about 1,900 IU/mL, about 2,000 IU/mL, about
2,100 IU/mL,
about 2,200 IU/mL, about 2,300 IU/mL, about 2,400 IU/mL, about 2,500 IU/mL,
about 2,600
IU/mL, about 2,700 IU/mL, about 2,800 IU/mL, about 2,900 IU/mL, about 3,000
IU/mL, about
3,100 IU/mL, about 3,200 IU/mL, about 3,300 IU/mL, about 3,400 IU/mL, about
3,500 IU/mL,
about 3,600 IU/mL, about 3,700 IU/mL, about 3,800 IU/mL, about 3,900 IU/mL,
about 4,000
IU/mL, about 4,100 IU/mL, about 4,200 IU/mL, about 4,300 IU/mL, about 4,400
IU/mL, about
4,500 IU/mL, about 4,600 IU/mL, about 4,700 IU/mL, about 4,800 IU/mL, about
4,900 IU/mL,
about 5,000 IU/mL, about 5,100 IU/mL, about 5,200 IU/mL, about 5,300 IU/mL,
about 5,400
IU/mL, about 5,500 IU/mL, about 5,600 IU/mL, about 5,700 IU/mL, about 5,800
IU/mL, about
5,900 IU/mL, about 6,000 IU/mL, about 6,500 IU/mL, about 7,000 IU/mL, about
7,500 IU/mL,
about 8,000 IU/mL, about 8,500 IU/mL, about 9,000 IU/mL, about 9,500 IU/mL,
and about
10,000 IU/mL.
[00164] In an embodiment of the invention, the starting cell number of PBMCs
for the
expansion process is from about 25,000 to about 1,000,000, from about 30,000
to about 900,000,
from about 35,000 to about 850,000, from about 40,000 to about 800,000, from
about 45,000 to
about 800,000, from about 50,000 to about 750,000, from about 55,000 to about
700,000, from
about 60,000 to about 650,000, from about 65,000 to about 600,000, from about
70,000 to about
550,000, preferably from about 75,000 to about 500,000, from about 80,000 to
about 450,000,
from about 85,000 to about 400,000, from about 90,000 to about 350,000, from
about 95,000 to
about 300,000, from about 100,000 to about 250,000, from about 105,000 to
about 200,000, or
from about 110,000 to about 150,000. In an embodiment of the invention, the
starting cell
number of PBMCs is about 138,000, 140,000, 145,000, or more. In another
embodiment, the
starting cell number of PBMCs is about 28,000. In another embodiment, the
starting cell number
of PBMCs is about 62,000. In another embodiment, the starting cell number of
PBMCs is about
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338,000. In another embodiment, the starting cell number of PBMCs is about
336,000. In
another embodiment, the starting cell number of PBMCs is 1 million, 2 million,
3 million, 4
million, 5 million, 6 million, 7 million, 8 million, 9 million, 10 million or
more. In another
embodiment, the starting cell number of PBMCs is 1 million to 10 million, 2
million to 9 million,
3 million to 8 million, 4 million to 7 million, or 5 million to 6 million. In
another embodiment,
the starting cell number of PBMCs is about 4 million. In yet another
embodiment, the starting
cell number of PBMCs is at least about 4 million, at least about 5 million, or
at least about 6
million or more.
[00165] In an embodiment of the invention, the cells are grown in a GRex 24
well plate. In an
embodiment of the invention, a comparable well plate is used. In an
embodiment, the starting
material for the expansion is about 5x105 T-cells per well. In an embodiment
of the invention,
there are 1x106 cells per well. In an embodiment of the invention, the number
of cells per well is
sufficient to seed the well and expand the T-cells.
[00166] In an embodiment of the invention, the fold expansion of PBLs is from
about 20% to
about 100%, 25% to about 95%, 30% to about 90%, 35% to about 85%, 40% to about
80%, 45%
to about 75%, 50% to about 100%, or 25% to about 75%. In an embodiment of the
invention,
the fold expansion is about 25%. In another embodiment of the invention, the
fold expansion is
about 50%. In another embodiment, the fold expansion is about 75%.
[00167] In an embodiment of the invention, additional IL-2 may be added to the
culture on
one or more days throughout the process. In an embodiment of the invention,
additional IL-2 is
added on Day 4. In an embodiment of the invention, additional IL-2 is added on
Day 7. In an
embodiment of the invention, additional IL-2 is added on Day 11. In an other
embodiment,
additional IL-2 is added on Day 4, Day 7, and/or Day 11. In an embodiment of
the invention, the
cell culture medium may be changed on one or more days through the cell
culture process. In an
embodiment, the cell culture medium is changed on Day 4, Day 7, and/or Day 11
of the process.
In an embodiment of the invention, the PBLs are cultured with additional IL-2
for a period of 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. In an embodiment of the invention, PBLs are cultured for a
period of 3 days
after each addition of IL-2.
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[00168] In an embodiment, the cell culture medium is exchanged at least once
time during the
method. In an embodiment, the cell culture medium is exchanged at the same
time that
additional IL-2 is added. In another embodiment the cell culture medium is
exchanged on at
least one of Day 1, Day 2, Day 3, Day 4, Day 5, Day 6, Day 7, Day 8, Day 9,
Day 10, Day 11,
Day 12, Day 13, or Day 14. In an embodiment of the invention, the cell culture
medium used
throughout the method may be the same or different. In an embodiment of the
invention, the cell
culture medium is CM-2, CM-4, or AIM-V.
[00169] In an embodiment of the invention, T-cells may be restimulated with
antiCD3/antiCD28 antibodies on one or more days throughout the 14-day
expansion process. In
an embodiment, the T-cells are restimulated on Day 7. In an embodiment, GRex
10M flasks are
used for the restimulation step. In an embodiment of the invention, comparable
flasks are used.
[00170] In an embodiment of the invention, the DynaBeads are removed using a
DynaMagTm
Magnet, the cells are counted, and the cells are analyzed using phenotypic and
functional
analysis as further described in the Examples below. In an embodiment of the
invention,
antibodies are separated from the PBLs or MILs using methods known in the art.
In any of the
foregoing embodiments, magnetic bead-based selection of TILs, PBLs, or MILs is
used.
[00171] In an embodiment 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
an embodiment of
the invention, the incubation time is about 3 hours. In an embodiment of the
invention, the
temperature is about 37 Celsius. The non-adherent cells are then expanded
using the process
described above.
[00172] In an embodiment of the invention, the PBMCs are obtained from a
patient who has
been treated with ibrutinib or another ITK or kinase inhbitor, such ITK and
kinase inhibitors as
described elsewhere herein. In an embodiment of the invention, the ITK
inhibitor is a covalent
ITK inhibitor that covalently and irreversibly binds to ITK. In an embodiment
of the invention,
the ITK inhibitor is an allosteric ITK inhibitor that binds to ITK. In an
embodiment of the
invention, the PBMCs are obtained from a patient who has been treated with
ibrutinib or other
ITK inhbitor, including ITK inhibitors as described elsewhere herein, prior to
obtaining a PBMC
sample for use with any of the foregoing methods, including PBL Method 1, PBL
Method 2, or
PBL Method 3. In an embodiment of the invention, the ITK inhibitor treatment
has been
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administered at least 1 time, at least 2, times, or at least 3 times or more.
In an embodiment of
the invention, PBLs that are expanded from patients pretreated with ibrutinib
or other ITK
inhibitor comprise less LAG3+, PD-1+ cells than those expanded from patients
not pretreated
with ibrutinib or other ITK inhibitor. In an embodiment of the invention PBLs
that are expanded
from patients pretreated with ibrutinib or other ITK inhibitor comprise
increased levels of IFNy
production than those expanded from patients not pretreated with ibrutinib or
other ITK
inhibitor. In an embodiment of the invention, PBLs that are expanded from
patients pretreated
with ibrutinib or other ITK inhibitor comprise increased lytic activity at
lower Effector:Target
cell ratios than those expanded from patients not pretreated with ibrutinib or
other ITK inhibitor.
In an embodiment of the invention, patients pretreated with ibrutinib or other
ITK inhibitor have
higher fold-expansion as compared with untreated patients.
[00173] In an embodiment of the invention, the method includes a step of
adding an ITK
inhibitor to the cell culture. In an embodiment, the ITK inhibitor is added on
one or more of Day
0, Day 1, Day 2, Day 3, Day 4, Day 5, Day 6, Day 7, Day 8, Day 9, Day 10, Day
11, Day 12,
Day 13, or Day 14 of the process. In an embodiment, the ITK inhibitor is added
on the days
during the method when cell culture medium is exchanged. In an embodiment, the
ITK inhibitor
is added on Day 0 and when cell culture medium is exchanged. In an embodiment,
the ITK
inhibitor is added during the method when IL-2 is added. In an embodiment, the
ITK inhibitor is
added on Day 0, Day 4, Day 7, and optionally Day 11 of the method. In an
embodiment of the
invention, the ITK inhibitor is added at Day 0 and at Day 7 of the method. In
an embodiment of
the invention, the ITK inhibitor is one known in the art. In an embodiment of
the invention, the
ITK inhibitor is one described elsewhere herein.
[00174] In an embodiment of the invention, the ITK inhibitor is used in the
method at a
concentration of from about 0.1nM to about 5uM. In an embodiment, the ITK
inhibitor is used
in the method at a concentration of about 01M, 0.5nM, 1nM, 5nM, lOnM, 20nM,
30nM,
40nM, 50nM, 60nM, 70nM, 80nM, 90nM, 100nM, 150nM, 200nM, 250nM, 300nM, 350nM,
400nM, 450nM, 500nM, 550nM, 600nM, 650nM, 700nM, 750nM, 800nM, 850nM, 900nM,
950nM, luM, 2uM, 3uM, 4uM, or 5uM.
[00175] In an embodiment of the invention, the method includes a step of
adding an ITK
inhibitor when the PBMCs are derived from a patient who has no prior exposure
to an ITK
inhibitor treatment, such as ibrutinib.
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[00176] 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
another embodiment, the PBMCs are derived from a patient who is currently on
an ITK inhibitor
regimen, such as ibrutinib.
[00177] 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.
[00178] 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 another embodiment, 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.
[00179] In an embodment of the invention, at Day 0, cells are selected for
CD19+ and sorted
accordingly. In an embodiment of the invention, the selection is made using
antibody binding
beads. In an embodiment of the invention, pure T-cells are isolated on Day 0
from the PBMCs.
In an embodiment of the invention, at Day 0, the CD19+ B cells and pure T
cells are co-cultured
with antiCD3/antiCD28 antibodies for a minimum of 4 days. In an embodiment of
the invention,
on Day 4, IL-2 is added to the culture. In an embodiment of the invention, on
Day 7, the culture
is restimulated with antiCD3/antiCD28 antibodies and additional IL-2. In an
embodiment of the
invention, on Day 14, the PBLs are harvested.
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[00180] In an embodiment of the invention, for patients that are not pre-
treated with ibrutinib
or other ITK inhibitor, 10-15m1 of Buffy Coat will yield about 5x109PBMC,
which, in turn, will
yield about 5.5x107 starting cell material, and about 11x109PBLs at the end of
the expansion
process. In an embodiment of the invention, about 54x106 PBMCs will yield
about 6x105
starting material, and about 1.2x108 MTh (about a 205-fold expansion).
[00181] In an embodiment of the invention, for patients that are pre-treated
with ibrutinib or
other ITK inhibitor, the expansion process will yield about 20x109PBLs. In an
embodiment of
the invention, 40.3x106 PBMCs will yield about 4.7x105 starting cell material,
and about 1.6x108
PBLs (about a 338-fold expansion).
[00182] In an embodiment of the invention, the clinical dose of PBLs useful in
the present
invention for patients with chronic lymphocytic leukemia (CLL) is from about
0.1x109 to about
15x109 PBLs, from about 0.1x109 to about 15x109 PBLs, from about 0.12x109 to
about 12x109
PBLs, from about 0.15x109 to about 11x109 PBLs, from about 0.2x109 to about
10x109 PBLs,
from about 0.3x109 to about 9x109 PBLs, from about 0.4x109 to about 8x109
PBLs, from about
0.5x109 to about 7x109 PBLs, from about 0.6x109 to about 6x109 PBLs, from
about 0.7x109 to
about 5x109 PBLs, from about 0.8x109 to about 4x109 PBLs, from about 0.9x109
to about 3x109
PBLs, or from about ix i0 to about 2x109 PBLs.
[00183] 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.
Methods of Expanding Marrow Infiltrating Lymphocytes (MILs) from PBMCs Derived
from Bone Marrow
[00184] MIL Method 1. In an embodiment of the invention, a method for
expanding MILs from
PBMCs derived from bone marrow is described. In an embodiment of the
invention, the method
is performed over 14 days. In an embodiment, the method comprises obtaining
bone marrow
PBMCs and cryopreserving the PBMCs. On Day 0, the PBMCs are cultured with
antiCD3/antiCD28 antibodies (DynaBeadsg) in a 1:1 ratio (beads:cells) and IL-2
at 3000 IU/ml.
On Day 4, additional IL-2 is added to the culture at 3000 IU/ml. On Day 7, the
culture is again
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stimulated with antiCD3/antiCD28 antibodies (DynaBeads ) in a 1:1 ratio
(beads:cells), and
additional IL-2 at 3000 IU/ml is added to the culture. MILs are harvested on
Day 14, beads are
removed, and MILs are optionally counted and phenotyped.
[00185] In an embodiment of the invention, MTh Method 1 is performed as
follows: On Day 0,
a cryopreserved PBMC sample derived from bone marrow is thawed and the PBMCs
are
counted. The PBMCs are co-cultured in a GRex 24-well plate at 5x105 cells per
well with anti-
CD3/anti-CD28 antibodies (DynaBeads ) at a 1:1 ratio in about 8m1 per well of
CM-2 cell
culture medium (comprised of RPMI-1640, human AB serum, 1-glutamine, 2-
mercaptoethanol,
gentamicin sulfate, AIM-V media) in the presence of IL-2 at 3000IU/ml. On Day
4, the cell
culture media is exchanged with AIM-V supplemented with additional IL-2 at
3000IU/ml. On
Day 7, the expanded MILs are counted. lx106 cells per well are transferred to
a new GRex 24-
well plate and cultured with anti-CD3/anti-CD28 antibodies (DynaBeads ) at a
1:1 ratio in
about 8m1 per well of AIM-V media in the presence of IL-2 at 3000IU/ml. On Day
11, the cell
culture media is exchanged from AIM-V to CM-4 (comprised of AIM-V media, 2mM
Glutamax,
and 3000IU/ml IL2). On Day 14, the DynaBeads are removed using a DynaMag
Magnet
(DynaMagTm15) and the MILs are counted.
[00186] MIL Method 2. In an embodiment of the invention, the method is
performed over 7
days. In an embodiment, the method comprises obtaining PMBCs derived from bone
marrow
and cryopreserving the PBMCs. On Day 0, the PBMCs are cultured with with
antiCD3/antiCD28 antibodies (DynaBeads ) in a 3:1 ratio (beads:cells) and IL-2
at 3000 IU/ml.
MILs are harvested on Day 7, beads are removed, and MILs are optionally
counted and
phenotyped.
[00187] In an embodiment of the invention, MIL Method 2 is performed as
follows: On Day 0,
a cryopreserved PBMC sample is thawed and the PBMCs are counted. The PBMCs are
co-
cultured in a GRex 24-well plate at 5x105 cells per well with anti-CD3/anti-
CD28 antibodies
(DynaBeads ) at a 1:1 ratio in about 8m1 per well of CM-2 cell culture medium
(comprised of
RPMI-1640, human AB serum, 1-glutamine, 2-mercaptoethanol, gentamicin sulfate,
AIM-V
media) in the presence of IL-2 at 3000IU/ml. On Day 7, the DynaBeads are
removed using a
DynaMag Magnet (DynaMagTm15) and the MILs are counted.
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[00188] MIL Method 3. In an embodiment 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. IL-2 is added to the cell culture at 3000 IU/ml. On Day 3, the PBMCs
are cultured
with antiCD3/antiCD28 antibodies (DynaBeadsg) in a 1:1 ratio (beads:cells) and
IL-2 at 3000
IU/ml. On Day 4, additional IL-2 is added to the culture at 3000 IU/ml. On Day
7, the culture is
again stimulated with antiCD3/antiCD28 antibodies (DynaBeadsg) in a 1:1 ratio
(beads:cells),
and additional IL-2 at 3000 IU/ml is added to the culture. On Day 11, IL-2 is
added to the
culture at 3000 IU/ml. MILs are harvested on Day 14, beads are removed, and
MILs are
optionally counted and phenotyped.
[00189] In an embodiment of the invention, MIL Method 3 is performed as
follows: On Day 0,
the PBMCs are selected for CD45hiCD3+ cells (immune cell fraction) by sorting
method and
CD45lowCD3- fraction (AML blast cell fraction) is sonicated and a portion of
the sonicated cell
fraction is added back to the selected cell fraction. IL-2 is added to the
cell culture at 3000
IU/ml. On Day 3, the PBMCs are cultured with antiCD3/antiCD28 antibodies
(DynaBeadsg) in
a 1:1 ratio (beads:cells) and IL-2 at 3000 IU/ml. On Day 4, additional IL-2 is
added to the
culture at 3000 IU/ml. On Day 7, the culture is again stimulated with
antiCD3/antiCD28
antibodies (DynaBeadsg) in a 1:1 ratio (beads:cells), and additional IL-2 at
3000 IU/ml is added
to the culture. On Day 11, IL-2 is added to the culture at 3000 IU/ml. MILs
are harvested on
Day 14, beads are removed, and MILs are optionally counted and phenotyped.
[00190] In an embodiment of the invention, MIL Method 3 is performed as
follows: On Day 0,
a cryopreserved 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+).
A number of cells from the AML blast cell fraction that is about equal to the
number of cells
from the immune cell fraction (or MIL fraction) to be seeded on a Grex 24-well
plate is
suspended in 100u1 of media and sonicated. In this example, about 2.8x104 to
about 3.38x105
cells from the AML blast cell fraction is taken and suspended in 100u1 of CM2
media and then
sonicated for 30 seconds. The 100u1 of sonicated AML blast cell fraction is
added to the
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immune cell fraction in a Grex 24-well plate. The immune cells are present in
an amount of
about 2.8x104 to about 3.38x105 cells per well in about 8m1 per well of CM-2
cell culture
medium in the presence of IL-2 at 60001U/m1 and are cultured with the portion
of AML blast cell
fraction for about 3 days. On Day 3, anti-CD3/anti-CD28 antibodies
(DynaBeadsg) at a 1:1
ratio are added to the each well and cultured for about 1 day. On Day 4, the
cell culture media is
exchanged with AIM-V supplemented with additional IL-2 at 30001U/ml. On Day 7,
the
expanded MILs are counted. About 1.5x105 to 4x105 cells per well are
transferred to a new
GRex 24-well plate and cultured with anti-CD3/anti-CD28 antibodies
(DynaBeadsg) at a 1:1
ratio in about 8m1 per well of AIM-V medium in the presence of IL-2 at
30001U/ml. On Day 11,
the cell culture media is exchanged from AIM-V to CM-4 (supplemented with IL-2
at
30001U/m1). On Day 14, the DynaBeads are removed using a DynaMag Magnet
(DynaMagTm15) and the MILs are optionally counted.
[00191] In an embodiment of the invention, PBMCs are obtained from bone
marrow. In an
embodiment, the PBMCs are obtained from the bone marrow through apheresis,
aspiration,
needle biopsy, or other similar means known in the art. In an embodiment, the
PBMCs are fresh.
In another embodiment, the PBMCs are cryopreserved.
[00192] In an embodiment of the invention, the method is performed over about
7 days, about
8 days, about 9 days, about 10 days, about 11 days, about 12 days, about 13
days, or about 14
days. In another embodiment, the method is performed over about 7 days. In
another
embodiment, the method is performed over about 14 days.
[00193] In an embodiment of the invention, the PBMCs are cultured with
antiCD3/antiCD28
antibodies. In an embodiment, any available antiCD3/antiCD28 product is useful
in the present
invention. In an embodiment of the invention, the commercially available
product used are
DynaBeads . In an embodiment, the DynaBeads are cultured with the PBMCs in a
ratio of 1:1
(beads:cells). In another embodiment, the antibodies are DynaBeads cultured
with the PBMCs
in a ratio of 1.5:1, 2:1, 2.5:1, 3:1, 3.5:1, 4:1, 4.5:1, or 5:1 (beads:cells).
In any of the foregoing
embodiments, magnetic bead-based selection of an immune cell fraction (or MIL
fraction)
(CD3+CD33+CD2O+CD14+) or an AML blast cell fraction (non-CD3+CD33+CD2O+CD14+)
is
used. In an embodiment of the invention, the antibody culturing steps and/or
the step of
restimulating cells with antibody is performed over a period of from about 2
to about 6 days,
from about 3 to about 5 days, or for about 4 days. In an embodiment of the
invention, the
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antibody culturing step is performed over a period of about 2 days, 3 days, 4
days, 5 days, or 6
days.
[00194] In an embodiment of the invention, the ratio of the number of cells
from the AML blast
cell fraction to the number of cells from the immune cell fraction (or MTh
fraction) is about 0.1:1
to about 10:1. In another embodiment, the ratio is about 0.1:1 to about 5:1,
about 0.1:1 to about
2:1, or about 1:1. In an embodiment of the invention, the AML blast cell
fraction is optionally
disrupted to break up cell aggregation. In an embodiment, the AML blast cell
fraction is
disrupted using sonication, homogenization, cell lysis, vortexing, or
vibration. In another
embodiment, the AML blast cell fraction is disrupted using sonication. In an
embodiment of the
invention, the non-CD3+, non-CD33+, non-CD20+, non-CD14+ cell fraction (AML
blast
fraction) is lysed using a suitable lysis method, including high temperature
lysis, chemical lysis
(such as organic alcohols), enzyme lysis, and other cell lysis methods known
in the art.
[00195] In an embodiment of the invention, the cells from AML blast cell
fraction are
suspended at a concentration of from about 0.2x105 to about 2x105 cells per
100uL and added to
the cell culture with the immune cell fraction. In another embodiment, the
concentration is from
about 0.5x105 to about 2x105 cells per 100uL, from about 0.7x105 to about
2x105 cells per
100uL, from about 1 x105 to about 2x105 cells per 100uL, or from about 1.5x105
to about 2x105
cells per 100uL.
[00196] In an embodiment, the PBMC sample is cultured with IL-2. In an
embodiment of the
invention, the cell culture medium used for expansion of the MILs comprises IL-
2 at a
concentration selected from the group consisting of about 100 IU/mL, about 200
IU/mL, about
300 IU/mL, about 400 IU/mL, about 100 IU/mL, about 100 IU/mL, about 100 IU/mL,
about 100
IU/mL, about 100 IU/mL, about 500 IU/mL, about 600 IU/mL, about 700 IU/mL,
about 800
IU/mL, about 900 IU/mL, about 1,000 IU/mL, about 1,100 IU/mL, about 1,200
IU/mL, about
1,300 IU/mL, about 1,400 IU/mL, about 1,500 IU/mL, about 1,600 IU/mL, about
1,700 IU/mL,
about 1,800 IU/mL, about 1,900 IU/mL, about 2,000 IU/mL, about 2,100 IU/mL,
about 2,200
IU/mL, about 2,300 IU/mL, about 2,400 IU/mL, about 2,500 IU/mL, about 2,600
IU/mL, about
2,700 IU/mL, about 2,800 IU/mL, about 2,900 IU/mL, about 3,000 IU/mL, about
3,100 IU/mL,
about 3,200 IU/mL, about 3,300 IU/mL, about 3,400 IU/mL, about 3,500 IU/mL,
about 3,600
IU/mL, about 3,700 IU/mL, about 3,800 IU/mL, about 3,900 IU/mL, about 4,000
IU/mL, about
4,100 IU/mL, about 4,200 IU/mL, about 4,300 IU/mL, about 4,400 IU/mL, about
4,500 IU/mL,
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about 4,600 IU/mL, about 4,700 IU/mL, about 4,800 IU/mL, about 4,900 IU/mL,
about 5,000
IU/mL, about 5,100 IU/mL, about 5,200 IU/mL, about 5,300 IU/mL, about 5,400
IU/mL, about
5,500 IU/mL, about 5,600 IU/mL, about 5,700 IU/mL, about 5,800 IU/mL, about
5,900 IU/mL,
about 6,000 IU/mL, about 6,500 IU/mL, about 7,000 IU/mL, about 7,500 IU/mL,
about 8,000
IU/mL, about 8,500 IU/mL, about 9,000 IU/mL, about 9,500 IU/mL, and about
10,000 IU/mL.
[00197] In an embodiment of the invention, additional IL-2 may be added to the
culture on one
or more days throughout the method. In an embodiment of the invention,
additional IL-2 is
added on Day 4. In an embodiment of the invention, additional IL-2 is added on
Day 7. In an
embodiment of the invention, additional IL-2 is added on Day 11. In another
embodiment,
additional IL-2 is added on Day 4, Day 7, and/or Day 11. In an embodiment of
the invention, the
MILs are cultured with additional IL-2 for a period of 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.
In an embodiment
of the invention, MILs are cultured for a period of 3 days after each addition
of IL-2.
[00198] In an embodiment, the cell culture medium is exchanged at least once
time during the
method. In an embodiment, the cell culture medium is exchanged at the same
time that
additional IL-2 is added. In another embodiment the cell culture medium is
exchanged on at
least one of Day 1, Day 2, Day 3, Day 4, Day 5, Day 6, Day 7, Day 8, Day 9,
Day 10, Day 11,
Day 12, Day 13, or Day 14. In an embodiment of the invention, the cell culture
medium used
throughout the method may be the same or different. In an embodiment of the
invention, the cell
culture medium is CM-2, CM-4, or AIM-V. In an embodiment of the invention, the
cell culture
medium exchange step on Day 11 is optional.In an embodiment of the invention,
the starting cell
number of PBMCs for the expansion process is from about 25,000 to about
1,000,000, from
about 30,000 to about 900,000, from about 35,000 to about 850,000, from about
40,000 to about
800,000, from about 45,000 to about 800,000, from about 50,000 to about
750,000, from about
55,000 to about 700,000, from about 60,000 to about 650,000, from about 65,000
to about
600,000, from about 70,000 to about 550,000, preferably from about 75,000 to
about 500,000,
from about 80,000 to about 450,000, from about 85,000 to about 400,000, from
about 90,000 to
about 350,000, from about 95,000 to about 300,000, from about 100,000 to about
250,000, from
about 105,000 to about 200,000, or from about 110,000 to about 150,000. In an
embodiment of
the invention, the starting cell number of PBMCs is about 138,000, 140,000,
145,000, or more.
In another embodiment, the starting cell number of PBMCs is about 28,000. In
another
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embodiment, the starting cell number of PBMCs is about 62,000. In another
embodiment, the
starting cell number of PBMCs is about 338,000. In another embodiment, the
starting cell
number of PBMCs is about 336,000.
[00199] In an embodiment of the invention, the fold expansion of MILs is from
about 20% to
about 100%, 25% to about 95%, 30% to about 90%, 35% to about 85%, 40% to about
80%, 45%
to about 75%, 50% to about 100%, or 25% to about 75%. In an embodiment of the
invention,
the fold expansion is about 25%. In another embodiment of the invention, the
fold expansion is
about 50%. In another embodiment, the fold expansion is about 75%.
[00200] In an embodiment of the invention, MILs are expanded from 10-50 ml of
bone marrow
aspirate. In an embodiment of the invention, 10m1 of bone marrow aspirate is
obtained from the
patient. In another embodiment, 20m1 of bone marrow aspirate is obtained from
the patient. In
another embodiment, 30m1 of bone marrow aspirate is obtained from the patient.
In another
embodiment, 40m1 of bone marrow aspirate is obtained from the patient. In
another
embodiment, 50m1 of bone marrow aspirate is obtained from the patient.
[00201] In an embodiment of the invention, the number of PBMCs yielded from
about 10-50m1
of bone marrow aspirate is about 5x107 to about 10x107PBMCs. In another
embodiment, the
number of PMBCs yielded is about 7x107PBMCs.
[00202] In an embodiment of the invention, about 5x107 to about 10x107PBMCs,
yields about
0.5x106 to about 1.5x106 expansion starting cell material. In an embodiment of
the invention,
about lx 106 expansion starting cell material is yielded.
[00203] In an embodiment of the invention, the total number of MILs harvested
at the end of the
expansion period is from about 0.01x109 to about 1x109, from about 0.05x109 to
about 0.9x109,
from about 0.1x109 to about 0.85x109, from about 0.15x109 to about 0.7x109,
from about 0.2x109
to about 0.65x109, from about 0.25x109 to about 0.6x109, from about 0.3x109 to
about 0.55x109,
from about 0.35x109 to about 0.5x109, or from about 0.4x109 to about 0.45x109.
[00204] In an embodiment of the invention, 12x106PBMC derived from bone marrow
aspirate
yields approximately 1.4x105 starting cell material, which yields about
1.1x107 MILs at the end
of the expansion process.
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[00205] In an embodiment of the invention, the MILs expanded from bone marrow
PBMCs
using MIL Method 3 described above comprise a high proportion of CD8+ cells
and lower
number of LAG3+ and PD1+ cells as compared with MILs expanded using MIL Method
1 or
MIL Method 2. In an embodiment of the invention, PBLs expanded from blood PBMC
using
MIL Method 3 described above comprise a high proportion of CD8+ cells and
increased levels
of IFNy production as compared with PBLs expanded using MIL Method 1 or MIL
Method 2.
[00206] In an embodiment of the invention, the clinical dose of MILs useful
for patients with
acute myeloid leukemia (AML) is in the range of from about 4x108 to about
2.5x109 MILs. In
another embodiment, the number of MILs provided in the pharmaceutical
compositions of the
invention is 9.5x108 MILs. In another embodiment, the number of MILs provided
in the
pharmaceutical compositions of the invention is 4.1x108. In another
embodiment, the number of
MILs provided in the pharmaceutical compositions of the invention is 2.2x109.
[00207] 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.
Methods for Expansion of TILs Using the "2A Process"
[00208] In an embodiment of the present invention, the invention provides
devices and methods
to expand T cells derived from bone marrow and/or peripheral blood. In an
embodiment of the
invention, the T cells have a heightened tumor specificity from the bone
marrow
microenvironment in a polyclonal but highly tumor-specific manner. In an
embodiment, the
bone marrow microenvironment is used to sustain and expand the T-cells. In an
embodiment of
the invention, there is roughly a 25 to 100-fold expansion of TILs in a 7-day
or 14-day expansion
process. In an embodiment, the fold expansion of TILs is from about 30-90-
fold. In an
embodiment, the fold expansion is from about 35-85-fold. In an embodiment, the
fold expansion
is from about 40-80-fold. In an embodiment, the fold expansion is from about
45-75-fold. In
another embodiment, the fold expansion is from about 40-70-fold. In another
embodiment, the
fold expansion is from about 45-65-fold. In another embodiment, the fold
expansion is about 25-
fold, about 30-fold, about 35-fold, about 40-fold, about 45-fold, and 50-fold,
about 55-fold,
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about 60-fold, about 65-fold, about 70 fold, about 75-fold, about 80-fold,
about 85-fold, about
90-fold, about 95-fold, or about 100-fold expansion.
[00209] In an embodiment of the invention, the T-cell manufacturing process
does not require
any intervention to select for tumor specificity. In an embodiment of the
invention, the T-cell
manufacturing process does not require the presence of tumor in the marrow
and/or peripheral
blood at the time of T-cell expansion. In an embodiment, the T-cells are
expanded in the
presence of almost complete bone marrow.
[00210] In an embodiment, the invention provides a method for extracting T-
cells from bone
marrow and/or peripheral blood as described in the Examples, and in
particular, Example 21, set
forth in W02010/062742, which is incorporated herein by reference. In an
embodiment, the
invention provides a method for extracting T-cells from bone marrow and/or
peripheral blood as
described in, for example, Noonan, et al., 2005, Cancer Res. 65:2026-2034,
which is
incorporated herein by reference.
[00211] In an embodiment, methods for obtaining bone marrow and/or peripheral
blood that are
known to someone skilled in the art are useful in the present invention. In an
embodiment of the
invention, bone marrow and/or peripheral blood is obtained using needle
aspiration. In an
embodiment of the invention, bone marrow from a patient is aspirated into
heparin-containing
syringes and stored overnight at room temperature. In an embodiment of the
invention, after
storage, the contents of the syringes are pooled together into a sterile
container and quality
tested. The bone marrow is enriched for mononuclear cells (MNCs) using
lymphocyte
separation media (LSM) and centrifugation with a COBE Spectra. Cells in the
gradient are
collected down to the red blood cells and washed using HBSS. The MNCs are
cryopreserved
using a hetastarch-based cryoprotectant supplemented with 2% HSA and 5% DMSO,
reserving
some of the MNCs for quality control. The QC vial is thawed to determine the
CD3+ and
CD38+/138+ cell content of the MNC product. It is important to note that the
collection of bone
marrow is not a limitation to the present invention.
[00212] In an embodiment of the invention, bone marrow is aspirated and
fractionated on a
Lymphocyte Separation Medium density gradient and cells are collected almost
to the level of
the red cell pellet. In an embodiment, this fractionation method substantially
removes red blood
cells and neutrophils, providing nearly complete bone marrow. In an
embodiment, the resulting
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fractionated material is T-cells and tumor cells. In an embodiment of the
invention, the methods
may be practiced without a T-cell specific separation step, and without a
tumor cell separation
step, such as, for example, without labeling T-cells with antibodies or other
cell-type specific
detectable labels, and without sorting using fluorescence activated cell
sorting (FACS).
[00213] In an embodiment of the invention, the obtained bone marrow is
Ficolled or the
peripheral blood is suspended in serum-free conditions at 1 x 106 cells/mL in
AIM-V medium at
200uL/well.
[00214] In an embodiment of the invention, the bone marrow is collected from a
subject who is
not in complete remission. In an embodiment of the invention, the bone marrow
is collected
from a subject who is in complete remission.
[00215] In an embodiment of the invention, the bone marrow may be obtained and
frozen. In
an embodiment, the bone marrow may be obtained and immediately used to extract
T-cells.
[00216] In further embodiments and in accordance with any of the above, the
invention
provides a method of expanding TILs, the method comprising contacting a
population of TILs
comprising at least one TIL obtained from a liquid tumor. All discussion of
expanding TILs
herein are applicable to expansion of TILs obtained from bone marrow,
peripheral blood, and/or
a hematological malignancy, including a liquid tumor.
[00217] In an embodiment, the invention provides a process for the preparation
of a population
of tumor infiltrating lymphocytes (TILs) from a tumor obtained from a cancer,
the process
comprising the steps of:
(a) contacting a fragmented tumor comprising a first population of TILs with a
first cell
culture medium;
(b) performing an initial expansion (pre-REP) 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;
(c) performing a second expansion of the second population of TILs in a second
cell culture
medium to obtain a third population of TILs, wherein the third population of
TILs is at
least 50-fold greater in number than the second population of TILs after 7
days from the
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start of the second expansion; wherein the second cell culture medium
comprises IL-2,
OKT-3 (anti-CD3 antibody), irradiated allogeneic peripheral blood mononuclear
cells
(PBMCs); and wherein the second expansion is performed over a period of 14
days or
less;
(d) harvesting the third population of TILs; and
wherein the tumor is a liquid tumor, and wherein the cancer is a hematological
malignancy.
[00218] In an embodiment, the invention provides a process for expanding a
population of TILs
including a first pre-rapid expansion (pre-REP) process and then a second
expansion process
(which can be a rapid expansion process - REP), wherein the cell culture
medium used for
expansion comprises IL-2 at a concentration selected from the group consisting
of between 100
IU/mL and 10,000 IU/mL, between 200 IU/mL and 5,000 IU/mL, between 300 IU/mL
and 4,800
IU/mL, between 400 IU/mL and 4,600 IU/mL, between 500 IU/mL and 4,400 IU/mL,
between
600 IU/mL and 4,200 IU/mL, between 700 IU/mL and 4,000 IU/mL, between 800
IU/mL and
3,800 IU/mL, between 900 IU/mL and 3,600 IU/mL, between 1,000 IU/mL and 3,400
IU/mL,
between 1,100 IU/mL and 3,200 IU/mL, between 1,200 IU/mL and 3,000 IU/mL,
between 1,300
IU/mL and 2,800 IU/mL, between 1,400 IU/mL and 2,600 IU/mL, between 1,500
IU/mL and
2,400 IU/mL, between 1,600 IU/mL and 2,200 IU/mL, between 1,700 IU/mL and
2,000 IU/mL,
between 5,500 IU/mL and 9,500 IU/mL, between 6,000 IU/mL and 9,000 IU/mL,
between 6500
IU/mL and 8,500 IU/mL, between 7,000 IU/mL and 8,000 IU/mL, and between 7,500
IU/mL
and 8,000 IU/mL.
[00219] In an embodiment, the invention provides a process for expanding a
population of TILs
including a pre-rapid expansion (pre-REP) process and a rapid expansion
process (REP),
wherein the cell culture medium used for expansion comprises IL-2 at a
concentration selected
from the group consisting of about 100 IU/mL, about 200 IU/mL, about 300
IU/mL, about 400
IU/mL, about 100 IU/mL, about 100 IU/mL, about 100 IU/mL, about 100 IU/mL,
about 100
IU/mL, about 500 IU/mL, about 600 IU/mL, about 700 IU/mL, about 800 IU/mL,
about 900
IU/mL, about 1,000 IU/mL, about 1,100 IU/mL, about 1,200 IU/mL, about 1,300
IU/mL, about
1,400 IU/mL, about 1,500 IU/mL, about 1,600 IU/mL, about 1,700 IU/mL, about
1,800 IU/mL,
about 1,900 IU/mL, about 2,000 IU/mL, about 2,100 IU/mL, about 2,200 IU/mL,
about 2,300
IU/mL, about 2,400 IU/mL, about 2,500 IU/mL, about 2,600 IU/mL, about 2,700
IU/mL, about
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2,800 IU/mL, about 2,900 IU/mL, about 3,000 IU/mL, about 3,100 IU/mL, about
3,200 IU/mL,
about 3,300 IU/mL, about 3,400 IU/mL, about 3,500 IU/mL, about 3,600 IU/mL,
about 3,700
IU/mL, about 3,800 IU/mL, about 3,900 IU/mL, about 4,000 IU/mL, about 4,100
IU/mL, about
4,200 IU/mL, about 4,300 IU/mL, about 4,400 IU/mL, about 4,500 IU/mL, about
4,600 IU/mL,
about 4,700 IU/mL, about 4,800 IU/mL, about 4,900 IU/mL, about 5,000 IU/mL,
about 5,100
IU/mL, about 5,200 IU/mL, about 5,300 IU/mL, about 5,400 IU/mL, about 5,500
IU/mL, about
5,600 IU/mL, about 5,700 IU/mL, about 5,800 IU/mL, about 5,900 IU/mL, about
6,000 IU/mL,
about 6,500 IU/mL, about 7,000 IU/mL, about 7,500 IU/mL, about 8,000 IU/mL,
about 8,500
IU/mL, about 9,000 IU/mL, about 9,500 IU/mL, and about 10,000 IU/mL.
[00220] In an embodiment, the invention provides a process for expanding a
population of TILs
including a pre-rapid expansion (pre-REP) process. In an embodiment, the
invention provides a
pre-REP process of expanding a population of TILs, the pre-REP process
comprising the steps of
contacting the population of TILs obtained from a liquid tumor with a cell
culture medium,
wherein the cell culture medium further comprises IL-2 at an initial
concentration of between
1000 IU/mL and 6000 IU/mL.
[00221] In an embodiment, the invention provides a pre-REP process for
expanding a
population of TILs, the process comprising the steps of contacting the
population of TILs
obtained from a liquid tumor with a cell culture medium, wherein the cell
culture medium further
comprises IL-2 at an initial concentration of about 6000 IU/mL.
[00222] In an embodiment, REP can be performed in a gas permeable container
using the TILs
obtained from a liquid tumor according to the present disclosure by any
suitable method. 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, about 30 ng/mL of OKT-3, a monoclonal anti-
CD3 antibody
(commercially available from Ortho-McNeil, Raritan, NJ or Miltenyi Biotech,
Auburn, CA).
TILs can be rapidly expanded by further stimulation of the TILs in vitro with
one or more
antigens, 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 [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
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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.
[00223] In an embodiment, a method for expanding TILs may include using about
5000 mL to
about 25000 mL of cell culture medium, about 5000 mL to about 10000 mL of cell
culture
medium, or about 5800 mL to about 8700 mL of cell culture medium. In an
embodiment, a
method for expanding TILs may include using about 1000 mL to about 2000 mL of
cell medium,
about 2000 mL to about 3000 mL of cell culture medium, about 3000 mL to about
4000 mL of
cell culture medium, about 4000 mL to about 5000 mL of cell culture medium,
about 5000 mL to
about 6000 mL of cell culture medium, about 6000 mL to about 7000 mL of cell
culture
medium, about 7000 mL to about 8000 mL of cell culture medium, about 8000 mL
to about 9000
mL of cell culture medium, about 9000 mL to about 10000 mL of cell culture
medium, about
10000 mL to about 15000 mL of cell culture medium, about 15000 mL to about
20000 mL of
cell culture medium, or about 20000 mL to about 25000 mL of cell culture
medium. In an
embodiment, 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-
glutamine, 5011M
streptomycin sulfate, and 1011M 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 an
embodiment,
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.
[00224] In an embodiment, a second expansion is performed using a gas
permeable container.
Such embodiments allow for cell populations to expand from about 5 x 105
cells/cm2 to between
x 106 and 30 x 106 cells/cm2. In an embodiment, this expansion occurs without
feeding. In
an embodiment, this expansion occurs without feeding so long as medium resides
at a height of
about 10 cm in a gas-permeable flask. In an embodiment this is without feeding
but with the
addition of one or more cytokines. In an embodiment, the cytokine can be added
as a bolus
without any need to mix the cytokine with the medium. Such containers,
devices, and methods
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are known in the art and have been used to expand TILs, and include those
described in U.S.
Patent Application Publication No. US 2014/0377739 Al, International Patent
Application
Publication No. WO 2014/210036 Al, U.S. Patent Application Publication No. US
2013/0115617 Al, International Publication No. WO 2013/188427 Al, U.S. Patent
Application
Publication No. US 2011/0136228 Al, U.S. Patent No. 8,809,050, International
Patent
Application 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
Patent Application Publication No. WO 2012/129201 Al, U.S. Patent Application
Publication
No. US 2013/0102075 Al, U.S. Patent No. 8,956,860, International Patent
Application
Publication No. WO 2013/173835 Al, and 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, et al., I Immunotherapy 2012, 35, 283-
292, the disclosure of
which is incorporated by reference herein.
[00225] In an embodiment, the gas permeable container is a G-Rex 10 flask
(Wilson Wolf
Manufacturing Corporation, New Brighton, MN, USA). In an embodiment, the gas
permeable
container includes a 10 cm2 gas permeable culture surface. In an embodiment,
the gas permeable
container includes a 40 mL cell culture medium capacity. In an embodiment, the
gas permeable
container provides 100 to 300 million TILs after 2 medium exchanges.
[00226] In an embodiment, the gas permeable container is a G-Rex 100 flask
(Wilson Wolf
Manufacturing Corporation, New Brighton, MN, USA). In an embodiment, the gas
permeable
container includes a 100 cm2 gas permeable culture surface. In an embodiment,
the gas
permeable container includes a 450 mL cell culture medium capacity. In an
embodiment, the gas
permeable container provides 1 to 3 billion TILs after 2 medium exchanges.
[00227] In an embodiment, the gas permeable container is a G-Rex 100M flask
(Wilson Wolf
Manufacturing Corporation, New Brighton, MN, USA). In an embodiment, the gas
permeable
container includes a 100 cm2 gas permeable culture surface. In an embodiment,
the gas
permeable container includes a 1000 mL cell culture medium capacity. In an
embodiment, the
gas permeable container provides 1 to 3 billion TILs without medium exchange.
[00228] In an embodiment, the gas permeable container is a G-Rex 100L flask
(Wilson Wolf
Manufacturing Corporation, New Brighton, MN, USA). In an embodiment, the gas
permeable
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container includes a 100 cm2 gas permeable culture surface. In an embodiment,
the gas
permeable container includes a 2000 mL cell culture medium capacity. In an
embodiment, the
gas permeable container provides 1 to 3 billion TILs without medium exchange.
[00229] In an embodiment, the gas permeable container is a G-Rex 24 well plate
(Wilson Wolf
Manufacturing Corporation, New Brighton, MN, USA). In an embodiment, the gas
permeable
container includes a plate with wells, wherein each well includes a 2 cm2 gas
permeable culture
surface. In an embodiment, the gas permeable container includes a plate with
wells, wherein
each well includes an 8 mL cell culture medium capacity. In an embodiment, the
gas permeable
container provides 20 to 60 million cells per well after 2 medium exchanges.
[00230] In an embodiment, the gas permeable container is a G-Rex 6 well plate
(Wilson Wolf
Manufacturing Corporation, New Brighton, MN, USA). In an embodiment, the gas
permeable
container includes a plate with wells, wherein each well includes a 10 cm2 gas
permeable culture
surface. In an embodiment, the gas permeable container includes a plate with
wells, wherein
each well includes a 40 mL cell culture medium capacity. In an embodiment, the
gas permeable
container provides 100 to 300 million cells per well after 2 medium exchanges.
[00231] In an embodiment, 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 an embodiment, the cell medium in the first
and/or second gas
permeable container lacks beta-mercaptoethanol (BME).
[00232] In an embodiment, 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 therein; obtaining TILs from the tumor tissue sample;
expanding the
number of TILs in a second gas permeable container containing cell medium
therein for a
duration of about 14 to about 42 days, e.g., about 28 days.
[00233] In an embodiment, the cell culture medium comprises IL-2. In a
preferred
embodiment, the cell culture medium comprises about 3000 IU/mL of IL-2. In an
embodiment,
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 an embodiment,
the cell
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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.
[00234] In an embodiment, the cell culture medium comprises OKT-3 antibody. In
a preferred
embodiment, the cell culture medium comprises about 30 ng/mL of OKT-3
antibody. In an
embodiment, 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 pg/mL of OKT-3 antibody. In an
embodiment,
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.
[00235] In an embodiment, 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.
U.S. Patent Application Publication No. 2005/0106717 Al, the disclosures of
which are
incorporated herein by reference. In an embodiment, TILs are expanded in gas-
permeable bags.
In an embodiment, 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
an
embodiment, 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 an embodiment, 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, about 10 L, about 11 L, about 12 L, about 13 L, about 14 L,
about 15 L, about 16
L, about 17 L, about 18 L, about 19 L, about 20 L, about 25 L, and about 30 L.
In an
embodiment, the cell expansion system includes a gas permeable cell bag with a
volume range
selected from the group consisting of between 50 and 150 mL, between 150 and
250 mL,
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between 250 and 350 mL, between 350 and 450 mL, between 450 and 550 mL,
between 550 and
650 mL, between 650 and 750 mL, between 750 and 850 mL, between 850 and 950
mL, and
between 950 and 1050 mL. In an embodiment, the cell expansion system includes
a gas
permeable cell bag with a volume range selected from the group consisting of
between 1 L and 2
L, between 2 L and 3 L, between 3 L and 4 L, between 4 L and 5 L, between 5 L
and 6 L,
between 6 L and 7 L, between 7 L and 8 L, between 8 L and 9 L, between 9 L and
10 L, between
L and 11 L, between 11 L and 12 L, between 12 L and 13 L, between 13 L and 14
L, between
14 Land 15 L, between 15 Land 16 L, between 16 Land 17 L, between 17 Land 18
L, between
18 L and 19 L, and between 19 L and 20 L. In an embodiment, the cell expansion
system
includes a gas permeable cell bag with a volume range selected from the group
consisting of
between 0.5 L and 5 L, between 5 L and 10 L, between 10 L and 15 L, between 15
L and 20 L,
between 20 L and 25 L, and between 25 L and 30 L. In an embodiment, the cell
expansion
system utilizes a rocking time of about 30 minutes, about 1 hour, about 2
hours, about 3 hours,
about 4 hours, about 5 hours, about 6 hours, about 7 hours, about 8 hours,
about 9 hours, about
10 hours, about 11 hours, about 12 hours, about 24 hours, about 2 days, about
3 days, about 4
days, about 5 days, about 6 days, about 7 days, about 8 days, about 9 days,
about 10 days, about
11 days, about 12 days, about 13 days, about 14 days, about 15 days, about 16
days, about 17
days, about 18 days, about 19 days, about 20 days, about 21 days, about 22
days, about 23 days,
about 24 days, about 25 days, about 26 days, about 27 days, and about 28 days.
In an
embodiment, the cell expansion system utilizes a rocking time of between 30
minutes and 1
hour, between 1 hour and 12 hours, between 12 hours and 1 day, between 1 day
and 7 days,
between 7 days and 14 days, between 14 days and 21 days, and between 21 days
and 28 days. In
an embodiment, the cell expansion system utilizes a rocking rate of about 2
rocks/minute, about
5 rocks/minute, about 10 rocks/minute, about 20 rocks/minute, about 30
rocks/minute, and about
40 rocks/minute. In an embodiment, the cell expansion system utilizes a
rocking rate of between
2 rocks/minute and 5 rocks/minute, 5 rocks/minute and 10 rocks/minute, 10
rocks/minute and 20
rocks/minute, 20 rocks/minute and 30 rocks/minute, and 30 rocks/minute and 40
rocks/minute.
In an embodiment, the cell expansion system utilizes a rocking angle of about
2 , about 3 , about
4 , about 5 , about 6 , about 7 , about 8 , about 9 , about 10 , about 11 ,
and about 12 . In an
embodiment, the cell expansion system utilizes a rocking angle of between 2
and 3 , between 3
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and 4 , between 4 and 5 , between 5 and 6 , between 6 and 7 , between 7
and 8 , between 8
and 9 , between 9 and 10 , between 10 and 11 , and between 11 and 12 .
[00236] In an embodiment, a method of expanding TILs obtained from a liquid
tumor 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.
[00237] In an embodiment, the invention provides a method of expanding a
population of TILs
from a liquid tumor, the method comprising the steps as described in Jin, et
at.,
Immunotherapy 2012, 35, 283-292, the disclosure of which is incorporated by
reference herein.
For example, the tumor or portion thereof may be placed in enzyme media and
mechanically
dissociated for approximately 1 minute. The mixture may then be incubated for
30 minutes at 37
C in 5% CO2 and then mechanically disrupted again for approximately 1 minute.
After
incubation for 30 minutes at 37 C in 5% CO2, the tumor or portion thereof may
be mechanically
disrupted a third time for approximately 1 minute. If after the third
mechanical disruption, large
pieces of tissue are present, 1 or 2 additional mechanical dissociations may
be applied to the
sample, with or without 30 additional minutes of incubation at 37 C in 5%
CO2. 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 may be performed to remove
these cells. TIL
cultures were initiated in 24-well plates (Costar 24-well cell culture
cluster, flat bottom; Corning
Incorporated, Corning, NY), each well may be seeded with lx106tumor digest
cells or one tumor
fragment approximately 1 to 8 mm3 in size in 2 mL of complete medium (CM) with
IL-2 (6000
IU/mL; Chiron Corp., Emeryville, CA). CM comprises Roswell Park Memorial
Institute (RPMI)
1640 buffer with GlutaMAX, supplemented with 10% human AB serum, 25 mM Hepes,
and 10
mg/mL gentamicin. Cultures may be initiated in gas-permeable flasks with a 40
mL capacity
and a 10 cm2 gas-permeable silicon bottom (G-Rex 10; Wilson Wolf
Manufacturing, New
Brighton, each flask may be loaded with 10-40x106 viable tumor digest cells or
5-30 tumor
fragments in 10-40 mL of CM with IL-2. G-Rex 10 and 24-well plates may be
incubated in a
humidified incubator at 37 C in 5% CO2 and 5 days after culture initiation,
half the media may
be removed and replaced with fresh CM and IL-2 and after day 5, half the media
may be
changed every 2-3 days. A second expansion protocol (REP) of TILs may be
performed using
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T-175 flasks and gas-permeable bags or gas-permeable G-Rex flasks, as
described elsewhere
herein, using TILs obtains from the liquid tumors of the present disclosure.
For REP in T-175
flasks, 1x106 TILs may be suspended in 150 mL of media in each flask. The TIL
may be
cultured in a 1 to 1 mixture of CM and AIM-V medium (50/50 medium),
supplemented with
3000 IU/mL of IL-2 and 30 ng/mL of anti-CD3 antibody (OKT-3). The T-175 flasks
may be
incubated at 37 C in 5% CO2. Half the media may be changed on day 5 using
50/50 medium
with 3000 IU/mL of IL-2. On day 7, cells from 2 T-175 flasks may be combined
in a 3L bag and
300 mL of AIM-V with 5% human AB serum and 3000 IU/mL of IL-2 may be added to
the 300
mL of TIL suspension. The number of cells in each bag may be counted every day
or two days,
and fresh media may be added to keep the cell count between 0.5 and 2.0x106
cells/mL. For
REP in 500 mL capacity flasks with 100 cm2 gas-permeable silicon bottoms
(e.g., G-Rex 100,
Wilson Wolf Manufacturing, as described elsewhere herein), 5x106 or 10x106
TILs may be
cultured in 400 mL of 50/50 medium, supplemented with 3000 IU/mL of IL-2 and
30 ng/mL of
anti-CD3 antibody (OKT-3). The G-Rex100 flasks may be incubated at 37 C in 5%
CO2. On
day five, 250 mL of supernatant may be removed and placed into centrifuge
bottles and
centrifuged at 1500 rpm (491 g) for 10 minutes. The obtained TIL pellets may
be resuspended
with 150 mL of fresh 50/50 medium with 3000 IU/mL of IL-2 and added back to
the G-Rex 100
flasks. When TIL are expanded serially in G-Rex 100 flasks, on day seven the
TIL in each G-
Rex100 are suspended in the 300 mL of media present in each flask and the cell
suspension may
be divided into three 100 mL aliquots that may be used to seed 3 G-Rex100
flasks. About 150
mL of AIM-V with 5% human AB serum and 3000 IU/mL of IL-2 may then be added to
each
flask. G-Rex 100 flasks may then be incubated at 37 C in 5% CO2, and after
four days, 150 mL
of AIM-V with 3000 IU/mL of IL-2 may be added to each G-Rex 100 flask. After
this, the REP
may be completed by harvesting cells on day 14 of culture.
[00238] In an embodiment, a method of expanding or treating a cancer includes
a step wherein
TILs are obtained from a patient tumor sample. A patient tumor sample may be
obtained using
methods known in the art. For example, TILs may be cultured from enzymatic
tumor digests and
tumor fragments (about 1 to about 8 mm3 in size) from sharp dissection. 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
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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.
[00239] In an embodiment, the second/REP expansion process for TILs may be
performed
using T-175 flasks and gas permeable bags as previously described (Tran, et
at., I Immunother.
2008, 3/, 742-51; Dudley, et al., I Immunother. 2003, 26, 332-42) or gas
permeable cultureware
(G-Rex flasks, commercially available from Wilson Wolf Manufacturing
Corporation, New
Brighton, MN, USA). For TIL expansion in T-175 flasks, 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 (international units) per mL of IL-
2 and 30 ng
per ml of anti-CD3 antibody (e.g., OKT-3). The T-175 flasks may be 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. 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 IL-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.
[00240] In an embodiment, for second/REP TIL expansions in 500 mL capacity gas
permeable
flasks with 100 cm2 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 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 (OKT-3). 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 (revolutions per minute; 491 x
g) for 10 minutes.
The TIL pellets may be re-suspended with 150 mL of fresh medium with 5% human
AB serum,
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3000 IU per mL of IL-2, and added back to the original G-Rex 100 flasks. When
TILs are
expanded serially in G-Rex 100 flasks, on day 7 the TILs in each G-Rex 100
flask 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.
[00241] In an embodiment, TILs may be prepared as follows. 2 mm3 tumor
fragments are
cultured in complete media (CM) comprised of AIM-V medium (Invitrogen Life
Technologies,
Carlsbad, CA) supplemented with 2 mM glutamine (Mediatech, Inc. Manassas, VA),
100 U/mL
penicillin (Invitrogen Life Technologies), 100 [tg/mL streptomycin (Invitrogen
Life
Technologies), 5% heat-inactivated human AB serum (Valley Biomedical, Inc.
Winchester, VA)
and 600 IU/mL rhIL-2 (Chiron, Emeryville, CA). For enzymatic digestion of
liquid tumors,
tumor specimens are diced into RPMI-1640, washed and centrifuged at 800 rpm
for 5 minutes at
15-22 C, and resuspended in enzymatic digestion buffer (0.2 mg/mL Collagenase
and 30
units/ml of DNase in RPMI-1640) followed by overnight rotation at room
temperature. TILs
established from fragments may be grown for 3-4 weeks in CM and expanded fresh
or
cryopreserved in heat-inactivated HAB serum with 10% dimethylsulfoxide (DMSO)
and stored
at -180 C until the time of study. Tumor associated lymphocytes (TAL) obtained
from ascites
collections were seeded at 3 x 106 cells/well of a 24 well plate in CM. TIL
growth was inspected
about every other day using a low-power inverted microscope.
An Exemplary Embodiment of the TIL Manufacturing Process (the "2A Process")
[00242] An exemplary TIL manufacturing/expansion process known as process
2A is
schematically illustrated in FIG. 22. In certain aspects, the present methods
produce 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
younger TILs have been described in the literature, for example Donia, at al.,
Scandinavian
Journal of Immunology, 75:157-167 (2012); Dudley et al., Clin Cancer Res,
16:6122-6131
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(2010); Huang etal., J Immunother , 28(3):258-267 (2005); Besser etal., Clin
Cancer Res,
19(17):0F1-0F9 (2013); Besser et al., J Immunother 32:415-423 (2009); Robbins,
et al., J
Immunol 2004; 173:7125-7130; Shen etal., J Immunother, 30:123-129 (2007);
Zhou, etal., J
Immunother, 28:53-62 (2005); and Tran, etal., J Immunother, 31:742-751 (2008),
all of which
are incorporated herein by reference in their entireties.
[00243] As discussed herein, the present invention can include a step relating
to the
restimulation of cyropreserved 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.
[00244] In some embodiments, the TILs may be cryopreserved. Once thawed, they
may also be
restimulated to increase their metabolism prior to infusion into a patient.
[00245] In some embodiments, the first expansion (including processes referred
to as the
preREP) is shortened in comparison to conventional expansion methods to 7-14
days and the
second expansion (including processes referred to as the REP) is shortened to
7-14 days, as
discussed in detail below as well as in the examples and figures.
[00246] FIG. 23 illustrates an exemplary 2A process. As illustrated in FIG. 23
and further
explained in detail below, in some embodiments, the first expansion (Step B)
is shortened to 11
days and the second expansion (Step D) is shortened to 11 days. In some
embodiments, the
combination of the first and second expansions (Step B and Step D) is
shortened to 22 days, as
discussed in detail herein. As will be appreciated, the process illustrated in
FIG. 23 and described
below is exemplary and the methods described herein encompass alterations and
additions to the
described steps as well as any combinations. An exemplary embodiment of this
process is
described in U.S. Patent Application No. U52018/0282694 Al, which is herein
incorporated by
reference in its entirety.
STEP A: Obtain patient tumor sample
[00247] In general, TILs are initially obtained from a patient tumor sample
("primary TILs")
and then expanded into a larger population for further manipulation as
described herein,
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optionally cyropreserved, restimulated as outlined herein and optionally
evaluated for phenotype
and metabolic parameters as an indication of TIL health.
[00248] A patient tumor sample may be obtained using methods known in the art,
generally via
surgical resection, needle biopsy, apheresis 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, 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 some embodiments, useful TILs are obtained from
malignant
melanoma tumors, as these have been reported to have particularly high levels
of TILs. In some
embodiments, the tumor is greater than about 1.5 cm but less than about 4 cm.
In some
embodiments, the tumor is less than 4 cm.
[00249] 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
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.
[00250] In general, the harvested cell suspension is called a "primary cell
population" or a
"freshly harvested" cell population.
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[00251] In an embodiment, TILs can be initially cultured from enzymatic tumor
digests and
tumor fragments obtained from patients.
[00252] In some embodiments, the TILs, are obtained from tumor fragments. In
some
embodiments, the tumor fragment is obtained 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 mm3 and 8 mm3. 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 10 mm3. In some
embodiments, about the tumor fragment is about 8-27 mm3. In some embodiments,
about the
tumor fragment is about 10-25 mm3. In some embodiments, about the tumor
fragment is about
15-25 mm3. In some embodiments, the tumor fragment is about 8-20 mm3. In some
embodiments, the tumor fragment is about 15-20 mm3. In some embodiments, the
tumor
fragment is about 8-15 mm3. In some embodiments, the tumor fragment is about 8-
10 mm3.
[00253] In some embodiments, the number of tumor fragments is about 40 to
about 50 tumor
fragments. In some embodiments, the number of tumor fragments is about 40
tumor fragments.
In some embodiments, the number of tumor fragments is about 50 tumor
fragments. In some
embodiments, the tumor fragment size is about 8-27 mm3 and there are less than
about 50 tumor
fragments.
[00254] 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, 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
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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.
STEP B: First Expansion
[00255] After dissection or digestion of tumor fragments in Step A, 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 serum with 6000 IU/mL of IL-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 x 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. In some
embodiments, this primary
cell population is cultured for a period of about 11 days, resulting in a bulk
TIL population,
generally less than or equal to about 200x106 bulk TIL cells.
[00256] In a preferred embodiment, expansion of TILs may be performed using an
initial bulk
TIL expansion step (Step B as pictured in FIG. 23, 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 (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.
[00257] In embodiments where TIL cultures are initiated in 24-well plates, for
example, using
Costar 24-well cell culture cluster, flat bottom (Corning Incorporated,
Corning, NY, each well
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can be seeded with 1 x 106 tumor digest cells or one tumor fragment in 2mL of
complete
medium (CM) with IL-2 (6000 IU/mL; Chiron Corp., Emeryville, CA). In some
embodiments,
the tumor fragment is between about 1 mm3 and 10 mm3.
[00258] In some embodiments, CM for Step B consists of RPMI 1640 with
GlutaMAX,
supplemented with 10% human AB serum, 25mM HEPES, and 10 mg/mL gentamicin. In
embodiments where cultures are initiated in gas-permeable flasks with a 40 mL
capacity and a
10cm2 gas-permeable silicon bottom (for example, G-Rex10; Wilson Wolf
Manufacturing, New
Brighton, MN) (Fig. 1), each flask was loaded with 10-40 x 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% CO2 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.
[00259] In an embodiment, the cell culture medium further comprises IL-2. In a
preferred
embodiment, the cell culture medium comprises about 3000 IU/mL of IL-2. In an
embodiment,
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 an embodiment,
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.
[00260] In some embodiments, the first expansion (including processes referred
to as the pre-
REP; Step B) process is shortened to 3-14 days, as discussed in the examples
and figures. In
some embodiments, the first expansion of Step B is shortened to 7-14 days, as
discussed in the
Examples and shown in Figures 4 and 5. In some embodiments, the first
expansion of Step B is
shortened to 10-14 days, as discussed in the Examples. In some embodiments,
the first
expansion of Step B is shortened to 11 days, as discussed in the Examples.
[00261] In some embodiments, IL-2, IL-7, IL-15, and IL-21 as well as
combinations thereof can
be included during Step B processes as described herein.
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[00262] In some embodiments, Step B 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 GREX-10 or a GREX-100.
STEP C: First Expansion to Second Expansion Transition
[00263] In some embodiments, the bulk TIL population from Step B can be
cryopreserved
immediately, using methods known in the art and described herein.
Alternatively, the bulk TIL
population can be subjected to a second expansion (REP) and then cryopreserved
as discussed
below.
[00264] In some embodiments, the Step B TILs are not stored and the Step B
TILs proceed
directly to Step D. In some embodiments, the transition occurs in a closed
system, as further
described herein.
STEP D: Second Expansion
[00265] In some embodiments, the TIL cell population is expanded in number
after harvest and
initial bulk processing (i.e., after Step A and Step B). This 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). The second expansion is generally accomplished using
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, the second
expansion can include
scaling-up in order to increase the number of TILs obtained in the second
expansion.
[00266] In an embodiment, REP and/or the second expansion can be performed in
a gas
permeable container using the methods of the present disclosure. 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, about 30 ng/ml of OKT3, a mouse monoclonal anti-CD3 antibody
(commercially
available from Ortho-McNeil, Raritan, NJ or Miltenyi Biotech, Auburn, CA).
TILs can be
rapidly expanded further stimulation of the TILs in vitro with one or more
antigens, including
antigenic portions thereof, such as epitope(s), of the cancer, which can be
optionally expressed
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from a vector, such as a human leukocyte antigen A2 (HLA-A2) binding peptide,
e.g., 0.311M
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.
[00267] In an embodiment, the cell culture medium further comprises IL-2. In a
preferred
embodiment, the cell culture medium comprises about 3000 IU/mL of IL-2. In an
embodiment,
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 an embodiment,
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.
[00268] In an embodiment, the cell culture medium comprises OKT3 antibody. In
a preferred
embodiment, the cell culture medium comprises about 30 ng/mL of OKT3 antibody.
In an
embodiment, 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 pg/mL of OKT3 antibody. In an
embodiment,
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 OKT3 antibody.
[00269] In some embodiments, IL-2, IL-7, IL-15, and IL-21 as well as
combinations thereof can
be included during the second expansion in Step D processes as described
herein.
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[00270] In some embodiments, the second expansion can be conducted in a
supplemented cell
culture medium comprising IL-2, OKT-3, and antigen-presenting feeder cells.
[00271] In some embodiments the antigen-presenting feeder cells (APCs) are
PBMCs. In an
embodiment, 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 an
embodiment, 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 an embodiment, the ratio of TILs to PBMCs in
the rapid
expansion and/or the second expansion is between 1 to 100 and 1 to 200.
[00272] In an embodiment, 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 IL-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 GRex
flasks and gas
permeable containers as more fully discussed below.
[00273] In some embodiments, the second expansion (also 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.
[00274] In an embodiment, REP and/or the second expansion may be performed
using T-175
flasks and gas permeable bags as previously described (Tran, et at., I
Immunother. 2008, 3/,
742-51; Dudley, et at., I Immunother. 2003, 26, 332-42) or gas permeable
cultureware (G-Rex
flasks). For TIL rapid expansion and/or second expansion in T-175 flasks, 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 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. 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 IL-2 was added to the 300 ml of TIL suspension.
The number of
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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.
[00275] In an embodiment, REP and/or the second expansion 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, 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-Rexl 00 flask. The cells may be
harvested on
day 14 of culture.
[00276] In an embodiment, 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 IL-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 GRex
flasks and gas
permeable containers as more fully discussed below.
[00277] In an embodiment, REP and/or the second expansion 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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[00278] REP and/or the second expansion of TIL can be performed using T-175
flasks and gas-
permeable bags as previously described (Tran KQ, Zhou J, Durflinger KH, et
al., 2008, J
Immunother , 31:742-751, and Dudley ME, Wunderlich JR, Shelton TE, et al.
2003, J
Immunother , 26:332-342) or gas-permeable G-Rex flasks. In some embodiments,
REP and/or
the second expansion is performed using flasks. In some embodiments, REP is
performed using
gas-permeable G-Rex flasks. For TIL REP and/or the second expansion in T-175
flasks, about 1
x 106 TIL are 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 1 to 100
and the cells were cultured in a 1 to 1 mixture of CM and AIM-V medium (50/50
medium),
supplemented with 3000 IU/mL of IL-2 and 30 ng/mL of anti-CD3. The T-175
flasks are
incubated at 37 C in 5% CO2. 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 106 cells/mL.
[00279] For TIL REP and/or the second expansion in 500 mL capacity flasks with
100 cm2 gas-
permeable silicon bottoms (G-Rex100,Wilson Wolf), about 5 x 106 or 10x 106 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-Rex100
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 IU/ mL
of IL-2 and added back to the original G-Rex100 flasks. In embodiments where
TILs are
expanded serially in G-Rex100 flasks, on day 7 the TIL in each G-Rex100 are
suspended in the
300mL of media present in each flask and the cell suspension was divided into
three 100mL
aliquots that are used to seed 3 G-Rex100 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-Rex100 flasks are
incubated at
37 C in 5% CO2 and after 4 days 150 mL of AIM-V with 3000 IU/mL of IL-2 is
added to each
G-Rex100 flask. The cells are harvested on day 14 of culture.
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Feeder Cells and Antigen Presenting Cells
[00280] In an embodiment, the second expansion procedures described herein
(Step D,
including 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.
[00281] In general, the allogenic PBMCs are inactivated, either via
irradiation or heat treatment,
and used in the REP procedures, as described in the examples, in particular
example 14, which
provides an exemplary protocol for evaluating the replication incompetence of
irradiate
allogeneic PBMCs.
[00282] 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).
[00283] 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 30ng/m1 OKT3 antibody and 3000 IU/ml IL-2.
[00284] 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.
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[00285] In an embodiment, artificial antigen presenting cells are used in the
REP stage as a
replacement for, or in combination with, PBMCs.
Cytokines
[00286] 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.
[00287]
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 generally outlined in U.S. Patent Application Publication
No. US
2017/0107490 Al, International Publication No. WO 2015/189356, U.S. Patent
Application
Publication No. US 2017/0107490 Al, and International Publication No. WO
2015/189357, each
of which is 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.
Anti-CD3 Antibodies
[00288] In some embodiments, the culture media used in expansion methods
described herein
(including REP) also includes 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(ab')2 fragments, with the
former being generally
preferred; see, e.g., Tsoukas et al., I Immunol. 1985, 135, 1719, hereby
incorporated by
reference in its entirety.
[00289] 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 is
used (commercially available from Ortho-McNeil, Raritan, NJ or Miltenyi
Biotech, Auburn,
CA).
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STEP E: Harvest TILs
[00290] After the second expansion step, cells can be harvested. In some
embodiments the
TILs are harvested after one, two, three, four or more second expansion steps.
[00291] 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 be employed with the present process. In some embodiments, TILs
are harvest
using an automated system. In some embodiments, TILs are harvest using a semi-
automated
system. In some embodiments, TILs are harvested using a semi-automated system.
In some
embodiments, the TILs from the second expansion are harvested using a semi-
automated
machine. In some embodiments, the LOVO system is employed (commercially
available from
Benchmark Electronics, for example). In some embodiments, the harvesting step
includes wash
the TILs, formulating the TILs, and/or aliquoting the TILs. In some
embodiments, the cells are
optionally frozen after harvesting or as part of harvesting.
STEP F: Final Formulation/ Transfer to Infusion Bag
[00292] After Steps A through E are complete, cells are transferred to a
container for use in
administration to a patient.
[00293] In an embodiment, TILs expanded using APCs of the present disclosure
are
administered to a patient as a pharmaceutical composition. In an embodiment,
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.
Additional expansion steps
[00294] As will be appreciated, any of the steps A through F described above
can be repeated
any number of times and may in addition be conducted in different orders than
described above.
[00295] In some embodiments, one or more of the expansion steps may be
repeated prior to the
Final Formulation Step F. Such additional expansion steps may include the
elements of the first
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and/or second expansion steps described above (e.g., include the described
components in the
cell culture medium). The additional expansion steps may further include
additional elements,
including additional components in the cell culture medium that are
supplemented into the cell
culture medium before and/or during the additional expansion steps.
[00296] In further embodiments, any of the expansion steps described in FIG.
23 and in the
above paragraphs may be preceded or followed by a cryopreservation step in
which the cells
produced during an expansion step are preserved using methods known in the art
for storage
until needed for the remaining steps of the manufacturing/expansion process.
Pharmaceutical Compositions, Dosages, and Dosing Regimens for TILs, MILs, and
PBLs
[00297] In an embodiment, the invention provides a therapeutic population of
TILs prepared
by any method of expanding TILs described herein, optionally modified to
express a chimeric
antigen receptor (CAR) and/or express a modified T-cell receptor and/or
suppress or reduce
expression of one or more immune checkpoint genes in a transient or stable
manner as described
herein.
[00298] In another embodiment, the invention provides a therapeutic population
of MILs
prepared by any method of expanding MILs described herein, optionally modified
to express a
chimeric antigen receptor (CAR) and/or express a modified T-cell receptor
and/or suppress or
reduce expression of one or more immune checkpoint genes as described herein.
[00299] In another embodiment, the invention provides a therapeutic population
of PBLs
prepared by any method of expanding PBLs described herein, optionally modified
to express a
chimeric antigen receptor (CAR) and/or express a modified T-cell receptor
and/or suppress or
reduce expression of one or more immune checkpoint genes as described herein.
[00300] In another embodiment, the invention provides a pharmaceutical
composition
comprising a therapeutic population of TILs prepared by any method of
expanding TILs
described herein, optionally modified to express a chimeric antigen receptor
(CAR) and/or
express a modified T-cell receptor and/or suppress or reduce expression of one
or more immune
checkpoint genes as described herein, and a pharmaceutically acceptable
carrier.
[00301] In another embodiment, the invention provides a pharmaceutical
composition
comprising a therapeutic population of MILs prepared by any method of
expanding MILs
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described herein, optionally modified to express a chimeric antigen receptor
(CAR) and/or
express a modified T-cell receptor and/or suppress or reduce expression of one
or more immune
checkpoint genes as described herein, and a pharmaceutically acceptable
carrier.
[00302] In another embodiment, the invention provides a pharmaceutical
composition
comprising a therapeutic population of PBLs prepared by any method of
expanding PBLs
described herein, optionally modified to express a chimeric antigen receptor
(CAR) and/or
express a modified T-cell receptor and/or suppress or reduce expression of one
or more immune
checkpoint genes as described herein, and a pharmaceutically acceptable
carrier.
[00303] In an embodiment, TILs expanded using methods of the present
disclosure are
administered to a patient as a pharmaceutical composition. In an embodiment,
the
pharmaceutical composition is a suspension of TILs in a sterile buffer. TILs
expanded using
methods of the present disclosure may be administered by any suitable route as
known in the art.
Preferably, 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 administration.
[00304] Any suitable dose of TILs can be administered. Preferably, from about
2.3 x101 to
about 13.7x1010 TILs are administered, with an average of around 7.8x1010
TILs, particularly if
the cancer is a hematological malignancy. In an embodiment, about 1.2x101 to
about 4.3x10'
of TILs are administered.
[00305] In some embodiments, the number of the TILs provided in the
pharmaceutical
compositions of the invention is about 1 x106, 2x106, 3x106, 4x106, 5x106,
6x106, 7x106, 8x106,
9x106, 1x107, 2x107, 3x107, 4x107, 5x107, 6x107, 7x107, 8x107, 9x107, 1x108,
2x108, 3x108,
4x108, 5x108, 6x108, 7x108, 8x108, 9x108, 1x109, 2x109, 3x109, 4x109, 5x109,
6x109, 7x109,
8x109, 9x109, 1 x101 , 2x1010, 3x1010, 4x1010, 5x1010, 6x1010, 7x1010, 8x1010,
9x1-10 ,
u 1 x10",
2x10n,
3x10", 4x10", 5x10", 6x10", 7x10", 8x10", 9x10", lx1012, 2x1012, 3x1042,
4x1012,
5x1012,
6x1012, 7x101-2, 8x1012, 9x1012,
lx i0'3, 2x1013, 3x1013, 4x1013, 5x1013, 6x1013, 7x1013,
8x1013, and 9x1013. In an embodiment, the number of the TILs provided in the
pharmaceutical
compositions of the invention is in the range of 1x106 to 5x106, 5x106 to
1x107, 1x107 to 5x107,
5x107 t0 1x108, 1x108 to 5x108, 5x108 to 1x109, 1x109 to 5x109, 5x109 to
1x1010,
1X101 I0
5x1010, 5x101 to 1xi's'',
5x10" to 1x10'2, 1x, rsIV12
to 5 x 1012, and 5 x 1012 to 1 x 1013. In an
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embodiment of the invention, the number of TILs provided in the pharmaceutical
compositions
of the invention is in the range of from about 4x108 to about 2.5x109. In
another embodiment,
the number of TILs provided in the pharmaceutical compositions of the
invention is 9.5x108. In
another embodiment, the number of TILs provided in the pharmaceutical
compositions of the
invention is 4.1x108. In another embodiment, the number of TILs provided in
the
pharmaceutical compositions of the invention is 2.2x109.
[00306] In an embodiment of the invention, the number of TILs provided in the
pharmaceutical compositions of the invention is in the range of from about
0.1x109 to about
15x109 TILs, from about 0.1x109 to about 15x109 TILs, from about 0.12x109 to
about 12x109
TILs, from about 0.15x109 to about 11x109 TILs, from about 0.2x109 to about
10x109 TILs, from
about 0.3x109 to about 9x109 TILs, from about 0.4x109 to about 8x109 TILs,
from about 0.5x109
to about 7x109 TILs, from about 0.6x109 to about 6x109 TILs, from about
0.7x109 to about 5x109
TILs, from about 0.8x109 to about 4x109 TILs, from about 0.9x109 to about
3x109 TILs, or from
about 1x109 to about 2x109 TILs.
[00307] 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.
[00308] In 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%,
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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.
[00309] 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.
[00310] 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.
[00311] In 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.
[00312] 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, 0.0009 g, 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,
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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,
8 g, 8.5 g, 9 g, 9.5 g, or 10
g.
[00313] 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.
[00314] 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.
[00315] In some embodiments, an effective dosage of TILs is about lx106,
2x106, 3x106,
4x106, 5x106, 6x106, 7x106, 8x106, 9x106, 1x107, 2x107, 3x107, 4x107, 5x107,
6x107, 7x107,
8x107, 9x107, 1x108, 2x108, 3x108, 4x108, 5x108, 6x108, 7x108, 8x108, 9x108,
1x109, 2x109,
3x109, 4x109, 5x109, 6x109, 7x109, 8x109, 9x109, lx1010, 2x1010, 3x1010,
4x10i0
,
5x1010,
6x101 , 7x101 , 8x101 , 9x101 , lx10", 2x10", 3x10", 4x10", 5x10", 6x10",
.7x1nn,
u 8x10",
9x10", lx1012, 2x1012, 3x1012, 4x1012, 5x1012, 6x1012, 7x1012, 8x1012, 9x1n12,
u lx
i0'3, 2x1013,
3 x1013, 4x1013, 5x1013, 6x1013, 7x1013, 8x1013, and 9x1013. In some
embodiments, an effective
dosage of TILs is in the range of lx106 to 5x106, 5x106 to 1x107, 1x107 to
5x107, 5x107 to
1x108, 1x108 to 5x108, 5x108 to 1x109, 1x109 to 5x109, 5x109 to 1x1010, ix-io
iu to 5x101 ,
5x101 to 1xi's'',
u 5x1011 to lx1012, ix, nIV12
to 5x1012, and 5x1012 to lx1013.
[00316] In an embodiment of the invention, the clinical dose of MILs useful
for patients with
acute myeloid leukemia (AML) is in the range of from about 4x108 to about
2.5x109MILs. In
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another embodiment, the number of MILs provided in the pharmaceutical
compositions of the
invention is 9.5x108 MILs. In another embodiment, the number of MILs provided
in the
pharmaceutical compositions of the invention is 4.1x108. In another
embodiment, the number of
MILs provided in the pharmaceutical compositions of the invention is 2.2x109.
[00317] In 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 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.
[00318] In 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.
[00319] 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 direct injection
into tumor, or by inhalation.
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Optional Genetic Engineering of TILs, PBLs, and/or MILs
[00320] In some embodiments, the expanded TILs, PBLs, and/or MILs 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, PBLs, and/or
MILs of the
present invention are treated with transcription factors (TFs) and/or other
molecules capable of
transiently altering protein expression in the TILs, PBLs, and/or MILs. 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, PBLs, and/or MILs.
[00321] In certain embodiments, the method comprises genetically editing a
population of TILs,
PBLs, and/or MILs. In certain embodiments, the method comprises genetically
editing the first
population of TILs, PBLs, and/or MILs, the second population of TILs, PBLs,
and/or MILs
and/or the third population of TILs, PBLs, and/or MILs.
[00322] 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, PBLs,
and/or MILs 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.
[00323] In some embodiments, the expanded TILs, PBLs, and/or MILs of the
present invention
undergo transient alteration of protein expression. In some embodiments, the
transient alteration
of protein expression occurs at any time before, during, or after the
expansion process. In some
embodiments, the transient alteration of protein expression occurs at any step
within the
expansion process. In some embodiments, the transient alteration of protein
expression occurs in
the bulk TIL, PBL, and/or MIL population prior to a first expansion. In some
embodiments, the
transient alteration of protein expression occurs during the first expansion.
In some
embodiments, the transient alteration of protein expression occurs after the
first expansion,
including, for example in the TIL, PBL, and/or MIL population in transition
between the first
and second expansion (e.g. the second population of TILs, PBLs, and/or MILs as
described
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herein. In some embodiments, the transient alteration of protein expression
occurs in the bulk
TIL, PBL, and/or MTh population prior to second expansion. In some
embodiments, the
transient alteration of protein expression occurs during the second expansion,
including, for
example in the TIL, PBL, and/or MTh population being expanded (e.g. the third
population of
TILs, PBLs, and/or MILs). In some embodiments, the transient alteration of
protein expression
occurs after the second expansion.
[00324] In an embodiment, a method of transiently altering protein expression
in a population
of TILs, PBLs, and/or MILs 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 an embodiment, a method of transiently altering
protein expression in
population of TILs, PBLs, and/or MILs 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 an
embodiment, a method
of transiently altering protein expression in a population of TILs, PBLs,
and/or MILs 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 N-[1-(2,3-
dioleyloxy)propy1]-
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, 10,
520-525 and Felgner, 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 an embodiment, a method
of transiently
altering protein expression in a population of TILs, PBLs, and/or MILs
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.
[00325] 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 T cells. TSCMs generally display the long-term survival, self-renewal,
and
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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 (Gattinoni et at. Nat
Med 2009, 2011;
Gattinoni, Nature Rev. Cancer, 2012; Cieri et al. Blood 2013). 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.
[00326] 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.
[00327] 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.
[00328] 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,
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IL-21, NOTCH 1/2 ICD, TIM3, LAG3, TIGIT, TGFO, CCR2, CCR4, CCR5, CXCR1, CXCR2,
CSCR3, CCL2 (MCP-1), CCL3 (MIP-1a), CCL4 (MIP1-0), CCL5 (RANTES), CXCL1/CXCL8,
CCL22, CCL17, CXCL1/CXCL8, VHL, CD44, PIK3CD, SOCS1, and/or cAMP protein
kinase
A (PKA). In some embodiments, the transient alteration of protein expression
targets a gene
selected from the group consisting of PD-1, TGFBR2, CCR4/5, CBLB (CBL-B),
CISH, CCRs
(chimeric co-stimulatory receptors), IL-2, IL-12, IL-15, IL-21, NOTCH 1/2 ICD,
TIM3, LAG3,
TIGIT, TGFP, CCR2, CCR4, CCR5, CXCR1, CXCR2, CSCR3, CCL2 (MCP-1), CCL3 (MIP-
la), CCL4 (MIP113), CCL5 (RANTES), CXCL1/CXCL8, CCL22, CCL17, CXCL1/CXCL8,
VHL, CD44, PIK3CD, SOCS1, 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 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 IL-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 TGFP. 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
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embodiments, the transient alteration of protein expression targets CCL4
(MIP143). 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 some
embodiments, the transient
alteration of protein expression targets PIK3CD. In some embodiments, the
transient alteration of
protein expression targets SOCS1. In some embodiments, the transient
alteration of protein
expression targets cAMP protein kinase A (PKA).
[00329] 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 overexpressed by transient protein expression includes a receptor with
a ligand that
includes but is not limited to CCL2 (MCP-1), CCL3 (MW-la), CCL4 (MIP113), CCL5
(RANTES), CXCL1, CXCL8, CCL22, and/or CCL17.
[00330] In some embodiments, the transient alteration of protein expression
results in a decrease
and/or reduced expression of PD-1, CTLA-4, TIM-3, LAG-3, TIGIT, TGFf3R2,
and/or TGFO
(including resulting in, for example, TGFP pathway blockade). 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.
[00331] 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.
[00332] 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
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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.
[00333] 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,
[00334] In some embodiments, the transient alteration of protein expression
results in decreased
and/or reduced expression of cAMP protein kinase A (PKA).
[00335] 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, CISH, TGFOR2, 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, CISH, TGFOR2, 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, CISH, TGFOR2, 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, LAG-3, CISH, CBLB, TIM3, 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 LAG3, CISH, CBLB, TIM3, and combinations
thereof. 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 LAG3 and CISH. In
some
embodiments, the transient alteration of protein expression results in
decreased and/or reduced
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expression of LAG3 and CBLB. 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 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.
[00336] 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
gammaretroviral or lentiviral method into the first population of TILs, PBLs,
and/or MILs,
second population of TILs, PBLs, and/or MILs, or harvested population of TILs,
PBLs, and/or
MILs (e.g., the expression of the adhesion molecule is increased).
[00337] 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, CISH, TGFOR2, 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, LAG3, TIM3, CISH, CBLB, and combinations thereof, and increased
and/or enhanced
expression of CCR2, CCR4, CCR5, CXCR2, CXCR3, CX3CR1, and combinations
thereof.
[00338] 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
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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%.
[00339] 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 expression of at
least about 95%.
In some embodiments, there is an increase in expression of at least about 99%.
[00340] In some embodiments, transient alteration of protein expression is
induced by treatment
of the TILs, PBLs, and/or MILs with transcription factors (TFs) and/or other
molecules capable
of transiently altering protein expression in the TILs, PBLs, and/or MILs. 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 (Sharei et at.
PNAS 2013, as well
as Sharei et al. PLOS ONE 2015 and Greisbeck et al. J. Immunology vol. 195,
2015) have been
described; see, for example, International Patent Publications WO
2013/059343A1, WO
2017/008063A1, and WO 2017/123663A1, all of which are incorporated by
reference herein in
their entireties. Such methods as described in International Patent
Publications WO
2013/059343A1, WO 2017/008063A1, and WO 2017/123663A1 can be employed with the
present invention in order to expose a population of TILs, PBLs, and/or MILs
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
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increased expression of tumor antigens and/or an increase in the number of
tumor antigen-
specific T cells in the population of TILs, PBLs, and/or MILs, 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,
PBLs, and/or MILs, as described herein.
[00341] In some embodiments, the transcription factor (TF) includes but is not
limited to TCF-
1, NOTCH 1/2 ICD, 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.
[00342] 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, PBLs,
and/or MILs including the step of stable incorporation of genes for production
of one or more
proteins. In certain embodiments, the method comprises a step of genetically
modifying a
population of TILs, PBLs, and/or MILs. In certain embodiments, the method
comprises
genetically modifying the first population of TILs, PBLs, and/or MILs, the
second population of
TILs, PBLs, and/or MILs and/or the third population of TILs, PBLs, and/or
MILs. In an
embodiment, a method of genetically modifying a population of TILs, PBLs,
and/or MILs
includes the step of retroviral transduction. In an embodiment, a method of
genetically
modifying a population of TILs, PBLs, and/or MILs includes the step of
lentiviral transduction.
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Lentiviral transduction systems are known in the art and are described, e.g.,
in Levine, et at.,
Proc. Nat'l Acad. Sci. 2006, 103, 17372-77; Zufferey, et al., Nat. Biotechnol.
1997, 15, 871-75;
Dull, et al., 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 an embodiment, a method
of genetically
modifying a population of TILs, PBLs, and/or MILs includes the step of gamma-
retroviral
transduction. Gamma-retroviral 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 an embodiment, a method of genetically
modifying a
population of TILs, PBLs, and/or MILs includes the step of transposon-mediated
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 at.,
Mot. Therapy 2010, 18,
674-83 and U.S. Patent No. 6,489,458, the disclosures of each of which are
incorporated by
reference herein.
[00343] In some embodiments, transient alteration of protein expression is a
reduction in
expression 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. In some embodiments, the method comprises transient alteration of
protein expression in
a population of TILs, PBLs, and/or MILs, comprising the use of 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. Methods of using sdRNA
have been
described in Khvorova and Watts, Nat. Biotechnol. 2017, 35, 238-248; Byrne, et
at., I Ocul.
Pharmacol. Ther. 2013, 29, 855-864; and Ligtenberg, et at., Mot. Therapy,
2018, 26, 1482-1493,
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the disclosures of which are incorporated by reference herein. In an
embodiment, 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 M/10,000 TILs, PBLs, and/or MILs in medium. In certain
embodiments, the
method comprises delivery sdRNA to a TILs, PBLs, and/or MILs population
comprising
exposing the TILs, PBLs, and/or MILs population to sdRNA at a concentration of
1 M/10,000
TILs, PBLs, and/or MILs in medium for a period of between 1 to 3 days. In an
embodiment,
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 10 M/10,000 TILs, PBLs,
and/or MILs in
medium. In an embodiment, 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 50 M/10,000
TILs, PBLs, and/or MILs in medium. In an embodiment, 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 TILs, PBLs, and/or MILs and 50
M/10,000 TILs,
PBLs, and/or MILs in medium. In an embodiment, 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 TILs, PBLs, and/or MILs and 50
M/10,000 TILs,
PBLs, and/or MILs 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.
[00344] In some embodiments, sdRNA is inserted into a population of TILs,
PBLs, and/or MILs
during manufacturing. In some embodiments, the sdRNA encodes RNA that
interferes with
NOTCH 1/2 ICD, PD-1, CTLA-4 TIM-3, LAG-3, TIGIT, TGFO, 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
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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%.
[00345] 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, PBLs, and/or MILs as described herein. The combination of backbone
modifications
with asymmetric siRNA structure and a hydrophobic ligand (see, for eample,
Ligtenberg, et at.,
Mol. Therapy, 2018 and US20160304873) 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.
[00346] In some embodiments, over 95% transfection efficiency of TILs, PBLs,
and/or MILs
and a reduction in expression of the target by various specific sdRNA occurs.
In some
embodiments, 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 dyas, or 8 days or more. In some embodiments, the
reduction in
expression effect decreases at 10 days or more post sdRNA treatment of the
TILs, PBLs, and/or
MILs. 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 in TILs, PBLs, and/or MILs. In some embodiments, a
reduction in
expression in the PD-1/PD-L1 pathway allows for the TILs, PBLs, and/or MILs to
exhibit a
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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 sdRNA results in an increase TIL proliferation.
[00347] 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.
[00348] 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.
[00349] sdRNA (self-deliverable RNA) are a new class of covalently modified
RNAi
compounds that do not require a delivery vehicle to enter cells and have
improved pharmacology
compared to traditional siRNAs. "Self-deliverable RNA" or "sdRNA" is a
hydrophobically
modified RNA interfering-antisense hybrid, demonstrated to be highly
efficacious in vitro in
primary cells and in vivo upon local administration. Robust uptake and/or
silencing without
toxicity has been demonstrated. sdRNAs are generally asymmetric chemically
modified nucleic
acid molecules with minimal double stranded regions. sdRNA molecules typically
contain
single stranded regions and double stranded regions, and 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,
US20160304873, W02010033246, W02017070151, W02009102427, W02011119887,
W02010033247A2, W02009045457, W02011119852, all of which are incorporated by
reference herein in their entireties for all purposes. To optimize sdRNA
structure, chemistry,
targeting position, sequence preferences, and the like, a proprietary
algorithm has been
developed and utilized for sdRNA potency prediction (see, for example, US
20160304873).
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Based on these analyses, functional sdRNA sequences have been generally
defined as having
over 70% reduction in expression at 1 i.tM concentration, with a probability
over 40%.
[00350] 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 i.tM 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 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 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 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 2.0 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
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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 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 4.0 M.
[00351] In some emodiments, the 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 T-O-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 an additional particular embodiment, 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-ribose, 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- methoxyethoxy,
2'-allyloxy (-
OCH2CH=CH2), 2'-propargyl, 2'-propyl, ethynyl, ethenyl, propenyl, and cyano
and the like. In
one embodiment, the sugar moiety can be a hexose and incorporated into an
oligonucleotide as
described (Augustyns, K., et al., Nucl. Acids. Res. 18:4711 (1992)).
[00352] In some embodiments, the double-stranded 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 oligonucleotide
of the invention is
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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.
[00353] In some embodiments, a double-stranded 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
another embodiment, a double-stranded 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 oligonucleotide. In some embodiments, the double-stranded
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.
[00354] In some embodiments, the oligonucleotide can be substantially
protected from
nucleases e.g., by modifying the 3' or 5' linkages (e.g., U.S. Pat. No.
5,849,902 and WO
98/13526). 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.
[00355] In some embodiments, at least a portion of the contiguous
polynucleotides within the
sdRNA are linked by a substitute linkage, e.g., a phosphorothioate linkage.
[00356] 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, 500 enhancements in cellular uptake.
In some
embodiments, at least one of the C or U residues includes a hydrophobic
modification. In some
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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.
[00357] In some embodiments, the sdRNA or sd-rxRNAs exhibit enhanced endosomal
release
of sd-rxRNA molecules 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
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 sdRNA
comprises 2-12
phosphorothioate intemucleotide linkages (referred to as phosphorothioate
modifications). In
some embodiments, 6-8 phosphorothioate intemucleotide linkages are employed.
In some
embodiments, the sdRNA compounds of the invention also include a unique
chemical
modification pattern, which provides stability and is compatible with RISC
entry.
[00358] 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.
[00359] In some embodiments, at least 30% of the nucleotides in the sdRNA or
sd-rxRNA are
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 sdRNA or sd-rxRNA are modified. In some embodiments, 100% of the
nucleotides in the
sdRNA or sd-rxRNA are modified.
[00360] 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,
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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.
[00361] In some embodiments, the sdRNA molecules have increased stability. In
some
instances, a chemically modified sdRNA or sd-rxRNA 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 sd-rxRNA
has a half-life in media that is longer than 12 hours.
[00362] In some embodiments, the 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 sdRNA has
no 2'F
modification and yet are characterized by equal efficacy in cellular uptake
and tissue penetration.
[00363] 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
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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 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 al9 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.
[00364] The self-deliverable RNAi technology provides a method of directly
transfecting cells
with the RNAi agent, 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, PBLs, and/or MILs of the present invention. The sdRNAi methods allow
direct delivery of
chemically synthesized compounds to a wide range of primary cells and tissues,
both ex-vivo and
in vivo. The sdRNAs described in some embodiments of the invention herein are
commercially
available from Advirna LLC, Worcester, MA, USA.
[00365] The sdRNA are formed as hydrophobically-modified siRNA-antisense
oligonucleotide
hybrid structures, and are disclosed, for example in Byrne et al., December
2013, J. Ocular
Pharmacology and Therapeutics, 29(10): 855-864, incorporated by reference
herein in its
entirety.
[00366] In some embodiments, the sdRNA oligonucleotides can be delivered to
the TILs, PBLs,
and/or MILs described herein using sterile electroporation. In certain
embodiments, the method
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comprises sterile electroporation of a population of TILs, PBLs, and/or MILs
to deliver sdRNA
oligonucleotides.
[00367] In some embodiments, the oligonucleotides can be delivered to the
cells in combination
with a transmembrane delivery system. In some embodimets, 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 comprises use of a transmembrane delivery system to
deliver sdRNA
oligonucleotides to a population of TILs, PBLs, and/or MILs.
[00368] 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,
PBLs, and/or MILs described herein, including through passive uptake by TILs,
PBLs, and/or
MILs. The sdRNA can be added to the TILs, PBLs, and/or MILs 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 E, 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.
Mroeover, sdRNA can be added after thawing from any cryopreservation step in
Step F. In an
embodiment, one or more sdRNAs targeting genes as described herein, including
PD-1, LAG-3,
TIM-3, CISH, and CBLB, may be added to cell culture media comprising TILs,
PBLs, and/or
MILs 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, 1 [tM to 100 [tM, and 1 [tM to 100 M. In
an
embodiment, one or more sdRNAs targeting genes as described herein, including
PD-1, LAG-3,
TIM-3, CISH, and CBLB, may be added to cell culture media comprising TILs,
PBLs, and/or
MILs and other agents at amounts selected from the group consisting of 0.1 [tM
sdRNA/10,000
TILs, PBLs, and/or MILs/100 pL media, 0.5 [tM sdRNA/10,000 TILs, PBLs, and/or
MILs /100
pL media, 0.75 [tM sdRNA/10,000 TILs, PBLs, and/or MILs /100 pL media, 1 [tM
sdRNA/10,000 TILs, PBLs, and/or MILs /100 pL media, 1.25 [tM sdRNA/10,000
TILs, PBLs,
and/or MILs /100 pL media, 1.5 [tM sdRNA/10,000 TILs, PBLs, and/or MILs /100
pL media, 2
[tM sdRNA/10,000 TILs, PBLs, and/or MILs /100 pL media, 5 [tM sdRNA/10,000
TILs, PBLs,
and/or MILs /100 pL media, or 10 [tM sdRNA/10,000 TILs, PBLs, and/or MILs /100
pL media.
In an embodiment, one or more sdRNAs targeting genes as described herein,
including PD-1,
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LAG-3, TIM-3, CISH, and CBLB, may be added to TIL 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.
[00369] Oligonucleotide compositions of the invention, including sdRNA, can be
contacted
with TILs, PBLs, and/or MILs 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, PBLs, and/or MILs 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, PBLs, and/or MILs. The TILs, PBLs, and/or MILs may be a first
population, a second
population and/or a third population as described herein.
[00370] 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 by transfection, e.g., using cationic, anionic, or neutral
lipid compositions or
liposomes using methods known in the art (see, e.g., WO 90/14074; WO 91/16024;
WO
91/17424; U.S. Pat. No. 4,897,355; Bergan et a 1993. Nucleic Acids Research.
21:3567).
[00371] In some embodiments, more than one sdRNA is used to reduce expression
of a target
gene. In some embodiments, one or more of PD-1, TIM-3, CBLB, LAG3 and/or CISH
targeting
sdRNAs are used together. In some embodiments, a PD-1 sdRNA is used with one
or more of
TIM-3, CBLB, LAG3 and/or CISH in order to reduce expression of more than one
gene target.
In some embodiments, a LAG3 sdRNA is used in combination with a CISH targeting
sdRNA to
reduce gene expression of both targets. In some embodiments, the sdRNAs
targeting one or
more of PD-1, TIM-3, CBLB, LAG3 and/or CISH herein are commercially available
from
Advirna LLC, Worcester, MA, USA.
[00372] In some embodiments, the sdRNA targets a gene selected from the group
consisting of
PD-1, LAG3, TIM3, CTLA-4, TIGIT, CISH, TGFOR2, PKA, CBLB, BAFF (BR3), and
combinations thereof. In some embodiments, the sdRNA targets a gene selected
from the group
consisting of PD-1, LAG3, TIM3, CTLA-4, TIGIT, CISH, TGFOR2, PKA, CBLB, BAFF
(BR3),
and combinations thereof. In some embodiments, one sdRNA targets PD-1 and
another sdRNA
targets a gene selected from the group consisting of LAG3, TIM3, CTLA-4,
TIGIT, CISH,
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TGFOR2, PKA, CBLB, BAFF (BR3), and combinations thereof In some embodiments,
the
sdRNA targets a gene selected from PD-1, LAG-3, CISH, CBLB, TIM3, and
combinations
thereof. In some embodiments, the sdRNA targets a gene selected from PD-1 and
one of LAG3,
CISH, CBLB, TIM3, and combinations thereof. In some embodiments, one sdRNA
targets PD-1
and one sdRNA targets LAG3. In some embodiments, one sdRNA targets PD-1 and
one sdRNA
targets CISH. In some embodiments, one sdRNA targets PD-1 and one sdRNA
targets CBLB.
In some embodiments, one sdRNA targets LAG3 and one sdRNA targets CISH. In
some
embodiments, one sdRNA targets LAG3 and one sdRNA targets CBLB. In some
embodiments,
one sdRNA targets CISH and one sdRNA targets CBLB. In some embodiments, one
sdRNA
targets TIM3 and one sdRNA targets PD-1. In some embodiments, one sdRNA
targets TIM3
and one sdRNA targets LAG3. In some embodiments, one sdRNA targets TIM3 and
one
sdRNA targets CISH. In some embodiments, one sdRNA targets TIM3 and one sdRNA
targets
CBLB.
[00373] As discussed above, embodiments of the present invention provide TILs,
PBLs, and/or
MILs 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, PBLs, and/or MILs 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, PBLs, and/or MILs into a therapeutic population, wherein the
methods
comprise gene-editing the TILs, PBLs, and/or MILs. There are several gene-
editing
technologies that may be used to genetically modify a population of TILs,
PBLs, and/or MILs,
which are suitable for use in accordance with the present invention.
[00374] In some embodiments, the method comprises a method of genetically
modifying a
population of TILs, PBLs, and/or MILs which include the step of stable
incorporation of genes
for production of one or more proteins. In an embodiment, a method of
genetically modifying a
population of TILs, PBLs, and/or MILs includes the step of retroviral
transduction. In an
embodiment, a method of genetically modifying a population of TILs, PBLs,
and/or MILs
includes the step of lentiviral transduction. Lentiviral transduction systems
are known in the art
and are described, e.g., in Levine, et al., Proc. Nat 1 Acad. Sci. 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.
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Patent No. 6,627,442, the disclosures of each of which are incorporated by
reference herein. In
an embodiment, a method of genetically modifying a population of TILs, PBLs,
and/or MILs
includes the step of gamma-retroviral transduction. Gamma-retroviral
transduction systems are
known in the art and are described, e.g., Cepko and Pear, Cur. Prot. Mot.
Biol. 1996, 9.9.1-
9.9.16, the disclosure of which is incorporated by reference herein. In an
embodiment, a method
of genetically modifying a population of TILs, PBLs, and/or MILs includes the
step of
transposon-mediated 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 at., 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.
[00375] In an embodiment, the method comprises a method of genetically
modifying a
population of TILs, PBLs, and/or MILs e.g. a first population, a second
population and/or a third
population as described herein. In an embodiment, a method of genetically
modifying a
population of TILs, PBLs, and/or MILs includes the step of stable
incorporation of genes for
production or inhibition (e.g., silencing) of one ore more proteins. In an
embodiment, a method
of genetically modifying a population of TILs, PBLs, and/or MILs 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. 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 are incorporated by reference herein, may be used. In an
embodiment, the
electroporation method is a sterile electroporation method. In an embodiment,
the
electroporation method is a pulsed electroporation method. In an embodiment,
the
electroporation method is a pulsed electroporation method comprising the steps
of treating TILs,
PBLs, and/or MILs with pulsed electrical fields to alter, manipulate, or cause
defined and
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controlled, permanent or temporary changes in the TILs, PBLs, and/or MILs,
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, PBLs,
and/or MILs, 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
an embodiment, the
electroporation method is a pulsed electroporation method comprising the steps
of treating TILs,
PBLs, and/or MILs with pulsed electrical fields to alter, manipulate, or cause
defined and
controlled, permanent or temporary changes in the TILs, PBLs, and/or MILs,
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, PBLs,
and/or MILs, wherein at least two of the at least three pulses differ from
each other in pulse
amplitude. In an embodiment, the electroporation method is a pulsed
electroporation method
comprising the steps of treating TILs, PBLs, and/or MILs with pulsed
electrical fields to alter,
manipulate, or cause defined and controlled, permanent or temporary changes in
the TILs, PBLs,
and/or MILs, 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, PBLs, and/or MILs, wherein at least two of
the at least three
pulses differ from each other in pulse width. In an embodiment, the
electroporation method is a
pulsed electroporation method comprising the steps of treating TILs, PBLs,
and/or MILs with
pulsed electrical fields to alter, manipulate, or cause defined and
controlled, permanent or
temporary changes in the TILs, PBLs, and/or MILs, 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, PBLs,
and/or MILs,
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
an embodiment, the
electroporation method is a pulsed electroporation method comprising the steps
of treating TILs,
PBLs, and/or MILs with pulsed electrical fields to induce pore formation in
the TILs, PBLs,
and/or MILs, comprising the step of applying a sequence of at least three DC
electrical pulses,
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having field strengths equal to or greater than 100 V/cm, to TILs, PBLs,
and/or MILs, 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, PBLs, and/or
MILs is maintained. In an embodiment, a method of genetically modifying a
population of TILs,
PBLs, and/or MILs 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 at., 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 an embodiment, a method of
genetically
modifying a population of TILs, PBLs, and/or MILs 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)propy1]-n,n,n-trimethylammonium
chloride (DOTMA)
and dioleoyl phophotidylethanolamine (DOPE) in filtered water, are known in
the art and are
described in Rose, et at., Biotechniques 1991, /0, 520-525 and Felgner, et
at., 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 an embodiment, a method of genetically modifying a
population of TILs,
PBLs, and/or MILs 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, PBLs, and/or MILs may be a first
population, a
second population and/or a third population of TILs, PBLs, and/or MILs as
described herein.
[00376] 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
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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.
[00377] 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 at., Nature
Medicine, 2015, Vol. 21, No. 2.
[00378] 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, PBLs, and/or MILs into a therapeutic population may
be carried out
in accordance with any embodiment of the methods described herein (e.g., GEN 3
process) or as
described in PCT/US2017/058610, PCT/US2018/012605, or PCT/US2018/012633,
wherein the
method further comprises gene-editing at least a portion of the TILs, PBLs,
and/or MILs by one
or more of a CRISPR method, a TALE method or a ZFN method, in order to
generate TILs,
PBLs, and/or MILs that can provide an enhanced therapeutic effect. According
to an
embodiment, gene-edited TILs, PBLs, and/or MILs can be evaluated for an
improved therapeutic
effect by comparing them to non-modified TILs, PBLs, and/or MILs in vitro,
e.g., by evaluating
in vitro effector function, cytokine profiles, etc. compared to unmodified
TILs, PBLs, and/or
MILs. In certain embodiments, the method comprises gene editing a population
of TILs, PBLs,
and/or MILs using CRISPR, TALE and/ or ZFN methods.
[00379] In some embodiments of the present invention, electroporation is used
for delivery of a
gene editing system, such as CRISPR, TALEN, and ZFN 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
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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 AgilePulse 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.
[00380] A method for expanding TILs, PBLs, and/or MILs into a therapeutic
population may be
carried out in accordance with any embodiment of the methods described herein
(e.g., process
GEN 3) or as described in PCT/US2017/058610, PCT/US2018/012605, or
PCT/US2018/012633, wherein the method further comprises gene-editing at least
a portion of
the TILs, PBLs, and/or MILs 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, PBLs, and/or MILs.
Alternatively, the
use of a CRISPR 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, PBLs, and/or MILs.
[00381] 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.
[00382] 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
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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
use 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).
[00383] Non-limiting examples of genes that may be silenced or inhibited by
permanently gene-
editing TILs, PBLs, and/or MILs via a CRISPR method include PD-1, CTLA-4, LAG-
3,
HAVCR2 (TIM-3), Cish, TGFO, PKA, CBL-B, PPP2CA, PPP2CB, PTPN6, PTPN22, PDCD1,
BTLA, CD160, TIGIT, CD96, CRTAM, LAIR1, SIGLEC7, SIGLEC9, CD244, TNFRSF10B,
TNFRSF10A, 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, and
GUCY1B3.
[00384] Non-limiting examples of genes that may be enhanced by permanently
gene-editing
TILs, PBLs, and/or MILs via a CRISPR method include CCR2, CCR4, CCR5, CXCR2,
CXCR3,
CX3CR1, IL-2, IL12, IL-15, and IL-21.
[00385] 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, which are incorporated by reference herein. Resources for carrying
out CRISPR
methods, such as plasmids for expressing CRISPR/Cas9 and CRISPR/Cpfl, are
commercially
available from companies such as GenScript.
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[00386] In an embodiment, genetic modifications of populations of TILs, PBLs,
and/or MILs,
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.
[00387] A method for expanding TILs, PBLs, and/or MILs into a therapeutic
population may be
carried out in accordance with any embodiment of the methods described herein
(e.g., process
2A) or as described in PCT/US2017/058610, PCT/US2018/012605, or
PCT/US2018/012633,
wherein the method further comprises gene-editing at least a portion of the
TILs, PBLs, and/or
MILs 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, PBLs, and/or
MILs. 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, PBLs, and/or MILs.
[00388] TALE stands for "Transcription Activator-Like Effector" proteins,
which include
TALENs ("Transcription Activator-Like Effector Nucleases"). 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 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
FokI monomers in
close proximity to dimerize and produce a targeted double-strand break.
[00389] 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
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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 which are incorporated by reference herein.
[00390] Non-limiting examples of genes that may be silenced or inhibited by
permanently
gene-editing TILs, PBLs, and/or MILs via a TALE method include PD-1, CTLA-4,
LAG-3,
HAVCR2 (TIM-3), Cish, TGFP, PKA, CBL-B, PPP2CA, PPP2CB, PTPN6, PTPN22, PDCD1,
BTLA, CD160, TIGIT, CD96, CRTAM, LAIR1, SIGLEC7, SIGLEC9, CD244, TNFRSF10B,
TNFRSF10A, 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, and
GUCY1B3.
[00391] Non-limiting examples of genes that may be enhanced by permanently
gene-editing
TILs, PBLs, and/or MILs via a TALE method include CCR2, CCR4, CCR5, CXCR2,
CXCR3,
CX3CR1, IL-2, IL12, IL-15, and IL-21.
[00392] 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.
[00393] A method for expanding TILs, PBLs, and/or MILs into a therapeutic
population may be
carried out in accordance with any embodiment of the methods described herein
(e.g., process
GEN 3) or as described in PCT/U52017/058610, PCT/U52018/012605, or
PCT/U52018/012633, wherein the method further comprises gene-editing at least
a portion of
the TILs, PBLs, and/or MILs 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, PBLs, and/or MILs.
Alternatively, the use of a
zinc finger 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,
PBLs, and/or MILs.
[00394] An individual zinc finger contains approximately 30 amino acids in a
conserved f3f3a
configuration. Several amino acids on the surface of the a-helix typically
contact 3 bp in the
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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.
[00395] 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;
Sangamo Biosciences
(Richmond, CA, USA) has developed a propriety platform (CompoZrg) for zinc-
finger
construction in partnership with Sigma-Aldrich (St. Louis, MO, USA).
[00396] Non-limiting examples of genes that may be silenced or inhibited by
permanently gene-
editing TILs, PBLs, and/or MILs via a zinc finger method include PD-1, CTLA-4,
LAG-3,
HAVCR2 (TIM-3), Cish, TGFO, PKA, CBL-B, PPP2CA, PPP2CB, PTPN6, PTPN22, PDCD1,
BTLA, CD160, TIGIT, CD96, CRTAM, LAIR1, SIGLEC7, SIGLEC9, CD244, TNFRSF10B,
TNFRSF10A, 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, and
GUCY1B3.
[00397] Non-limiting examples of genes that may be enhanced by permanently
gene-editing
TILs, PBLs, and/or MILs via a zinc finger method include CCR2, CCR4, CCR5,
CXCR2,
CXCR3, CX3CR1, IL-2, IL12, IL-15, and IL-21.
[00398] 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,
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6,794,136, 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, which
are incorporated
by reference herein.
[00399] 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, et al.,Mol.
Therapy, 2015, 23
1380-1390, the disclosure of which is incorporated by reference herein.
Chimeric Antigen Receptors and Genetically-Modified T-Cell Receptors
[00400] In some embodiments, the TILs, PBLs, and/or MILs are optionally
genetically
engineered to include additional functionalities, including, but not limited
to, a high-affinity T
cell receptor (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
certain embodiments, the method comprises genetically engineering a population
of TILs, PBLs,
and/or MILs to include a high-affinity T cell receptor (TCR), e.g., a TCR
targeted at a tumor-
associated antigen such as MAGE-1, RER2, or NY-ESO-1, or a CAR which binds to
a tumor-
associated cell surface molecule (e.g., mesothelin) or lineage-restricted cell
surface molecule
(e.g., CD19). In certain embodiments, the method comprises genetically
engineering a
population of TILs, PBLs, and/or MILs to include a CAR specific for CD19,
CD20, CD19 and
CD20 (bispecific), CD30, CD33, CD123, PSMA, mesothelin, CE7, HER2/neu BCMA,
EGFRvIII, HER2/CMV, IL13Ra2, human C4 folate receptor-alpha (aFR), or GD2.
[00401] In some embodiments, the TILs, PBLs, and/or MILs expanded according to
the
methods of the present invention are genetically modified to target antigens
through expression
of CARs. In some embodiments, the TILs, PBLs, and/or MILs of the present
invention are
transduced with an expression vector comprising a nucleic acid encoding a CAR
comprising a
single chain variable fragment antibody fused with at least one endodomain of
a T-cell signaling
molecule. In some embodiments, the transducing step takes place at any time
during the
expansion process. In some embodiments, the transducing step takes place after
the expanded
cells are harvested. In some embodiments, the TILs, PBLs, and/or MILs of the
present invention
include a polynucleotide capable of expression of a CAR.
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[00402] In one embodiment, the CARs or nucleotides encoding CARs are prepared
and
transduced according to the disclosure in U.S. Patent Nos. 9,328,156;
8,399,645; 7,446,179;
6,410,319; 7,446,190, and U.S. Patent Application Publication Nos. US
2015/0038684; US
2015/0031624; US 2014/0301993 Al; US 2014/0271582 Al; US 2015/0051266 Al; US
2014/0322275 Al; and US 2014/0004132 Al, the disclosures of each of which is
incorporated
by reference herein. In U.S. Patent No. 9,328,156, CAR-T cells are prepared to
treat patients
with B-cell lymphomas, and particularly CLL, and the embodiments discussed
therein are useful
in the present invention. For example, a CAR-T cell expressing a CD19 antigen
binding domain,
a transmembrane domain, a 4-1BB costimulatory signaling region, and a CD3 zeta
signaling
domain is useful in the present invention. In an embodiment of the invention,
the CAR comprises
a target-specific binding element, or antibody binding domain, a transmembrane
domain, and a
cytoplasmic domain. Hematopoietic tumor antigens (for the antimbody binding
domain) are
well known in the art and include, for example, CD19, CD20, CD22, ROR1,
Mesothelin,
CD33/IL3Ra, c-Met, PSMA, Glycolipid F77, EGFRvIII, GD-2, NY-ES0-1 TCR, MAGE A3
TCR, and the like. In an embodiment, the transmembrane domain comprises the
alpha, beta or
zeta chain of the T-cell receptor, CD28, CD3 epsilon, CD45, CD4, CD5, CD8,
CD9, CD16,
CD22, CD33, CD37, CD64, CD80, CD86, CD134, CD137, CD154, and may be synthetic.
In an
embodiment, the cytoplasmic or signaling domain comprise a portion or all of
the TCR zeta, FcR
gamma, FcR beta, CD3 gamma, CD3 delta, CD3 epsilon, CD3 zeta, CD5, CD22,
CD79a,
CD79b, or CD66d domains. In an embodiment of the invention, the cytoplasmic or
signaling
domain may also include a co-stimulatory molecule, for example, CD27, CD28, 4-
1BB
(CD137), 0X40, CD30, CD40, PD-1, ICOS, lymphocyte function-associated antigen-
1 (LFA-1),
CD2, CD7, LIGHT, NKG2C, B7-H3, MC, or a ligand that specifically binds with
CD83, and the
like.
[00403] In an embodiment of the invention, the CAR modified TILs, PBLs, and/or
MILs
comprise an antigen binding domain, a costimulatory signaling region, and a
CD3 zeta signaling
domain. In an embodiment of the invention, the CAR-modified TILs, PBLs, and/or
MILs
comprise a CD19-directed antigen binding domain, a 4-1BB or CD28 costimulatory
signaling
region, and a CD3 zeta signaling domain. In an embodiment of the invention,
the CAR-modified
TILs include a suicide switch (such as a Caspase-9/rimiducid) or an activation
switch (such as an
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inducible MyD88/CD40 activation switch). In an embodiment of the invention,
the CAR-
modified TILs are modified using a lentiviral vector expressing a CAR.
[00404] In some embodiments of the invention, the TILs, PBLs, and/or MILs
expanded
according to the methods of the present invention are used in a method to
modify signaling in the
cells using modified T-cell receptors (TCRs), including genetically altered
TCRs. In some
embodiments, the TILs, PBLs, and/or MILs of the present invention are modified
to include
additional functionalities, including, but not limited to, a high-affinity T
cell receptor (TCR),
e.g., a TCR targeted at a tumor-associated antigen such as MAGE-1, MAGE-3,
MAGE-A3,
MAGE-A4, MAGE-A10, MART-1, CEA, gp100, alpha-fetoprotein (AFP), HER2, PRAME,
CT83, SSX2, or NY-ES0-1. Methods for modifying TCRs and methods for creating
artificial
TCRs are known in the art, and are disclosed, for example, in U.S. Patent Nos.
6,811,785;
7,569,664; 7,666,604; 8,143,376; 8,283,446; 9,181,527; 7,329,731; 7,070,995;
7,265,209;
8,361,794; and 8,697,854; and U.S. Patent Application Publication Nos. US
2017/0051036 Al;
US 2010/0034834 Al; US 2011/0014169 Al; US 2016/0200824 Al; and US
2002/0058253 Al,
the disclosures of each of which are incorporated by reference herein. In some
embodiments of
the invention, the TILs, PBLs, and/or MILs expanded according to the methods
of the present
invention are used in a method to modify signaling in the cells using modified
TCRs against a
tumor-associated antigen. In some embodiments of the invention, the TILs,
PBLs, and/or MILs
expanded according to the methods of the present invention are used in a
method to modify
signaling in the cells using modified TCRs, including genetically altered TCRs
wherein the TILs,
PBLs, and/or MILs are modified to reduce the presence of endogenous TCRs.
[00405] In some embodiments of the invention, the TILs, PBLs, and/or MILs
expanded
according to the methods of the present invention comprise transiently or
stably modified TCRs,
such as TCRs modified to be specific for a cancer testis antigen, such as a
MAGE-A antigen. In
some embodiments, the TILs, PBLs, and/or MILs may include at least one TCR
comprising a
modified complementarity determining region (CDR). In some embodiments, the
TILs, PBLs,
and/or MILs may include at least one TCR comprising a modified CDR2, with
retention of the
wild type sequences in the beta chain to increase the TCR affinity. In some
embodiments, the
TILs, PBLs, and/or MILs may include TCRs which are mutated relative to the
native TCR a
chain variable domain and/or 0 chain variable domain (see FIG. 1 b and SEQ ID
NO: 2) in at
least one CDR (such as CDR2), variable domain framework region, or other
hypervariable
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regions in the variable domains of the TCRs (such as the hypervariable 4 (HV4)
regions), such
that the mutants produce a high affinity TCR. The TILs, PBLs, and/or MILs may
include at least
one TCR anchored to the membrane by a transmembrane sequence, said TCR
comprising an
interchain disulfide bond between extracellular constant domain residues which
is not present in
native TCRs, as described in U.S. Patent No. 8,361,794, the disclosure of
which is incorporated
by reference herein. In some embodiments, the TILs, PBLs, and/or MILs may
include at least
one TCR having the property of binding to a specific human leukocyte antigen
(HLA)-A1
complex and comprising a specified wild type TCR which has specific mutations
in the TCR
alpha variable domain and/or the TCR beta variable domain to increase
affinity. In some
embodiments of the invention, the TILs, PBLs, and/or MILs expanded according
to the methods
of the present invention comprise a stably modified TCR with increased
affinity to NY-ES0-1,
MART-1, CEA, gp100, alpha-fetoprotein (AFP), HER2, PRAME (preferentially-
expressed
antigen in melanoma), CT83, 55X2, MAGE-1, MAGE-3, MAGE-A3, MAGE-A4, or MAGE-
A10.
Immune Checkpoints
[00406] In an embodiment of the present invention, one or more immune
checkpoint genes
may be modified. Immune checkpoints are molecules expressed by lymphocytes
that regulate an
immune response via inhibitory or stimulatory pathways. In the case of cancer,
immune
checkpoint pathways are often activated to inhibit the anti-tumor response,
i.e., the expression of
certain immune checkpoints by malignant cells inhibits the anti-tumor immunity
and favors the
growth of cancer cells. See, e.g., Marin-Acevedo et al., Journal of Hematology
& Oncology
(2018) 11:39. Thus, certain inhibitory checkpoint molecules serve as targets
for
immunotherapies of the present invention. According to particular embodiments,
cells are
modified through CAR or TCR to block or stimulate certain immune checkpoint
pathways and
thereby enhance the body's immunological activity against tumors.
[00407] The most broadly studied checkpoints include programmed cell death
receptor-1 (PD-
1) and cytotoxic T lymphocyte-associated molecule-4 (CTLA-4), which are
inhibitory receptors
on immune cells that inhibit key effector functions (e.g., activation,
proliferation, cytokine
release, cytoxicity, etc.) when they interact with an inhibitory ligand.
Numerous checkpoint
molecules, in addition to PD-1 and CTLA-4, have emerged as potential targets
for
immunotherapy, as discussed in more detail below.
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[00408] Non-limiting examples of immune checkpoint genes that may be silenced
or inhibited
include PD-1, CTLA-4, LAG-3, HAVCR2 (TIM-3), Cish, TGFP, PKA, CBL-B, PPP2CA,
PPP2CB, PTPN6, PTPN22, PDCD1, BTLA, CD160, TIGIT, BAFF (BR3), CD96, CRTAM,
LAIR1, SIGLEC7, SIGLEC9, CD244, TNFRSF10B, TNFRSF10A, 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, and GUCY1B3. For example, immune checkpoint
genes that may be silenced or inhibited may be selected from the group
comprising PD-1,
CTLA-4, LAG-3, TIM-3, Cish, TGFP, and PKA. BAFF (BR3) is described in Bloom,
et al., J.
Immunother., 2018, in press. According to another example, immune checkpoint
genes that may
be silenced or inhibited in TILs of the present invention may be selected from
the group
comprising PD-1, LAG-3, TIM-3, CTLA-4, TIGIT, CISH, TGFPR2, PRA, CBLB, BAFF
(BR3),
and combinations thereof.
PD-1
[00409] One of the most studied targets for the induction of checkpoint
blockade is the
programmed death receptor (PD1 or PD-1, also known as PDCD1), a member of the
CD28 super
family of T-cell regulators. Its ligands, PD-Li and PD-L2, are expressed on a
variety of tumor
cells, including melanoma. The interaction of PD-1 with PD-Li inhibits T-cell
effector function,
results in T-cell exhaustion in the setting of chronic stimulation, and
induces T-cell apoptosis in
the tumor microenvironment. PD1 may also play a role in tumor-specific escape
from immune
surveillance.
[00410] According to particular embodiments, expression of PD1 in TILs is
silenced or
reduced in accordance with compositions and methods of the present invention.
CTLA-4
[00411] CTLA-4 expression is induced upon T-cell activation on activated T-
cells, and
competes for binding with the antigen presenting cell activating antigens CD80
and CD86.
Interaction of CTLA-4 with CD80 or CD86 causes T-cell inhibition and serves to
maintain
balance of the immune response. However, inhibition of the CTLA-4 interaction
with CD80 or
CD86 may prolong T-cell activation and thus increase the level of immune
response to a cancer
antigen.
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[00412] According to particular embodiments, expression of CTLA-4 in TILs is
silenced or
reduced in accordance with compositions and methods of the present invention.
LAG-3
[00413] Lymphocyte activation gene-3 (LAG-3, CD223) is expressed by T cells
and natural
killer (NK) cells after major histocompatibility complex (MHC) class II
ligation. Although its
mechanism remains unclear, its modulation causes a negative regulatory effect
over T cell
function, preventing tissue damage and autoimmunity. LAG-3 and PD-1 are
frequently co-
expressed and upregulated on TILs, leading to immune exhaustion and tumor
growth. Thus,
LAG-3 blockade improves anti-tumor responses. See, e.g., Marin-Acevedo et al.,
Journal of
Hematology & Oncology (2018) 11:39.
[00414] According to particular embodiments, expression of LAG-3 in TILs is
silenced or
reduced in accordance with compositions and methods of the present invention.
TIM-3
[00415] T cell immunoglobulin-3 (TIM-3) is a direct negative regulator of T
cells and is
expressed on NK cells and macrophages. TIM-3 indirectly promotes
immunosuppression by
inducing expansion of myeloid-derived suppressor cells (MDSCs). Its levels
have been found to
be particularly elevated on dysfunctional and exhausted T-cells, suggesting an
important role in
malignancy.
[00416] According to particular embodiments, expression of TIM-3 in TILs is
silenced or
reduced in accordance with compositions and methods of the present invention.
Cish
[00417] Cish, a member of the suppressor of cytokine signaling (SOCS) family,
is induced by
TCR stimulation in CD8+ T cells and inhibits their functional avidity against
tumors. Genetic
deletion of Cish in CD8+ T cells may enhance their expansion, functional
avidity, and cytokine
polyfunctionality, resulting in pronounced and durable regression of
established tumors. See,
e.g., Palmer et al., Journal of Experimental Medicine, 212 (12): 2095 (2015).
[00418] According to particular embodiments, expression of Cish in TILs is
silenced or
reduced in accordance with compositions and methods of the present invention.
TGFP
[00419] The TGFP signaling pathway has multiple functions in regulating cell
growth,
differentiation, apoptosis, motility and invasion, extracellular matrix
production, angiogenesis,
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and immune response. TGFP signaling deregulation is frequent in tumors and has
crucial roles in
tumor initiation, development and metastasis. At the microenvironment level,
the TGFP pathway
contributes to generate a favorable microenvironment for tumor growth and
metastasis
throughout carcinogenesis. See, e.g., Neuzillet et al., Pharmacology &
Therapeutics, Vol. 147,
pp. 22-31 (2015).
[00420] According to particular embodiments, expression of TGFP in TILs, PBLs,
and/or
MILs is silenced or reduced in accordance with compositions and methods of the
present
invention.
PKA
[00421] Protein Kinase A (PKA) is a well-known member of the serine-threonin
protein
kinase superfamily. PKA, also known as cAMP-dependent protein kinase, is a
multi-unit protein
kinase that mediates signal transduction of G-protein coupled receptors
through its activation
upon cAMP binding. It is involved in the control of a wide variety of cellular
processes from
metabolism to ion channel activation, cell growth and differentiation, gene
expression and
apoptosis. Importantly, PKA has been implicated in the initiation and
progression of many
tumors. See, e.g., Sapio et al., EXCLI Journal; 2014; 13: 843-855.
[00422] According to particular embodiments, expression of PKA in TILs is
silenced or
reduced in accordance with compositions and methods of the present invention.
CBLB
[00423] CBLB (or CBL-B) is a E3 ubiquitin-protein ligase and is a negative
regulator of T
cell activation. Bachmaier, et al., Nature, 2000, 403, 211-216; Wallner, et
al., Clin. Dev.
Immunol. 2012, 692639.
[00424] According to particular embodiments, expression of CBLB in TILs is
silenced or
reduced in accordance with compositions and methods of the present invention.
[00425] Overexpression of Co-Stimulatory Receptors or Adhesion Molecules
[00426] According to additional embodiments, one or more immune checkpoint
genes are
enhanced. Non-limiting examples of immune checkpoint genes that may exhibit
enhanced
expression include certain chemokine receptors and interleukins, such as CCR2,
CCR4, CCR5,
CXCR2, CXCR3, CX3CR1, IL-2, IL-4, IL-7, IL-15, and IL-21.
CCRs
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[00427] For adoptive T cell immunotherapy to be effective, T cells need to be
trafficked
properly into tumors by chemokines. A match between chemokines secreted by
tumor cells,
chemokines present in the periphery, and chemokine receptors expressed by T
cells is important
for successful trafficking of T cells into a tumor bed.
[00428] According to particular embodiments, an increase in the expression of
certain
chemokine receptors in the TILs, such as one or more of CCR2, CCR4, CCR5,
CXCR2, CXCR3
and CX3CR1 is contemplated. Over-expression of CCRs may help promote effector
function
and proliferation of TILs following adoptive transfer.
[00429] According to particular embodiments, expression of one or more of
CCR2, CCR4,
CCR5, CXCR2, CXCR3 and CX3CR1 is enhanced.
[00430] In an embodiment, CCR4 and/or CCR5 adhesion molecules are inserted
into a TIL
population using a gamma-retroviral or lentiviral method as described herein.
In an
embodiment, CXCR2 adhesion molecule are inserted into a TIL population using a
gamma-
retroviral or lentiviral method as described in Forget, et al., Frontiers
Immunology 2017, 8, 908
or Peng, et al., Clin. Cancer Res. 2010, 16, 5458, the disclosures of which
are incorporated by
reference herein.
Interleukins
[00431] According to additional embodiments, gene-editing methods of the
present invention
may be used to increase the expression of certain interleukins, such as one or
more of IL-2, IL-4,
IL-7, IL-15, and IL-21. Certain interleukins have been demonstrated to augment
effector
functions of T cells and mediate tumor control.
[00432] According to particular embodiments, expression of one or more of IL-
2, IL-4, IL-7,
IL-15, and IL-21 is enhanced in accordance with compositions and methods of
the present
invention.
[00433] Aptly, the population of TILs, PBLs, and/or MILs may be a first
population, a second
population and/or a third population as described herein.
Methods of Treating Cancers
[00434] The compositions and combinations of TILs, PBLs, and/or MILs (and
populations
thereof) described above can be used in a method for treating
hyperproliferative disorders. In a
preferred embodiment, they are for use in treating cancers. In a preferred
embodiment, the
invention provides a method of treating a cancer, wherein the cancer is a
hematological
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malignancy, such as a liquid tumor. In a preferred embodiment, the invention
provides a method
of treating a cancer, wherein the cancer is a hematological malignancy
selected from the group
consisting of acute myeloid leukemia (AML), mantle cell lymphoma (MCL),
follicular
lymphoma (FL), diffuse large B cell lymphoma (DLBCL), activated B cell (ABC)
DLBCL,
germinal center B cell (GCB) DLBCL, chronic lymphocytic leukemia (CLL), CLL
with
Richter's transformation (or Richter's syndrome), small lymphocytic leukemia
(SLL), non-
Hodgkin's lymphoma (NHL), Hodgkin's lymphoma, relapsed and/or refractory
Hodgkin's
lymphoma, B cell acute lymphoblastic leukemia (B-ALL), mature B-ALL, Burkitt's
lymphoma,
Waldenstrom's macroglobulinemia (WM), multiple myeloma, myelodysplatic
syndromes,
myelofibrosis, chronic myelocytic leukemia, follicle center lymphoma, indolent
NHL, human
immunodeficiency virus (HIV) associated B cell lymphoma, and Epstein¨Barr
virus (EBV)
associated B cell lymphoma, including subpopulations of patients with the
foregoing diseases
that are refractory to, intolerant to, or relapsed from treatment with a BTK
inhibitor, including
ibrutinib.
[00435] In an embodiment of the present invention, CLL patients who have been
pretreated
with ibrutinib represent a subpopulation of patients that can be successfully
treated with the
PBLs of the present invention. In particular, CLL patients who have been
pretreated with
ibrutinib, and who are no longer responsive to ibrutinib treatment, represent
a subpopulation of
patients that can be successfully treated with the PBLs of the present
invention. In another
embodiment, CLL patients who have been pretreated with ibrutinib and who have
developed
Richter's transformation (or Richter's syndrome), represent a subpopulation of
patients that can
be successfully treated with the PBLs of the present invention. In another
embodiment, CLL
patients who have been pretreated with ibrutinib, who have developed Richter's
transformation
(or Richter's syndrome) and who are no longer responsive to ibrutinib
treatment, represent a
subpopulation of patients that can be successfully treated with the PBLs of
the present invention.
[00436] In an embodiment, the invention provides a method of treating a
cancer, wherein the
cancer is a hematological malignancy that responds to therapy with PD-1 and/or
PD-Li
inhibitors including pembrolizumab, nivolumab, durvalumab, avelumab, or
atezolizumab.
[00437] In an embodiment, the invention provides a method of treating a cancer
in a patient
with a population of tumor infiltrating lymphocytes (TILs) comprising the
steps of:
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(a) obtaining a tumor from the patient by resection, biopsy, needle
aspiration, or apheresis,
the tumor comprising a first population of TILs;
(b) optionally fragmenting or dissociating the tumor to obtain tumor fragments
and
contacting the tumor or tumor fragments with a first cell culture medium;
(c) performing an initial 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;
(d) performing a second expansion of the second population of TILs in a second
cell culture
medium to obtain a third population of TILs, wherein the third population of
TILs is at
least 50-fold greater in number than the second population of TILs after 7
days from the
start of the second expansion; wherein the second cell culture medium
comprises IL-2,
OKT-3 (anti-CD3 antibody), and irradiated allogeneic peripheral blood
mononuclear
cells (PBMCs); and wherein the second expansion is performed over a period of
14 days
or less;
(e) harvesting the third population of TILs; and
(f) administering a therapeutically effective portion of the third population
of TILs to the
patient;
wherein the tumor is a liquid tumor, and wherein the cancer is a hematological
malignancy.
[00438] In an embodiment, the invention provides a method of treating a cancer
in a patient
with a population of tumor infiltrating lymphocytes (TILs) comprising the
steps of:
(a) obtaining a tumor from the patient by resection, biopsy, needle
aspiration, or apheresis,
the tumor comprising a first population of TILs;
(b) optionally fragmenting or dissociating the tumor to obtain tumor fragments
and
contacting the tumor or tumor fragments with a first cell culture medium;
(c) performing an initial 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;
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(d) performing a second expansion of the second population of TILs in a second
cell culture
medium to obtain a third population of TILs, wherein the third population of
TILs is at
least 50-fold greater in number than the second population of TILs after 7
days from the
start of the second expansion; wherein the second cell culture medium
comprises IL-2,
OKT-3 (anti-CD3 antibody), and irradiated allogeneic peripheral blood
mononuclear
cells (PBMCs); and wherein the second expansion is performed over a period of
14 days
or less;
(e) harvesting the third population of TILs; and
(f) administering a therapeutically effective portion of the third population
of TILs to the
patient;
wherein the tumor is a liquid tumor, and wherein the cancer is a hematological
malignancy
selected from the group consisting of acute myeloid leukemia (AML), mantle
cell lymphoma
(MCL), follicular lymphoma (FL), diffuse large B cell lymphoma (DLBCL),
activated B cell
(ABC) DLBCL, germinal center B cell (GCB) DLBCL, chronic lymphocytic leukemia
(CLL), CLL with Richter's transformation (or Richter's syndrome), small
lymphocytic
leukemia (SLL), non-Hodgkin's lymphoma (NHL), Hodgkin's lymphoma, relapsed
and/or
refractory Hodgkin's lymphoma, B cell acute lymphoblastic leukemia (B-ALL),
mature B-
ALL, Burkitt's lymphoma, Waldenstrom's macroglobulinemia (WM), multiple
myeloma,
myelodysplatic syndromes, myelofibrosis, chronic myelocytic leukemia, follicle
center
lymphoma, indolent NHL, human immunodeficiency virus (HIV) associated B cell
lymphoma, and Epstein¨Barr virus (EBV) associated B cell lymphoma.
[00439] In an embodiment, the invention provides a method of treating a cancer
in a patient
with a population of tumor infiltrating lymphocytes (TILs) comprising the
steps of:
(a) pre-treating the patient with a regimen comprising a kinase inhibitor or
an ITK inhibitor;
(b) obtaining a tumor from the patient by resection, biopsy, needle
aspiration, or apheresis,
the tumor comprising a first population of TILs;
(c) optionally fragmenting or dissociating the tumor to obtain tumor fragments
and
contacting the tumor or tumor fragments with a first cell culture medium;
(d) performing an initial expansion of the first population of TILs in the
first cell culture
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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;
(e) performing a second expansion of the second population of TILs in a second
cell culture
medium to obtain a third population of TILs, wherein the third population of
TILs is at
least 50-fold greater in number than the second population of TILs after 7
days from the
start of thesecond expansion; wherein the second cell culture medium comprises
IL-2,
OKT-3 (anti-CD3 antibody), and irradiated allogeneic peripheral blood
mononuclear
cells (PBMCs); and wherein the second expansion is performed over a period of
14 days
or less;
(f) harvesting the third population of TILs; and
(g) administering a therapeutically effective portion of the third population
of TILs to the
patient;
wherein the tumor is a liquid tumor, and wherein the cancer is a hematological
malignancy.
[00440] In an embodiment, the invention provides a method of treating a cancer
in a patient
with a population of tumor infiltrating lymphocytes (TILs) comprising the
steps of:
(a) pre-treating the patient with a regimen comprising a kinase inhibitor or
an ITK inhibitor;
(b) obtaining a tumor from the patient by resection, biopsy, needle
aspiration, or apheresis,
the tumor comprising a first population of TILs;
(c) optionally fragmenting or dissociating the tumor to obtain tumor fragments
and
contacting the tumor or tumor fragments with a first cell culture medium;
(d) performing an initial 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;
(e) performing a second expansion of the second population of TILs in a second
cell culture
medium to obtain a third population of TILs, wherein the third population of
TILs is at
least 50-fold greater in number than the second population of TILs after 7
days from the
start of the second expansion; wherein the second cell culture medium
comprises IL-2,
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OKT-3 (anti-CD3 antibody), and irradiated allogeneic peripheral blood
mononuclear
cells (PBMCs); and wherein the second expansion is performed over a period of
14 days
or less;
(f) harvesting the third population of TILs; and
(g) administering a therapeutically effective portion of the third population
of TILs to the
patient;
wherein the tumor is a liquid tumor, and wherein the cancer is a hematological
malignancy
selected from the group consisting of acute myeloid leukemia (AML), mantle
cell lymphoma
(MCL), follicular lymphoma (FL), diffuse large B cell lymphoma (DLBCL),
activated B cell
(ABC) DLBCL, germinal center B cell (GCB) DLBCL, chronic lymphocytic leukemia
(CLL), CLL with Richter's transformation (or Richter's syndrome), small
lymphocytic
leukemia (SLL), non-Hodgkin's lymphoma (NHL), Hodgkin's lymphoma, relapsed
and/or
refractory Hodgkin's lymphoma, B cell acute lymphoblastic leukemia (B-ALL),
mature B-
ALL, Burkitt's lymphoma, Waldenstrom's macroglobulinemia (WM), multiple
myeloma,
myelodysplatic syndromes, myelofibrosis, chronic myelocytic leukemia, follicle
center
lymphoma, indolent NHL, human immunodeficiency virus (HIV) associated B cell
lymphoma, and Epstein¨Barr virus (EBV) associated B cell lymphoma.
[00441] In an embodiment of the invention, TILs are expanded using MIL Method
1 and
administered to a patient in accordance with the present invention.
[00442] In an embodiment of the invention, TILs are expanded using MIL Method
2 and
administered to a patient in accordance with the present invention to treat
cancer.
[00443] In an embodiment of the invention, TILs are expanded using MIL Method
3 and
administered to a patient in accordance with the present invention to treat
cancer.
[00444] In an embodiment of the invention, TILs expanded using MIL Method 1,
MIL
Method 2, or MIL Method 3 are administered to a patient in accordance with the
present
invention to treat AML.
[00445] In an embodiment of the invention, TILs are expanded using PBL Method
2 and
administered to a patient in accordance with the present invention to treat
cancer.
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[00446] In an embodiment of the invention, TILs are expanded using PBL Method
2 and
administered to a patient in accordance with the present invention to treat
cancer.
[00447] In an embodiment of the invention, TILs are expanded using PBL Method
2 and
administered to a patient in accordance with the present invention to treat
cancer.
[00448] In an embodiment of the invention, TILs expanded using PBL Method 1,
PBL
Method 2, or PBL Method 3 are administered to a patient in accordance with the
present
invention to treat CLL.
[00449] In any of the foregoing embodiments of the invention, pre-treatment
with a kinase
inhibitor is described. In an embodiment, the kinase inhibitor is selected
from the group
consisting of imatinib, dasatinib, ibrutinib, bosutinib, nilotinib, erlotinib,
or other kinase
inhibitors, tyrosine kinase inhibitors, or serine/threonine kinase inhibitors
known in the art. In an
embodiment, pre-treatment regimens with a kinase inhbitor are as known in the
art and/or as
prescribed by a physician.
[00450] In any of the foregoing embodiments of the invention, pre-treatement
with an IL-2-
inducible T-cell kinase (ITK) inhibitor is described. Interleukin-2-inducible
T cell kinase (ITK)
is a non-receptor tyrosine kinase expressed in T-cells and regulates various
pathways. Any ITK
inhibitor known in the art may be used in embodiments of the present invention
(see, for
example, Lo, et at., Expert Opinion on Therapeutic Patents, 20:459-469 (2010);
Vargas, et at.,
Scandinavian Journal of Immunology, 78(2):130-139 (2013); W02015112847;
W02016118951;
W02007136790, U520120058984A1, and U.S. Patent Nos. 9,531,689 and 9,695,200;
all of
which are incorporated by reference herein in their entireties). In an
embodiment of the
invention, the ITK inhibitor is a covalent ITK inhibitor that covalently and
irreversibly binds to
ITK. In an embodiment of the invention, the ITK inhibitor is an allosteric ITK
inhibitor that
binds to ITK. In an embodiment of the invention, the ITK inhibitor is selected
from the group
consisting of aminothiazole-based ITK inhibitors, 5-aminomethylbenzimdazoles-
based ITK
inhibitors, 3-Aminopyrid-2-ones-based ITK inhibitors, (4 or 5-aryl)pyrazolyl-
indole-based ITK
inhibitors, benzimidazole-based ITK inhibitors, aminobenzimidazole-based ITK
inhibitors,
aminopyrimidine-based ITK inhibitors, aminopyridine-based ITK inhibitors,
diazolodiazine-
based ITK inhibitors, triazole-based ITK inhibitors, 3-aminopyride-2-ones-
based ITK inhibitors,
indolylindazole-based ITK inhibitors, indole-based ITK inhibitors, aza-indole-
based ITK
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inhibitors, pyrazolyl-indole-based inhibitors, thienopyrazole-based ITK
inhibitors, heterocyclic
ITK inhibitors, and ITK inhibitors targeting cysteine-442 in the ATP pocket
(such as ibrutinib),
aza-benzimidazole-based ITK inhibitors, benzothiazole-based ITK inhibitors,
indole-based ITK
inhibitors, pyridone-based ITK inhibitors, sulfoximine-substituted pyrimidine
ITK inhibitors,
arylpyridinone-based ITK inhibitors, and any other ITK inhibitors known in the
art. In an
embodiment of the invention, pre-treatment regimens with an ITK inhibitor are
as known in the
art and/or as prescribed by a physician. In an embodiment of the invention,
the ITK inhibitor is
selected from the group consisting of:
ibrutinib,
e
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PCT/US2018/060183
.0
N.0õ.......
N
N
0..----'- BMS509744,
H
N .--
0 N / \
* N
- N N
0
CTA056,
N
N i \
HN_
K
C.:H GSK2250665A,
o
11
"12 H
.----1'----.
-.-----...-----N.-----,.-----
0 PF06465469,
137
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PCT/US2018/060183
....-::=-",,,
L 1 1
hE
õ::,..s.,...---.....y..--,..sr," -7:c-k,_.--- \ 1 skµ ",;;=
11 .>=N
1-1
)
- 0
...
...
H
I \\N
'''==,-'5;::-."- N
) il
... (/
..., \
OH
\>4
H
/ -- ,,
N
N-I `,...;.....
/ 'µ., , I
7 (:µ
/
i
11'--
/---- µ.
i \
CS N -----%
\
\ .1 \-- ..... \ /1----\\\
\:>---:=\, I
'----- NE-3
t ......,
;
in
138
CA 03083118 2020-05-20
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PCT/US2018/060183
H -N
il / 1
illq .. I:// .. \''''''\
...s,
\\_/'
Nke \ is.
ri..---*--,,,,:;.,- .õ.....,
-.. .b.,a
1
I0
0 õ......
,,,,, ,..3.,,,,e......¨,s, ,-----d N-----q
..., `-=:::1 R ii \ e \
I i Cii '____j ,
1 i 1
/7
I ../. 6 (43-4885121
........,,,
/ -
i
-
i
4 H
''''''''' 'N"'s,..-:" -N;Zs,\----- '''', "===' Nii
1 ..
.. :1
.,
...---
''''µ,...õ.õ--'e
,0-
==3'¨'"--,
/7 li
\
\ ,,N
3 4
139
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PCT/US2018/060183
OH
HO
\
N
N:\rj
= j
N NH
0
140
CA 03083118 2020-05-20
WO 2019/103857
PCT/US2018/060183
N N
a
Ii N t
=
NH
6
NH
0
NH
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µ 11/41 0
1
S ' N L.
-"
11 ,
11
t1/41
,...--." Ns,õµõ,,,,,,,....,õ--..T..."'" ...õ..,,
1 0
s ds.....,.-y
i
N
--
\
HN µ -
N/
:
\ ________________________________________________________
NN.,...,
OH
H214
.....7¨ ICs
I
1. .....-<,
I-X)
142
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PCT/US2018/060183
NH2
/r-----:------
t / -----\\'o
i
\\ i
A ,
i
H i
?
N
1 i
'''',...=:,-, N
r'''' .1 1,.
i...0
N
HN ................... <1 11
_.....i.
0
õ,,-----1,
HC 4t µ
\
I .=%,
HOr .-----N...õ..õ4õ, õ,,õ
s,
H3c. .N ,,,," 'Nr=,,
")./ 'N..:1\ õ,,, 19
H
g .,i ttf
e,....._¨.
N '
H
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CA 03083118 2020-05-20
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CHs
I
4 F
liCY--;
J `...
N "e
1-11C ---- H 11 \ 'S.' l,r1 F
N ,,, '
tkr.
H
Clia
CH
/
1,1
14.C
1 ir¨
ip\
\ - f
1 N 0
i
t,
II
0
N, ,,.......1.õ.NHIVie
0
I 4.....
40 N NH
H 0
0
e,,,..
I
N
C.?'
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CA 03083118 2020-05-20
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Np
H
0.14 =
0
0
N S
1
H0.1
and combinations thereof. In an embodiment of the invention, the ITK inhibitor
is selected from
the group consisting of imatinib, dasatinib (BMS-354825), Sprycel [N-(2-chloro-
6-
methylpheny1)-2-(6-(4-(2-hydroxyethyl)-piperazin-1-y1)-2-meth-ylpyrimidin-4-
ylamino)thiazole-
5-carboxamide), ibrutinib ((1-{(3R)-3-[4-amino-3-(4-phenoxypheny1)-1H-
pyrazolo[3,4-
d]pyrimidin-1-yl]piperidin-1-yl}prop-2-en-1-one), bosutinib, nilotinib,
erlotinib, 1H-
pyrazolo[4,3-c]cinnolin-3-ol, CTA056 (7-benzy1-1-(3-(piperidin-1-yl)propy1)-2-
(4-(pyridin-4-
y1)pheny1)-1H-imidazo[4,5-g]quinoxalin-6(5H)-one), Compound 10 (Boehringer
Ingelheim from
Moriarty, et al., Bioorg Med Chem Lett, 18:5537-40 (2008)), Compound 19
(Boehringer
Ingelheim from Moriarty, et al., Bioorg Med Chem Lett., 18:5537-40 (2008)),
Compound 27
(Boehringer Ingelheim from Moriarty, et at., Bioorg Med Chem Lett., 18:5537-40
(2008)),
Compound 26 (Boehringer Ingelheim from Winters, et at., Bioorg Med Chem Lett.,
18:5541-4
(2008)), Compound 37 (Boehringer Ingelheim from Cook, et at., Bioorg Med Chem
Lett.,
19:773-7 (2009)), Compound 41 (Boehringer Ingelheim from Cook, et al., Bioorg
Med Chem
Lett., 19:773-7 (2009)), Compound 48 (Boehringer Ingelheim from Cook, et al.,
Bioorg Med
Chem Lett., 19:773-7 (2009)), Compound 51 (Boehringer Ingelheim from Cook, et
al., Bioorg
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Med Chem Lett., 19:773-7 (2009)), Compound 10n (Boehringer Ingelheim from
Riethe, et al., .
Bioorg Med Chem Lett., 19:1588-91 (2009)), Compound 10o (Boehringer Ingelheim
from
Riethe, et at., Bioorg Med Chem Lett., 19:1588-91 (2009)), Compound 7v (Vertex
from
Charrier, et al., J Med Chem., 54:2341-50 (2011)), Compound 7w (Vertex from
Charrier, et al.,
J Med Chem., 54:2341-50 (2011)), Compound 7x (Vertex from Charrier, et at., J
Med Chem.,
54:2341-50 (2011)), Compound 7y (Vertex from Charrier, et al., J Med Chem.,
54:2341-50
(2011)), Compound 44 (Bayer Schering Pharma from vonBonin, et at., Exp
Dermatol., 20:41-7
(2011)), Compound 13 (Nycomed from Velankar, et at., Bioorg Med Chem., 18:4547-
59
(2010)), Compound 24 (Nycomed from Velankar, et al., Bioorg Med Chem., 18:4547-
59
(2010)), Compound 34 (Nycomed from Velankar, et al., Bioorg Med Chem., 18:4547-
59
(2010)), Compound 10o (Nycomed from Herdemann, et at., Bioorg Med Chem Lett.,
21:1852-6
(2011)), Compound 3 (Sanofi US from McLean, et at., Bioorg Med Chem Lett.,
22:3296-300
(2012)), Compound 7 (Sanofi US from McLean, et at., Bioorg Med Chem Lett.,
22:3296-300
(2012), and/or or other kinase inhibitors, tyrosine kinase inhibitors, or
serine/threonine kinase
inhibitors known in the art, as well as any combinations thereof.
[00451] In any of the foregoing embodiments, pre-treatment regimens comprising
ibrutinib
(commercially available as IMBRUVICA, and which has the chemical name 1-[(3R)-
344-
amino-3-(4-phenoxypheny1)-1H-pyrazolo[3,4-d]pyrimidin-1-y1]-1-piperidiny1]-2-
propen-1-one)
may include orally administering one 140 mg capsule q.d., orally administering
two 140 mg
capsules q.d., orally administering three 140 mg capsules q.d., or orally
administering four 140
mg capsules q.d., for a duration of about one day, two days, three days, four
days, five days, six
days, seven days, eight days, nine days, ten days, eleven days, twelve days,
two weeks, three
weeks, one month, two months, three months, four months, five months, or six
months. In the
foregoing embodiments, pre-treatment regimens comprising ibrutinib may also
comprise orally
administering an ibrutinib dose selected from the group consisting of 25 mg,
50 mg, 75 mg, 100
mg, 125 mg, 150 mg, 175 mg, 200 mg, 225 mg, 250 mg, 275 mg, 300 mg, 325 mg,
350 mg, 375
mg, 400 mg, 425 mg, 450 mg, and 500 mg, wherein the administering occurs once
daily, twice
daily, three times daily, or four times daily, and wherein the duration of
administration is
selected from the group consisting of about one day, two days, three days,
four days, five days,
six days, seven days, eight days, nine days, ten days, eleven days, twelve
days, two weeks, three
weeks, one month, two months, three months, four months, five months, and six
months.
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[00452] In any of the foregoing embodiments, the cancer to be treated is a
hematological
malignancy selected from the group consisting of acute myeloid leukemia (AML),
mantle cell
lymphoma (MCL), follicular lymphoma (FL), diffuse large B cell lymphoma
(DLBCL),
activated B cell (ABC) DLBCL, germinal center B cell (GCB) DLBCL, chronic
lymphocytic
leukemia (CLL), CLL with Richter's transformation (or Richter's syndrome),
small lymphocytic
leukemia (SLL), non-Hodgkin's lymphoma (NHL), Hodgkin's lymphoma, relapsed
and/or
refractory Hodgkin's lymphoma, B cell acute lymphoblastic leukemia (B-ALL),
mature B-ALL,
Burkitt's lymphoma, Waldenstrom's macroglobulinemia (WM), multiple myeloma,
myelodysplatic syndromes, myelofibrosis, chronic myelocytic leukemia, follicle
center
lymphoma, indolent NHL, human immunodeficiency virus (HIV) associated B cell
lymphoma,
and Epstein¨Barr virus (EBV) associated B cell lymphoma.
[00453] Efficacy of the methods and compositions described herein in treating,
preventing
and/or managing the indicated diseases or disorders can be tested using
various animal models
known in the art.
Non-Myeloablative Lymphodepletion with Chemotherapy
[00454] In an embodiment, the invention provides 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 present disclosure. In an embodiment,
the non-
myeloablative chemotherapy is one or more chemotherapeutic agents. In an
embodiment, 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 an embodiment, 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.
[00455] 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.
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[00456] 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 at., Cancer Immunol. Immunother. .
2011, 60, 75-85,
Muranski, et al., Nat. Cl/n. Pract. Oncol., 2006,3, 668-681, Dudley, et al., I
Cl/n. Oncol. 2008,
26, 5233-5239, and Dudley, et at., I Cl/n. Oncol. 2005, 23, 2346-2357, all of
which are
incorporated by reference herein in their entireties.
[00457] In some embodiments, the fludarabine is administered at a
concentration of 0.5
[tg/mL -10 [tg/mL fludarabine. In some embodiments, the fludarabine is
administered at a
concentration of 1 [tg/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/day 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.
[00458] In some embodiments, the mafosfamide, the active form of
cyclophosphamide, is
obtained at a concentration of 0.5 [tg/mL -10 [tg/mL by administration of
cyclophosphamide. In
some embodiments, mafosfamide, the active form of cyclophosphamide, is
obtained at a
concentration of 1 [tg/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.
[00459] In some embodiments, lymphodepletion is performed by administering the
fludarabine and the cyclophosphamide are 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.
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[00460] In an embodiment, 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. Several methods of
expanding TILs
obtained from bone marrow or peripheral blood are described herein. In an
embodiment of the
invention, 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 filve days. Several methods of expanding TILs obtained from bone marrow or
peripheral
blood are described herein.
EXAMPLES
[00461] The embodiments encompassed herein are 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 ¨ Expansion of TILs from Non-Hodgkin's Lymphomas
[00462] TILs were expanded from five non-Hodgkin's lymphoma tumors (one mantle
cell
lymphoma tumor, three follicular lymphoma tumors, and one ABC-type diffuse
large B cell
lymphoma tumor) with the pathologies given in FIG. 1, using IL-2 for 11 to 14
days in a pre-
REP stage, followed by subsequent REP for 14 days using IL-2, mitogenic anti-
CD3 antibody,
and irradiated allogeneic peripheral blood mononuclear cell (PBMC) feeders.
TILs were
successfully generated from all 5 lymphoma tumors with maximum expansion index
of 680 fold,
significantly higher than previously observed using other methods.
Schwartzentruber, et at.,
Blood 1993, 82, 1204-1211. Further, mean CD3+ T cell population was 95%
(versus 75% using
the method of Schwartzentruber, et at., Blood 1993, 82, 1204-1211).
[00463] Cell sorting and flow cytometry was performed using a Becton,
Dickinson & Co.
(BD) FACS CANTO II system. A marked relative increase in effector memory cells
that was
comparable to that in melanoma TILs was observed by flow cytometry analysis
(FIG. 2). A
significant increase in effector memory CD45RA+ (TEMRA) cells (p=0.0013; CD4,
CD8) and
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CD28+CD4+ (p= 0.008) subsets was observed in lymphoma compared with melanoma
TIL
cultures (FIG. 3).
[00464] Comparisons of phenotypic markers of T cell differentiation in CD4+
and CD8+
subsets are shown in FIG. 4 and FIG. 5, respectively. Comparisons of
phenotypic markers of T
cell exhaustion in CD4+ and CD8+ subsets are shown in FIG. 6 and FIG. 7,
respectively.
[00465] FIG. 8 illustrates a comparison of cell types between non-Hodgkin's
lymphoma TILs
and melanoma TILs. An increasing trend in the number of CD4+ T cells in
lymphoma TILs
compared to melanoma TILs is shown.
[00466] FIG. 9 illustrates bioluminescent redirected lysis assay (BRLA)
results. Minimal
cytolytic activity of TIL measured by BRLA as LU50/106 at 4 hrs ranged from <1-
6 LU50 and at
24hrs, 1-39 LU50 in lymphoma TIL compared to melanoma TIL (11-75 LU50, 4hrs).
[00467] FIG. 10 illustrates interferon-y (IFN- y) enzyme-linked immunosorbent
assay
(ELISA) results for lymphoma TILs versus melanoma TILs. Showing comparable
results.
ELIspot assay results for the lymphoma TILs are shown in FIG. 11 and are
compared to results
of the same assay for melanoma TILs in FIG. 12. In the ELIspot assay, a wide
range of IFN-y
production by lymphoma TILs was observed upon stimulation with phorbol 12-
myristate 13-
acetate/ionomycin, anti-CD3 antibody, or CD3/CD28/4-1BB beads, and IFN-y
produced by
some lymphoma TILs under these conditions was comparable to the IFN-y produced
by
melanoma TILs, and in several cases, IFN-y production in lymphoma TILs was
much higher.
[00468] FIG. 13 illustrates the results of a NANOSTRING NCOUNTER analysis
(Nanostring
Technologies, Inc., Seattle, WA), showing that lymphoma TILs express higher
levels of RORC
IL17A (TH17 phenotype) and GATA3 (Th2 phenotype) compared to melanoma TILs.
This
finding is consistent with the observation that lymphoma-reactive T cells are
primarily TH2 and
TH17.
[00469] Overall, the results provide evidence that TIL cell therapy may be
used to treat
patients with lymphoma.
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Example 2 ¨ Phenotypic and Functional Characterization of Marrow Infiltrating
Lymphocytes (MIL) Grown from Bone Marrow of ANIL Patients and Peripheral Blood
Lymphocytes (PBL) Grown from Peripheral Blood of AML Patients
[00470] Samples of bone marrow and as available a related blood sample were
obtained from
patients with acute myeloid leukemia (ANIL), including patients pre-treated
with at least three
rounds of a regimen comprising ibrutinib (1-[(3R)-3-[4-amino-3-(4-
phenoxypheny1)-1H-
pyrazolo[3,4-d]pyrimidin-l-y1]-1-piperidiny1]-2-propen-1-one), accompanied by
information
about the patient's age, gender, stage, tumor type, site of cancer, treatment
history, a de-
identified pathology report, and any molecular tests performed (e.g., MSI
expression and
Raf/Ras expression). MILs and PBLs were expanded using one of MIL Method 1,
MIL Method
2, or MIL Method 3, or PBL Method 1, PBL Method 2, or PBL Method 3, and the
MILs and
PBLs were phenotypically and functionally characterized.
[00471] FIGS. 36A and 36B illustrate the fold expansion for MILs and PBLs.
FIG. 36A
shows the fold expansion for 3 patients (MILL MIL2, MIL3) and FIG. 36B shows
the fold
expansion for the matched PBLs for patients 2 and 3 (PBL2, PBL3). MIL1.1 was
expanded
using MIL Method 1, MIL1.2 was expanded using MIL Method 2, and MIL1.3 was
expanded
using MIL Method 3, and PBLs were expanded using PBL Method 3. MIL1 fold
expansion
shows 25 (MIL1.1), 50 (MIL1.2), and 75 (MIL1.3) fold increases for each sample
within MILl.
This preliminarily demonstrates that MIL Method 3 may be a preferred expansion
method.
MIL2 and MIL3 fold expansion data appears poor, possibly due to low starting
cell number. For
comparison, the starting cell number for sample 3 of patient MIL1 (MIL1.3) was
138,000 cells,
while the starting cells numbers for MIL2 and MIL 3 were 62,000 and 28,000
respectively. PBL
fold expansion shown in FIG. 36B for MIL2 and MIL3 was about 10-fold and 40-
fold,
respectively, with similar starting cell numbers (338,000 for PBL2 and 336,000
for PBL3).
[00472] FIGS. 37A and 37B illustrate the number of IFN-y producing cells for
MILs (FIG.
37A) and matched PBLs (FIG. 37B). MIL1.3, MIL2, and MIL3 show significant
increases in
IFN-y secretion, indicating that MIL Method 3 is a preferred expansion method.
The data for
PBLs is inconclusive.
[00473] FIGS. 38A-38F show TCRc43+, CD4+, and CD8+ subsets for MILs and PBLs.
FIGS. 38A and 38D show TCRab+ subsets for MILs (FIG. 38A) expanded using all 3
methods
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(MIL1.1, MIL1.2, MIL1.3) and for PBLs (FIG. 38D) expanded using PBL Method 3.
The data
show that TCRc43+ subsets are at almost 100% for all MILs and PBLs, which
indicates that the
expansion process was successful in expanding almost all T-cells. FIGS 38B and
38E show CD4
subsets are decreased for MTh expanded by MTh Method 3 (which correlates to
the increase in
CD8 subsets in FIG. 38C). PBL data in FIGS. 38E and 38F appear consistent with
the MIL1.3
data.
[00474] FIGS. 39A-D and 40 A-D show data for CD4 subsets in MTh (FIG. 39) and
PBL
(FIG. 40). FIGS. 39A and 40A show data for naïve (CCR7+/CD45RA+); FIGS. 39B
and 40B
show data for central memory t-cells (CM) (CCR7+/CD45RA-); FIGS. 39C and 40C
show data
for effector memory T-cells (EM) (CCR7-/CD45RA-); and FIGS. 39D and 40D show
data for
terminally differentiated effector memory cells (TEMRA) (CCR7-/CD45RA+). All
samples
expanded using MIL Method 3 (MIL1.3) and PBL Method 3 (PBL2 and PBL3) are
consistent
with CD4 subsets in the comparator, melanoma TIL.
[00475] FIGS. 41A-D and 42A-D show data for CD8 subsets in MTh (FIG. 41) and
PBL (FIG.
42). FIGS. 41A and 42A show data for naïve (CCR7+/CD45RA+); FIGS. 41B and 42B
show
data for central memory t-cells (CM) (CCR7+/CD45RA-); FIGS. 41C and 42C show
data for
effector memory T-cells (EM) (CCR7-/CD45RA-); and FIGS. 41D and 42D show data
for
terminally differentiated effector memory cells (TEMRA) (CCR7-/CD45RA+). The
samples
expanded using MIL Method 3 (MIL1.3) are consistent with CD4 subsets in the
comparator,
melanoma TIL. The data for PBL2 and PBL3 was used as a control.
[00476] FIGS.43A and 43B show data for CD4CD27 and CD8CD27 subsets for MILs
(FIG.
43A) and PBLs (FIG. 43B). FIGS. 44A and 44B show data for CD4CD28 and CD8CD28
subsets for MILs (FIG. 44A) and PBLs (FIG.44B). The data for PBLs is shown for
Day 0 and
Day 14 of the expansion process for each sample, as compared with melanoma
TIL. The data
for MILs is shown at Day 0 and Day 14 for MIL1.3 only, as compared with
melanoma TIL.
CD28 subsets in MIL and PBL are similar to melanoma TIL.
[00477] FIGS. 45A and 45B represent a comparison of PD1+ cells within each of
CD4 and
CD8 subsets for MILs (FIG. 45A) and PBLs (FIG. 45B). FIGS. 46A and 46B
represent a
comparison of LAG3+ cells within each of CD4 and CD8 subsets for MILs (FIG.
46A) and
PBLs (FIG. 46B). The data for both PD1+ and LAG3+ show a substantial decrease
in for the
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MIL1.3 sample over the Day 0 measurement, while MILL 1 and MIL1.2 appeared to
trend
toward an increase for both PD1 and LAG3 over Day 0. The PBL data was used as
a control.
[00478] The experiments from this Example demonstrate that MILs expanded with
MIL
Method 3 had a higher fold expansion, were highly functional, had a higher
proportion of CD8
subsets, and had less LAG3+ and PD1+ T cell subsets. The data also showed that
the memory
subsets were similar to melanoma TIL. The data also showed that cryopreserved
samples
appeared to have higher fold expansion as compared with fresh samples. Much of
the data for
the PBL samples appears to be inconclusive, likely based on the small sample
size.
Example 3 ¨ Methods of Expanding TILs and Treating Cancers with Expanded TILs
[00479] Bone marrow is obtained using needle aspiration. The bone marrow
sample is
aspirated into heparin-containing syringes and stored overnight at room
temperature. After
storage, the contents of the syringes are pooled together into a sterile
container and quality
tested. The bone marrow is enriched for mononuclear cells (MNCs) using
lymphocyte
separation media (LSM) and centrifugation with a COBE Spectra. Cells in the
gradient are
collected down to the red blood cells and washed using HBSS. The MNCs are
cryopreserved
using a hetastarch-based cryoprotectant supplemented with 2% HSA and 5% DMSO,
reserving
some of the MNCs for quality control. The QC vial is thawed to determine the
CD3+ and
CD38-7138+ cell content of the MNC product.
[00480] The bone marrow is aspirated and fractionated on a Lymphocyte
Separation Medium
density gradient and cells are collected almost to the level of the red cell
pellet. This
fractionation method substantially removes red blood cells and neutrophils,
providing nearly
complete bone marrow. The resulting fractionated material is T-cells and tumor
cells. The bone
marrow is Ficolled, and TILs are expanded using methods known in the art and
any method
described herein. For example, an exemplary method for expanding TILs is
depicted in FIG. 14.
An exemplary method for expanding TILs and treating a cancer patient with
expanded TILs is
shown in FIG. 15.
Example 4 ¨ Phenotypic and Functional Characterization of Tumor Infiltrating
Lymphocytes (TIL) Grown from Non-Hodgkin Lymphoma Tumors
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[00481] The goals of the experiments described in this Example include
determing whether
TIL with therapeutic potential can be isolated and cultured from NHL tumors
and to compare
characteristics of NHL-derived TIL with melanoma-dervied TIL.
[00482] Materials and methods for extraction and expansion of TILs from a
patient are as
described herein. Patients' TIL were extracted from a suppressive tumor
microenvironment by
surgical resection of a lesion, in this case, lymph tissue. TILs were expanded
using the
expansion processes disclosed herein to yield 109 to 1011 TILs.
[00483] NEIL-derived TILs (1 mantle cell lymphoma (MCL), 3 follicular
lymphomas (FL), 3
diffuse large B cell lymphomas (DLBCL)) were analyzed for markers of
differentiation against
melanoma-derived TILs using flow cytometry. TILs were analyzed for anti-CD56,
anti-TCRab,
anti-CD3, anti-CD4, anti-CD8, anti-CD27 and anti-CD28 antibodies. These
antibodies were
used as Differentiation Panel 1 (DF1). Anti-CD3, anti-CD4, anti-CD9, anti-
CD38, and anti-
HLA-DR, anti-CCR7, and anti-CD45RA antibodies were used as differentiation
panel 2 (DF2).
DF2 was used to identify the following T-cell subsets: Naïve (CCR7+/CD45RA+);
central
memory t-cells (CM) (CCR7+/CD45RA-); effector memory T-cells (EM) (CCR7-
/CD45RA-);
and terminally differentiated effector memory cells (TEMRA) (CCR7-/CD45RA+).
[00484] Figure 16 shows CD4 and CD8 T-cells in different cell subpopulations
in different
cancer types. Melanoma (square), mantle cell (shaded circle), diffuse large B
cell lymphoma
(open circle) and follicular lymphoma (black circle) cancer types were tested.
Figures 16A-16D
generally demonstrate a trend for lymphoma TIL to be more highly proliferative
and therefore
have higher anti-tumor activity as compared with melanoma TIL. Likewise,
Figure 17B shows
that CD4/CD28 expressing lymphoma T-cells have higher proliferative capacity
than CD4/CD28
expressing melanoma T-cells.
[00485] Interferon gamma (IFNy) production by TILs was measured by stimulating
TILs with
mAB-coated DynabeadsTM (CD3, CD28, and CD137), then using ELIspotTM
(Immunospot CTL)
and enumerated using ImmunospotTM S6 entry analyzer, and also by ELISA using
DuoSetTM
ELISA kit (R&D systems following the manufacturer's instructions.
[00486] Figures 18A and 18B demonstrate that IFNy production by NHL TIL and
Melanoma
TIL are similar, indicating a similar cytotoxicity functionality between the
two TIL types.
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[00487] Lytic potential of TILs was determined using bioluminescent Redirected
Lysis Assay
(BRLA). P815 cells transduced with lentiviral vector encoding eGFP and firefly
luciferase were
used as target cells. TILs and target cells were cocultured for 4 hours/24
hours in the presence of
OKT3. Luciferin was then added and cells were incubated for 5 minutes.
Bioluminescence was
measured using a luminometer. Perent survival and percent cytotoxicity were
calculated as
follows:
% Survival = (experimental survival - minimum) / (maximum signal- minimum
signal) x 100
%Cytotoxicity = 100 ¨ (% Survival)
[00488] Lytic potential of TILs was expressed as a lytic unit, LUSO, which
represents 50
percent cytotoxicity of target cells induced by effector cells.
[00489] TILs were assayed to determine their tumor-killing ability on both
autologous and
allogeneic tumors. TILs were mixed with autologous lymphoma cells or
allogeneic melanoma
cell lines (526 melanoma cell line) at different effector cell to target cell
ratios (E:T ratio) ¨
either 10:1, 20:1, 50:1, or 100:1. Tumor cells were labeled with CellTrace
Violet dye
(ThermoFisher) prior to coculture. After 24 hours, cells were stained with 7-
AAD to determine
cell death. The proportion of tumor cells killed by TILs were represented as 7-
AAD positive
tumor cells that were gated on CellTrace Violet dye versus CD19 for the
lymphoma cells and
CellTrace Violet Vs MCSP for melanoma cells.
[00490] Figure 19 shows that NHL TIL and melanoma TIL have similar cytotoxic
functionality against both allogeneic and autologous tumors at 4 hours (Figure
19A) and 24
hours (Figure 19B).
[00491] Gene expression analysis was also performed on the TILs using the
nCounter GX
Human Immunology V2 panel (NanoString, Seattle). 10Ong total RNA was assayed
per the
manufacturer's instructions. Data were normalized by scaling with geometric
mean of the buil-
in control gene probes for each sample. The data was mapped against and
compared to
melanoma gene expression.
[00492] Figure 21 demonstrates the results of the gene expression analysis.
The heat map
shows fold change in gene expression over melanoma TIL. IL17A and RORC
expression from
lymphoma-derived TIL had higher expression as compared with melanoma-derived
TIL.
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[00493] Overall, the results of this experiment demonstrated that the
functional characteristics
of lymphoma-derived TIL are similar to melanoma-derived TIL, indicating that
use of
lymphoma-derived TIL would be successful in treating lymphoma cancers.
Example 5 - Phenotypic and Functional Characterization of Peripheral Blood
Lymphocytes
kPBLs) Grown from Peripheral Blood of Patients With Chronic Lymphocytic
Leukemia (CLL).
[00494] PBMCs were collected from patients with CLL pre- and post- treatment
with three
rounds of ibrutinib.
[00495] T-cells were expanded using three different methods, PBL Method 1, PBL
Method 2,
and PBL Method 3, as described in Figure 24 and elsewhere herein. Certain
samples were
derived from Fresh PBMCs and certain samples were derived from cryopreserved
PBMCs.
Once the cells were expanded and harvested, they were phenotyped and
functionally
characterized using the methods described in Example 4, above, and elsewhere
herein. The
goals of this Example were to determine an optimal expansion process for PBLs
and to
determine whether PBLs expanded from ibrutinib treated samples are more potent
than PBLs
expanded from untreated samples.
[00496] PBL fold expansion is shown in Figure 26. Results for PBLs expanded
using PBL
Method 1, PBL Method 2, and PBL Method 3 are shown. Untreated PBLs (PreRx PBL)
showed
a mean 179-fold expansion and ibrutinib treated PBLs (PostRx PBL) showed a
mean 306-fold
expansion. PBLs derived from Fresh PBMC (PBL) showed only a mean 82-fold
expansion. As
between PBLs and PostRx PBLs, p=0.006. As between PBLs and PreRx PBLs, p=0.3,
and as
between PreRx PBLs and PostRx PBLs, p=0.1. Overall, an increase in the mean
fold-expansion
is seen for all PostRx PBL groups over all groups in both PBLs and PreRx PBLs.
[00497] Figure 27 demonstrates interferon-gamma (IFN-y) producing cells in
PBL, PreRx
PBL, and PostRx PBL. For PBL, the mean number of IFN-y producing cells was
about 1864.
For PreRx PBL, the mean number of IFN-g producing cells was about 7530, and
for PostRx
PBL, the mean number of IFN-y producing cells was about 11984. As between PBLs
and
PostRx PBLs, p=0.006. As between PBLs and PreRx PBLs, p=0.006, and as between
PreRx
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PBLs and PostRx PBLs, p=0.01. Overall, a significant increase in the mean
number of IFN-y
producing cells is seen for all PostRx PBL groups over all groups in both PBLs
and PreRx PBLs.
[00498] Phenotypic characterization was performed on each of the samples. FIG.
28
represents the proportion of CD4+ and CD8+ T cell subsets in PreRx PBL and
PostRx PBL, and
uses melanoma TIL as a comparator. Here, the data show that CD4 subsets (shown
on the left)
were comparable between both PreRx PBL and PostRx PBL, regardless of which
method was
used to expand the cells. CD4 subsets in PreRx PBLs and PostRx PBLs were shown
to be higher
than melanoma TIL (p=0.0006 for each). CD8 subsets (shown on the right) were
lower in both
PreRX PBL and PostRX PBL, regardless of the process used to expand the cells.
CD8 subsets in
PreRx PBLs and PostRx PBLs were shown to be lower than melanoma TIL (p=0.0006
for each).
It is hypothesized that the lower CD8 subsets are merely a derivative of the
type of cancer (i.e.,
in CLL, CD4 subsets are typically expanded).
[00499] Figures 29A-29D represent a comparison between CD4 memory subset of
PreRx
PBLs and PostRx PBLs, using melanoma TIL as a comparator. Figure 29A shows
data for naïve
(CCR7+/CD45RA+); Figure 29B shows data for central memory t-cells (CM)
(CCR7+/CD45RA-); Figure 29C shows data for effector memory T-cells (EM) (CCR7-
/CD45RA-); and Figure 29D shows data for terminally differentiated effector
memory cells
(TEMRA) (CCR7-/CD45RA+). Figure 29 demonstrates that the CD4 memory subsets
for PreRx
PBLs and PostRx PBLs are comparable to that seen for melanoma TIL.
[00500]
Figures 30A-30D represent a comparison between CD8 memory subset of PreRx
PBLs and PostRx PBLs, using melanoma TIL as a comparator. Figure 30A shows
data for naïve
(CCR7+/CD45RA+); Figure 30B shows data for central memory t-cells (CM)
(CCR7+/CD45RA-); Figure30C shows data for effector memory T-cells (EM) (CCR7-
/CD45RA-); and Figure 30D shows data for terminally differentiated effector
memory cells
(TEMRA) (CCR7-/CD45RA+). Figure 30 demonstrates that the CD8 memory subsets
for PreRx
PBLs and PostRx PBLs are comparable to that seen for melanoma TIL.
[00501] Figures 31A and 31B represent a comparison of CD27 subsets of CD4
cells (Fig.
31A) and CD8 cells (Fig. 31B), using melanoma TIL as a comparator. CD4CD27
cell subsets
were significantly higher in both the PreRx PBL (p=0.03) and PostRx PBL
(p=0.02) as
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compared to melanoma TIL. CD8CD27 cell subsets were significantly higher in
both the PreRx
PBL (p=0.002) and PostRx PBL (p=0.001) as compared to melanoma TIL.
[00502] Figures 32A and 32B represent a comparison of CD28 subsets of CD4
cells (Fig.
30A) and CD8 cells (Fig. 30B), using melanoma TIL as a comparator. CD4CD28
cell subsets
and CD8CD28 cell subsets were shown to be comparable in both the PreRx PBL and
PostRx
PBL as compared to melanoma TIL.
[00503] Figures 33A and 33B represent a comparison of LAG3+ subsets within the
CD4+
(FIG. 33A) and CD8+ (FIG. 33B) populations for PreRx PBLs and PostRx PBLs. The
data
show a significant mean decrease in LAG3+ subsets in both CD4+ (p=0.06) and
CD8+ (p=0.01)
populations in the PostRx PBLs.
[00504] Figures 34A and 34B represent a comparison of PD1+ subsets within the
CD4+ (FIG.
34A) and CD8+ (FIG. 34B) populations for PreRx PBLs and PostRx PBLs. The data
show a
mean decrease in PD1+ subsets in both CD4+ and CD8+ populations in the PostRx
PBLs, but
the decrease was not significant.
[00505] Figures 35A and-35B show results of cytolytic activity of PreRx PBLs
(FIG. 35A)
and PostRx PBLs (FIG. 35B), measured using a Bioluminescent Redirecetd Lysis
Assay
(BRLA). The assay was performed using the Cell1TraceTm Violet Cell
Proliferation Kit
(Invitrogen) as follows: The Effector Cells, which are the PBLs, were labeled
with
carboxyfluorescein succinimidyl ester (CFSE). The Target Cells (autologous
CD19+ tumor
cells) were incubated with mitocyin C, and then labeled with CellTraceTm
Violet (CTV) in
accordance with the CellTraceTm Violet Cell Proliferation Kit instructions.
The Effector and
Target cells were incubated for 24 hours at ratios of 2:1, 5:1 and 20:1 (E:T
cells). The
countbright beads were added, the cells were stained with Annexin V ¨ PI, and
then analyzed for
CTV+/Annexin-V PI+ cells (which provides the number of dead cells). The PostRx
PBLs
appear to be more potent because less cells are required to kill 50% of the
target tumor cells (i.e.,
the LUSO is lower for PostRx PBLs than for PreRx PBLs).
[00506] The experiments performed in this Example demonstrated the following
results:
PBLs expanded from fresh CLL PBMCs showed lower fold expansion and
significantly less
IFN- production as compared to PBLs expanded from cryopreserved PBMCs (PreRx
PBLs and
PostRx PBLs); PostRx PBLs showed consistently higher fold expansion and
significant increase
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in IFN-LII production as compared with PreRx PBLs; and both PreRx PBLs and
PostRx PBLs
showed lytic activity against autologous (CD19+) tumor cells, although PostRx
PBLs had a
lower LUSO than PreRx PBLs.
[00507] Example 6 - An Exemplary Embodiment of Selecting and Expanding PBLs
from
PBMCs in CLL Patients
[00508] PBMCs are collected from patients (optionally pretreated with an ITK
inhibitor such
as ibrutinib) and either frozen prior to 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 6x106 IU/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 GlutaMAX4D),
then
diluting it with 100 mL (1:1) with AIM-V to make CM2. The CM2 is protected
from light, and
sealed tightly when not in use.
[00509] All of the following steps are performed under sterile cell culture
conditions. An
aliquot of 50 mL 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.
[00510] 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-
times with a pipette to break up the cell pellet. An additional 3-5 mL of CM2
medium is
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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 mL of the cell suspension from the tube for
cell counting
with an automatic cell counter, for example, a Nexcelom Cellometer K2. The
number of live
cells in the sample is determined and record.
[00511] 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% DMSO). Freeze one or two aliquots of the reserved cells in freezing
medium, each aliquot
consisting of 2-5 x 106 cells in 1 mL of freezing medium in a cryovial, 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.
[00512] 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 107 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 are aspirated from both samples,
disrupt the pellet, and
resuspend the cells in 1 mL of CM2 medium supplemented with 3000 IU/mL IL-2 to
each pellet,
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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.
[00513] 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.
[00514] The T-cells (PBLs) and beads are cultured together as follows. On day
0: In a G-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 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
cryopreservation medium using a Mr. Frosty cell freezer. The non-T-cell
fraction of cells are
frozen in CS10 cryopreservation 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.
[00515] 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 mL conical tube.
The wells are
washed with 1 mL 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 lx106 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
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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 IU/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.
[00516] 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.
[00517] 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). 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
transferred 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
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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 7 ¨ An Exemplary Embodiment of Selecting and Expanding PBLs from PBMCs
[00518] For the expansion of PBLs from PBMCs obtained from CLL patients or
patients with
other diseases described herein, including CLL patients having previously
received ibrutinb or an
ITK inhibitor or with ibrutinib-relapsed or refractory CLL, the following
procedure may be used.
All steps require the use of sterile technique in a biological safety cabinet
(BSC) or similar
enclosure.
[00519] On day 0, prepare 6 x 106 IU/mL IL-2. If aliquots are available, thaw
a fresh aliquot
and leave it at 4 C in refrigerator or on ice until ready to use. Prepare
small volume (e.g. 200
mL) of CM2 media. First prepare 100 mL of CM1 media, substituting GlutaMAX for
glutamine
in the procedure, then dilute it 1:1 with AIM V to make CM2. While performing
this
experiment, keep the CM2 warm in a 37 C water bath, protected from light,
with cap closed
tightly. When it is being used in the hood, do not leave the cap off or loose.
In a 37 C water
bath, warm an aliquot of CM2 (without IL-2) in a 50 mL conical tube to use for
thawing and
washing CLL sample. Remove the cryovial containing the CLL PBMC sample from
LN2
freezer storage, keeping sample on dry ice until ready to thaw, or use fresh
CLL PBMCs. Just
prior to beginning thaw, place warmed media aliquot into BSC. Add 5 mL of
media into a fresh,
sterile, labeled 50 mL conical tube. Thaw sample by placing the cryovial in 37
C water bath
until only a few ice crystals remain in the cryovial. Transfer cryovial
containing thawed samples
to the BSC. Using a sterile transfer pipet, add an equal volume of warmed
media dropwise to
CLL sample cryovial (-1 mL). Using the same transfer pipet, remove the sample
from the
cryovial and add it dropwise to the prepared 50 mL conical tube. Rinse tube
with an additional
1-2 mL of CM2 and transfer that to the 50 mL conical tube. Bring the volume to
15 mL, swirl
sample gently to rinse cells well, then spin sample in high speed centrifuge
to collect cell pellet,
at 400 x g for 5 min at room temperature. Return sample to BSC and aspirate
off supernatant
from pellet, being careful not to disturb cell pellet. Cap tube and scrape it
along a rough surface
(such as a tube rack) to help break up cell pellet. Using a 1 mL pipettor and
tip, add 1 mL of
fresh CM2 to the cell pellet and gently aspirate the cells up-and-down 5-10
times to break up cell
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pellet. Add an additional 3-5 mL of CM2 to cell suspension; pipet up-and-down
several times to
mix sample well. Record volume of cell suspension. Remove a representative
volume of cell
suspension from the tube for counting (e.g., 100 Using an automatic cell
counter, such as
Nexcelom Cellometer K2, count cells using appropriate procedure. Determine the
total number
of live cells in the sample. Reserve a minimum of 5 x 106 cells for
phenotyping and other
experiments. Spin reserved sample at 400x g for 5 min at room temperature to
collect pellet.
Freeze one or two aliquots of the reserved sample in freezing medium (sterile,
heat-inactivated
fetal bovine serum containing 20% DMSO). Slow-freeze cell sample in a Mr.
Frosty cell freezer
placed in a -80 C freezer. Transfer to LN2 storage after a minimum of 24 hours
at -80 C.
[00520] For the proceeding separation steps, work quickly, keeping the cells
cold; use pre-
cooled solutions. Purify the T-cell fraction of the CLL sample using Pan T-
cell Isolation Kit
(Miltenyi: Catalogue# 130-096-535). Prepare wash buffer prior to beginning
procedure. Wash
buffer: phosphate-buffered saline, pH 7.2, containing 0.5% bovine serum
albumin and 2 mM
EDTA; read pH and adjust if necessary; sterile filter; store at 4 C. Spin
sample at 400x g for 5
min to collect cell pellet. Aspirate off media supernatant and resuspend the
cell pellet in 40 tL
of wash buffer for every 107 total cells. Add 10 !IL of Pan T Cell Biotin-
Antibody Cocktail for
every 107 total cells. Mix sample well and incubate in the refrigerator or on
ice for 5 min. Add
30 !IL of wash buffer to sample for every 107 total cells. Add 20 tL of Pan T
Cell MicroBead
Cocktail for every 107 total cells. Mix well and incubate for 10 min in the
refrigerator or on ice.
Proceed to magnetic cell separation. Use LS column and QuadroMACS magnet for
this
procedure. Each LS column has a maximum capacity of 2 x 109 total cells.
Prepare LS column
for use; always wait until column reservoir is empty before proceeding to the
next step. Place LS
column in magnetic field of QuadroMACS magnet. Rinse LS column with 3 mL of
prepared,
cold wash buffer. Collect wash into a 15 mL conical tube. Discard wash. Place
fresh tube
labeled "T cell fraction" under LS column. Apply cell suspension onto the
column. Collect
flow-through containing the unlabeled cells ¨ this is the enriched T-cell
fraction. Wash column
with 3 mL of wash buffer. Collect the unlabeled cells that wash through the
column into the
same 15 mL conical "T cell fraction" tube. Cap tube and place on ice. Remove
LS column from
QuadroMACS magnet and place it onto a fresh 15-ml conical tube labeled "non-T
cell fraction."
Pipet 5 mL of wash buffer onto the column and immediately flush out the
magnetically labeled
non-T cells by firmly pushing plunger (provided with the LS column) into the
column. Place
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both "T-cell fraction" and "Non-T cell fraction" tubes into centrifuge and
spin at 400 x g for 5
min to collect cell pellet. Aspirate off supernatant from both samples, cap
tubes, and resuspend
each pellet by scraping tube against a rough surface. Using a 1 mL pipettor
and tips, add 1 mL
of CM2 medium supplemented with 3000 IU/ml IL-2 to each pellet. Resuspend each
pellet by
gently pipetting up-and-down 5-10 times to break pellets up further. Add 1-2
mL of fresh
medium to each sample, mixing each sample well. Remove a small representative
aliquot from
each sample (e.g., 50 L). Place cell samples into tissue culture incubator,
loosening cap. Count
cells; record counts and viability. Prepare a small amount of DynabeadsTM
Human T-Expander
CD3/CD28 for use. Vortex stock vial of CD3/CD28 Dynabeads for 30 sec at medium
speed on a
vortex mixer. Remove required aliquot of beads from stock vial to a sterile
1.5 mL microtube
Wash beads with bead wash solution by adding 1 mL of wash to the 1.5 mL
microtube
containing the beads. Tap tube to mix sample. Place tube containing beads onto
DynaMagTm-2
magnet and let tube sit for 30 sec while beads are drawn to magnet. Aspirate
off wash solution
from the side of the microtube opposite the DynaMag magnet. Remove microtube
from magnet
and place in a tube rack. Using a 1 mL pipettor and tip, add 1 ml of CM2
supplemented with IL-
2 to the beads. Transfer the bead solution to a fresh 15 mL (or 50 mL) conical
tube labeled
"beads, 500,000/mL." Bring beads to a final volume that will give a
concentration of 500,000
beads/ml (e.g., 10 x 106 beads brought to a final volume of 20 mL). Set up
cell culture as
follows, a minimum of 1 well per sample. More wells can be set up if there are
enough cells. In
a G-Rex 24-well plate, in a total of 7 mL per well, add 500,000 T cells,
500,000 CD3/CD28
Dynabeads (1 mL of 500,000 beads/mL suspension), and CM2 supplemented with
3000 IU/ml
IL-2. Place G-Rex 24-well plate into humidified 37 C, 5% CO2 incubator. If
there are enough
cells, retain a small portion of the T-cell fraction for repeat of REP, or for
other experiments,
freezing the sample in CS10 cryopreservation medium using a Mr. Frosty cell
freezer. Count the
non-T cell fraction of cells and freeze them in CS10 cryopreservation medium
using a Mr. Frosty
cell freezer.
[00521] On day 4, media exchange is performed as follows. Prepare a sufficient
volume of
CM4 (supplemented with 3000 IU/mL IL-2) to replace half the media from the
sample wells and
warm it to 37 C in water bath. Remove G-Rex 24-well plate from incubator to
BSC. Remove
half the volume of media from each well (3.5 mL). Add equivalent volume (3.5
mL) of fresh
media to each well. Return G-Rex 24-well plate to humidified, 37 C, 5% CO2
incubator.
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[00522] On day 7, expansion using REP is performed as follows. Prepare a small
volume of
CM4 (supplemented with 3000 IU/mL IL-2) to perform washes of culture wells.
Keep warm in
37 C water bath. Remove G-Rex 24-well plate from the incubator to BSC. Remove
half the
volume of media from each well and discard. Resuspend remaining cells and
transfer to a
labeled, sterile 15 mL conical tube. Wash well with 1 mL of prepared CM4 and
transfer wash
solution to the same 15 mL conical tube. Retain the G-Rex 24-well plate in the
tissue culture
hood ¨ unused wells of the same plate can be used for the expansion of the PBL
sample.
Remove a representative volume of cells and count using automated cell
counter. Determine
number of wells or culture vessels required to expand PBL. If < 1 x 106 total
live cells: Set up
expansion in one well of a G-Rex 24-well plate using 500,000 PBL and a 1:1
ratio of
DynabeadsTM Human T-Expander CD3/CD28 prepared as in Step 9.16 in 7 mL CM4
(supplemented with 3000 IU/mL IL-2). Freeze remainder of cells for back-up
expansion or for
phenotyping and other subsidiary procedures. If 1 x 106 or more total live
cells: set up
expansion in replicate wells in a G-Rex 24-well plate using 500,000 PBL per
well and a 1:1 ratio
of DynabeadsTM Human T-Expander CD3/CD28 prepared as in Step 9.16 in final
volume of 7
ml/well CM4 (supplemented with 3000 IU/mL IL-2). Alternately, with excess
sample, set up
expansion in a G-Rexl0M culture flask using 10-15 x 106 PBL per flask and a
1:1 ratio of
DynabeadsTM Human T-Expander CD3/CD28 prepared as in Step 9.16 in a final
volume of 100
ml/well CM4 (supplemented with 3000 IU/mL IL-2). Slow-freeze excess Day 7 PBL
samples in
labeled cryovials placed in a Mr. Frosty cell freezer at -80 C. Transfer to
LN2 storage after a
minimum 24 hours at -80 C. These samples can be used as back-up samples for
expansion or
for phenotyping and other subsidiary procedures. (Recommended minimum number
of cells to
retain on Day 7: 2 x 106 to 5 x 106.) Place culture plates or flasks into
humidified, 37 C, 5%
CO2 incubator.
[00523] On day 11, media exchange is performed as follows. Prepare a
sufficient volume of
CM4 (supplemented with 3000 IU/mL IL-2) to replace half of the volume in each
culture well or
vessel and keep it warm in a 37 C water bath. Remove the culture vessels from
the incubator to
the BSC. Remove half the media from each well or flask and discard. Add
equivalent volume to
each culture well or flask. Return culture vessels to humidified, 37 C, 5% CO2
incubator.
[00524] On day 14, REP harvest is performed as follows. Warm a small volume of
AIM V
media in a 37 C water bath to use for washes in the following steps. Transfer
to BSC when
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ready to harvest samples. Remove the culture vessels from the incubator to the
BSC. If culture
is in G-Rex 24-well plate, remove about half of the volume from each well and
discard. For
larger cultures, proceed to the "REP is complete" step in the next paragraph.
Mix sample with
serological pipet and transfer cells to labeled, sterile 15 mL conical tube.
Wash well with 1-2
mL of fresh, warmed media. Cap 15mL conical tube and place in DynaMagTm-15
Magnet.
Allow sample to sit for 1 min in magnet to allow magnetic beads to be drawn to
magnet. Using a
mL serological pipet, remove the cell suspension to a fresh, labeled 15 mL
conical tube.
Remove first 15-ml tube from magnet and wash the beads with a minimum of 2 ml
of fresh AIM
V. Place tube back on magnet and allow it to sit for 1 min. Using a 5 mL
serological pipet,
remove the wash media to the second labeled 15 mL conical tube. If more than
one well per
sample was prepared, all wells of the same condition can be combined after
washing the beads.
If culture is in G-Rex10M flask, reduce volume by aspiration to about 10 mL
total. Mix sample
using 10 mL serological pipet and transfer cells to labeled, sterile 15 mL
conical tube. Wash
flask with 2 mL of fresh, warmed media. Cap 15 mL conical tube and place in
DynaMagTm-15
Magnet. Allow sample to sit for 1 min in magnet to allow magnetic beads to be
drawn to
magnet. Using a 5 mL serological pipet, remove the cell suspension to a fresh,
labeled 15 mL
conical tube. Remove first 15 mL tube from magnet and wash the beads with a
minimum of 2
mL of fresh AIM V. Place tube back on magnet and allow it to sit for 1 min.
Using a 5 mL
serological pipet, remove the wash media to the second labeled 15 mL conical
tube. If more than
one flask per sample was prepared, all can be combined after washing the
beads. If cells appear
to be extremely dense, extra pre-warmed AIM V media can be added to the
culture. Remove a
representative volume of cells and count using automated cell counter. Record
cell number and
viability. Place tubes containing cells into humidified, 37 C, 5% CO2
incubator, with cap
loosened, while counting cells.
[00525] At this point, the REP is complete. Post-REP testing of PBL can be
done on fresh or
frozen samples. Freeze PBL samples in CS10 cryopreservation medium, or prepare
as needed in
alternative formulations for delivery to a patient. Lower concentrations of
cells (e.g., 5 x 106
cells/vial) can be used for phenotyping by flow cytometry and co-culture
assays, so it is
recommended to reserve 6-10 vials at low concentration, with the remainder at
a higher
concentration (30-50 x 106 cells/vial). The foregoing procedure may be scaled,
adjusted, or
optimized, and adapted as needed for regulatory compliance (including to
satisfy good
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manufacturing practices and International Conference on Harmonization
guidance, as adapted by
the U.S. Food and Drug Administration and other regulatory authorities), as
will be apparent to
the skilled artisan.
[00526] 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.
[00527] 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.
[00528] All 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.
[00529] 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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