Note : Les descriptions sont présentées dans la langue officielle dans laquelle elles ont été soumises.
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METHODS FOR CULTURING IMMUNE CELLS
CROSS-REFERENCE TO RELATED APPLICATIONS
[0001] This application claims priority benefit of ITS
Provisional Application Nos
63/273,138 filed October 28, 2021; 63/365,326 filed May 25, 2022; and
63/379,632 filed October
14, 2022, each of which is herein incorporated by reference in its entirety.
REFERENCE TO SEQUENCE LISTING SUBMITTED ELECTRONICALLY VIA EFS-WEB
[0002] The content of the electronically submitted sequence
listing in ASCII text file
(Name: 4385 110PCO2 Seqlisting ST26; Size: 7,976 bytes; and Date of Creation:
October 27,
2022) filed with the application is herein incorporated by reference in its
entirety.
FIELD
[0003] The present disclosure relates to compositions comprising
tumor infiltrating
lymphocytes (TILs) and methods of culturing the cells. In some aspects, the
methods disclosed
herein preferentially promote the enrichment of oligoclonal or polyclonal
tumor reactive (e.g.,
tumor specific) stem-like T-cells, e.g., TILs characterized by being less
differentiated. Cells
cultured using the methods disclosed herein can be used for various cell
therapies, including, but
not limited, to adoptive cell therapies such as autologous T cell therapies.
BACKGROUND
[0004] The use of immunotherapy strategies has demonstrated
considerable clinical
efficacy in the treatment of certain types of advanced cancer. However, in
spite of notable
successes, the vast majority of patients with advanced cancers still do not
benefit from
immunotherapy treatments and will eventually succumb to their illness. A key
limitation to cell
therapy techniques such as adoptive cell therapies including chimeric antigen
receptor (CAR) and
engineered TCR T cells is a lack of suitable tumor targets, which may
contribute to the lack of
widespread clinical responses observed in patients with solid cancers that
have been treated with,
e.g., CAR T cells.
[0005] Another approach that has had some success in mediating
clinical response in
patients with advanced cancer is the isolation, expansion, and infusion of
autologous tumor
infiltrating lymphocytes (TILs). TILs are heterogenous, with variable
compositions of tumor-
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reactive and irrelevant or suppressive T cells. The tumor-reactive populations
are frequently highly
antigen-experienced, resulting in cell products that are in a pre-
dysfunctional state with limited
functionality.
[0006] Traditional methods of culturing and expanding TILs have
been found to lead to
terminal differentiation of the TILs, resulting in poor persistence of the
TILs upon transfer to
patients. See, e.g., Rosenberg et al., Clinical Cancer Research 17(13): 4550-
557 (2011). Further,
current methods of expanding heterogenous TIL populations from tumor fragments
yield
populations of TILs with reduced polyclonality and the loss of many tumor-
dominant T cell clones
(see, e.g., Poschke, et al,, Clin. Cancer Res. (2020), which is incorporated
by reference herein in
its entirety). As a result, generating such TIL-derived infusion products
often results in loss of
tumor-specific T cells during expansion and an ill-defined mix of immune cells
at various states of
differentiation, which are ineffective at eradicating solid tumors. To be
curative, TILs with
enhanced self-renewing stem/effector properties are needed. Moreover, methods
have not yet been
described for obtaining an expanded population of less-differentiated TILs
with a high level of
clonal diversity that retain the ability to further divide and target and kill
cancer cells.
[0007] To date, these and other critical limitations have
curtailed the use of TILs as an
effective therapeutic, making it difficult to obtain a sufficient number of
tumor-reactive TILs for
use in T cell therapy. As such, there remains a need in the art for improved
methods of preparing
TIL compositions and therapies using the same.
BRIEF SUMMARY
[0008] Some aspects of the present disclosure are directed to a
method of culturing tumor
infiltrating lymphocytes (TILs) e.v vivo or in vitro comprising placing a
heterogeneous population
of TILs in a metabolic reprogramming medium ("MRM") comprising potassium ion
at a
concentration of about 30 mM to about 100 mM. In some aspects, the
heterogeneous population
of Tits is enriched in CD8+ TILs after being placed in the MR1\4.
[0009] Some aspects of the present disclosure are directed to a
method of increasing a
number or percentage of CD8 TILs ex vivo or in vitro comprising culturing a
heterogeneous
population of TILs in an MRM comprising potassium ion at a concentration of
about 30 mM to
about 100 mM.
[0010] Some aspects of the present disclosure are directed to a
method of preparing a
CD8 -enriched population of TILs, comprising culturing a heterogeneous
population of Tits ex
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vivo or in vitro in an MRM comprising potassium ion at a concentration of
about 30 mM to about
100 mM.
[0011] In some aspects, the heterogeneous population of TILs
comprises CD4+ TILs and
CDS+ TILs. In some aspects, the heterogeneous population of TILs is obtained
from one or more
tumor sample obtained from a subject. In some aspects, the tumor sample is
subjected to an initial
TIL culture. In some aspects, the initial TIL culture comprises culturing the
tumor sample in the
MRM.
[0012] In some aspects, the 1V1RM further comprises IL-2 during
the initial TIE, culture. In
some aspects, the MR1\4 further comprises IL-7, IL-15, IL-21, or any
combination thereof during
the initial TIL culture. In some aspects, the MRM comprises IL-2 and IL-21
during the initial TIL
culture. In some aspects, the initial TL culture lasts at least about 14-19
days. In some aspects, the
initial TIL culture lasts at least about 11 days. In some aspects, the initial
TIL culture lasts at least
about 14 days. In some aspects, the proportion of CD8+ TILs to non-CD8+ TILs
is increased
following the initial TIL culture, as compared to the proportion of CD8+ TILs
to non-CD8+ TILs
prior to the initial TIL culture. In some aspects, the TILs are stimulated
following the initial TIL
culture. In some aspects, the TILs are stimulated by culturing the TILs with a
CD3 agonist and/or
a CD28 agonist.
[0013] In some aspects, the tumor sample comprises a tumor
biopsy. In some aspects, the
tumor sample is fragmented prior to culturing. In some aspects, the tumor
sample is dissociated
prior to culturing.
[0014] In some aspects, following culture of the heterogeneous
population of TILs, at least
about 30%, at least about 35%, at least about 40%, at least about 45%, at
least about 50%, at least
about 55%, at least about 60%, at least about 65%, at least about 70%, at
least about 75%, or at
least about 80% of the TILs in the population are CD8+ TILs. In some aspects,
following culture
of the heterogeneous population of TILs, at least about 50% of the TILs in the
population are CD8+
TILs.
[0015] In some aspects, the MRM further comprises sodium ion,
calcium ion, glucose, or
any combination thereof.
[0016] In some aspects, the MR1\4 further comprises a cell
expansion agent. In some
aspects, the cell expansion agent comprises a GSK3B inhibitor, an ACLY
inhibitor, a PI3K
inhibitor, an AKT inhibitor, or any combination thereof. In some aspects, the
PI3K inhibitor
comprises LY294002, pictilisib, CAL101, IC87114, or any combination thereof In
some aspects,
the AKT inhibitor comprises MK2206, A443654, AKTi-VIII, or any combination
thereof
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[0017] In some aspects, the concentration of potassium ion is at
least about 30 mM, at least
about 35 mM, at least about 40 mM, at least about 45 mM, at least about 50 mM,
at least about 55
mM, at least about 60 mM, at least about 65 mM, at least about 70 mM, at least
about 75 mM, at
least about 80 mM, at least about 85 mM, at least about 90 mM, at least about
95 mM, or at least
about 100 mM. In some aspects, the concentration of potassium ion is about 30
mM to about 100
mM, about 30 mM to about 90 mM, about 30 mM to about 80 mM, about 30 mM to
about 70 mM,
about 30 mM to about 60 mM, about 30 mM to about 50 mM, about 40 mM to about
100 mM,
about 40 mM to about 90 mM, about 40 mM to about 80 mM, about 40 mM to about
70 mM, about
40 mM to about 60 mM, or about 40 mM to about 50 mM. In some aspects, the
concentration of
potassium ion is about 40 mM to about 90 mM. In some aspects, the
concentration of potassium
ion is about 50 mM to about 90 mM. In some aspects, the concentration of
potassium ion is about
50 mM to about 80 mM.
[0018] In some aspects, the MRM further comprises sodium ion. In
some aspects, the
concentration of the sodium ion is from about 25 mM to about 100 mM. In some
aspects, the
concentration of the sodium ion is from about 30 mM to about 40 mM, about 30
mM to about 50
mM, about 30 mM to about 60 mM, about 30 mM to about 70 mM, about 30 mM to
about 80 mM,
about 40 mM to about 50 mM, about 40 mM to about 60 mM, about 40 mM to about
70 mM, about
40 mM to about 80 mM, about 50 mM to about 55 mM, about 50 mM to about 60 mM,
about 50
mM to about 65 mM, about 50 mM to about 70 mM, about 50 mM to about 75 mM,
about 50 mM
to about 80 mM, about 55 mM to about 60 mM, about 55 mM to about 65 mM, about
55 mM to
about 70 mM, about 55 mM to about 75 mM, about 55 mM to about 80 mM, about 60
mM to about
65 mM, about 60 mM to about 70 mM, about 60 mM to about 75 mM, about 60 mM to
about 80
mM, about 70 mM to about 75 mM, about 70 mM to about 80 mM, or about 75 mM to
about 80
mM. In some aspects, the concentration of the sodium ion is about 30 mM, about
35 mM, about
40 mM, about 45 mM, about 50 mM, about 55 mM, about 60 mM, about 65 mM, about
70 mM,
about 75 mM, or about 80 mM. In some aspects, the concentration of the sodium
ion is about 55
mM. In some aspects, the concentration of the sodium ion is about 60 mM. In
some aspects, the
concentration of the sodium ion is about 65 mM.
[0019] In some aspects, the MRM further comprises glucose. In
some aspects, the
concentration of glucose is more than about 10 mM. In some aspects, the
concentration of glucose
is from about 10 mM to about 25 mM, about 10 mM to about 20 mM, about 15 mM to
about 25
mM, about 15 mM to about 20 mM, about 15 mM to about 19 mM, about 15 mM to
about 18 mM,
about 15 mM to about 17 mM, about 15 mM to about 16 mM, about 16 mM to about
20 mM, about
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16 mM to about 19 mM, about 16 mM to about 18 mM, about 16 mM to about 17 mM,
about 17
mM to about 20 mM, about 17 mM to about 19 mM, or about 17 mM to about 18 mM.
In some
aspects, the concentration of glucose is about 10 mM, about 11 mM, about 12
mM, about 13 mM,
about 14 mM, about 15 mM, about 16 mM, about 17 mM, about 18 mM, about 19 mM,
about 20
mM, about 21 mM, about 22 mM, about 23 mM, about 24 mM, or about 25 mM.
[0020] In some aspects, the MRM further comprises calcium ion.
In some aspects, the
concentration of calcium ion is more than about 0.4 mM. In some aspects, the
concentration of
calcium ion is from about 0.4 mM to about 2.5 mM, about 0.5 mM to about 2.0
mM, about 1.0
mM to about 2.0 mM, about 1.1 mM to about 2.0 mM, about 1.2 mM to about 2.0
mM, about 1.3
mM to about 2.0 mM, about 1.4 mM to about 2.0 mM, about 1.5 mM to about 2.0
mM, about 1.6
mM to about 2.0 mM, about 1.7 mM to about 2.0 mM, about 1.8 mM to about 2.0
mM, about 1.2
to about 1.3 mM, about 1.2 to about 1.4 mM, about 1.2 to about 1.5 mM, about
1.2 to about 1.6
mM, about 1.2 to about 1.7 mM, about 1.2 to about 1.8 mM, about 1.3 to about
1.4 mM, about 1.3
to about 1.5 mM, about 1.3 to about 1.6 mM, about 1.3 to about 1.7 mM, about
1.3 to about 1.8
mM, about 1.4 to about 1.5 mM, about 1.4 to about 1.6 mM, about 1.4 to about
1.7 mM, about 1.4
to about 1.8 mM, about 1.5 to about 1.6 mM, about 1.5 to about 1.7 mM, about
1.5 to about 1.8
mM, about 1.6 to about 1.7 mM, about 1.6 to about 1.8 mM, or about 1.7 to
about 1.8 mM. In some
aspects, the concentration of calcium ion is about 1.0 mM, about 1.1 mM, about
1.2 mM, about
1.3 mM, about 1.4 mM, about 1.5 mM, about 1.6 mM, about 1.7 mM, about 1.8 mM,
about 1.9
mM, or about 2.0 mM.
[0021] In some aspects, the MRM comprises about 40 mM to about
90 mM potassium ion
and (i) about 40 mM to about 80 mM sodium ion; (ii) about 10 mM to about 24 mM
glucose; (iii)
about 0.5 mM to about 2.8 mM calcium ion; or (iv) any combination of (i)-
(iii).
[0022] Some aspects of the present disclosure are directed to a
method of expanding TILs
obtained from a human subject comprising: culturing the TILs in an initial TIL
culture media;
culturing the TILs in a secondary TIL culture media; culturing the TILs in a
third (or final) TIL
culture media, wherein the initial TIL culture media, the secondary TIL
expansion media, and/or
the third TIL expansion media are MRM. In some aspects, the initial TIL
culture media and the
secondary TIL expansion media are hyperkalemic and the third TIL expansion
media are not
hyperkalemic. In some aspects, the initial TIL culture media further comprise
IL-2. In some
aspects, the initial TIL culture media further comprise IL-21. In some
aspects, the initial TIL
culture media further comprise a T cell supplement, a serum replacement,
glutamine, a glutamine
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substitute (e.g., Glutamax (L-alanine-L-glutamine)), non-essential amino
acids, an antibiotics (e.g.,
Penicillin, Streptomycin, or both), an anti-fungal agent (e.g., FUNGINTm),
and/or sodium pyruvate.
[0023] In some aspects, the Tits are cultured in the initial TIL
culture media for at least
about 9 days, at least about 10 days, at least about 11 days, at least about
12 days, at least about 13
days, at least about 1 week, at least about 2 weeks, or at least about 3
weeks. In some aspects, the
Tits are cultured in the initial TIL culture media until cell yield in the
initial culture reaches at
least about 1x105, at least about 2x105, at least about 3x105, at least about
4x105, at least about
5x105, at least about 6x105, at least about 7x105, at least about 8x105, at
least about 9x105, at least
about 1x106, at least about 2x106, at least about 3x106, at least about 4x106,
at least about 5x106,
at least about 6x106, at least about 7x106, at least about 8x106, at least
about 9x106, at least about
10x106, at least about 15x106, at least about 20 x106, at least about 25x106,
at least about 30x106,
at least about 35x106, at least about 40x106, at least about 45x106 or at
least about 50x106 cells per
fragment.
[0024] In some aspects, the Tits are stimulated with a CD3
agonist, a CD28 agonist, or
both in prior to the secondary TIL culture media. In some aspects, the Tits
are further stimulated
with a CD27 agonist in or prior to the secondary TIL culture media. In some
aspects, the Tits are
further stimulated with a 4-1BB agonist in or prior to the secondary TIL
culture media. In some
aspects, the Tits are cultured for at least about 7 days, at least about 8
days, at least about 9 days,
at least about 10 days, at least about 11 days, at least about 12 days, at
least about 13 days, at least
about 14 days, at least about 15 days, at least about 16 days, at least about
17 days, at least about
18 days, at least about 19 days, at least about 20 days, at least about 21
days, at least about 22 days,
at least about 23 days, at least about 24 days, at least about 25 days, or at
least about 26 days, after
the stimulation. In some aspects, the Tits are cultured in the secondary
culture media until cell
yield reaches at least about 1x107, at least about 2x107, at least about
3x107, at least about 4x107,
at least about 5x107, at least about 6x107, at least about 7x107, at least
about 8x107, at least about
9x107, at least about 10x107, at least about 11x107, at least about 12x107, at
least about 13x107, at
least about 14x107, at least about 15x107, at least about 16x107, at least
about 17x107, at least about
18x107, at least about 19x107, or at least about 20x107 cells.
[0025] In some aspects, the Tits are stimulated with a CD3
agonist, a CD28 agonist, a
CD27 agonist, and/or a 4-1BB agonist in the third TIL culture media. In some
aspects, the third
TIL culture media are not hyperkalemic. In some aspects, the Tits are cultured
in the third TIL
culture media for at least about 7 days, at least about 8 days, at least about
9 days, at least about 10
days, at least about 11 days, at least about 12 days, at least about 13 days,
at least about 14 days,
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at least about 15 days, at least about 16 days, at least about 17 days, at
least about 18 days, at least
about 19 days, at least about 20 days, or at least about 21 days.
[0026] Some aspects of the present disclosure are directed to a
method of increasing tumor
reactive, e.g., tumor specific, TILs comprising: culturing one or more tumor
fragments in initial
TIL culture media, which are hyperkalemic and comprise IL-2 and optionally 1L-
21, up to about
11 to 19 days thereby obtaining TILs from the tumor fragment; culturing the
TILs in a secondary
TIL culture media, which are hyperkalemic, after adding (i) a CD3 agonist and
(ii) a CD28 agonist,
a CD27 aognist, a 4-1BB agonist, or any combination thereof, for about 7 to at
least about 14 days;
culturing the TILs in a third TIL culture media, which are not hyperkalemic,
after adding (i) a CD3
agonist and (ii) a CD28 agonist, a CD27 agonist, a 4-1BB agonist, or any
combination thereof, for
about 14 days to at least about 21 days.
[0027] In some aspects, the TILs exhibit increased expression of
TCF7 following culture
in the MRM, relative to TCF7 expression in a population of TILs following
culture in a control
medium that is not hyperkalemic. In some aspects, the population of TILs
comprises an increased
proportion of CD8+ CD62L+ TILs following culture in the MR1VI, relative to the
proportion of
CDS+ CD62L TILs following culture in a control medium that is not
hyperkalemic. In some
aspects, the population of TILs comprises an increased proportion of CD8+ PDF'
TILs following
culture in the MRM, relative to the proportion of CD8+ PD1+ TILs following
culture in a control
medium that is not hyperkalemic. In some aspects, the methods provided herein
further comprise
administering the population of TILs in a subject having a tumor. In some
aspects, the tumor is
refractory to an immune checkpoint inhibitor. In some aspects, the immune
checkpoint inhibitor
comprises a PD-1 antagonist, a CTLA-4 antagonist, a TIM3 antagonist, a GITR
antagonist, a KIR
antagonist, a LAG3 antagonist, or any combination thereof. In some aspects,
the immune
checkpoint inhibitor comprises an anti-PD1 antibody, an anti-CTLA-4 antibody,
an anti-TIM3
antibody, an anti-GITR antibody, an anti-KIR antibody, an anti-LAG3 antibody,
or any
combination thereof. In some aspects, the tumor is relapsed. In some aspects,
the tumor is
m etastatic.
[0028] In some aspects, the heterogeneous population of TILs has
increased clonal
diversity after being placed in the MRM, as compared to the clonal diversity
of a heterogenous
population of TILs placed in a control medium.
[0029] In some aspects, the heterogeneous population of TILs
after being placed in the
MR_M has a clonal diversity that is at least about 99% to about 100%, at least
about 98% to about
100%, at least about 97% to about 100%, at least about 96% to about 100%, at
least about 95% to
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about 100%, at least about 94% to about 100%, at least about 93% to about
100%, at least about
92% to about 100%, at least about 91% to about 100%, at least about 90% to
about 100%, at least
about 85% to about 100%, at least about 80% to about 100%, at least about 75%
to about 100%,
at least about 70% to about 100%, at least about 65% to about 100%, at least
about 60% to about
100%, at least about 55% to about 100%, at least about 50% to about 100%, at
least about 45% to
about 100%, or at least about 40% to about 100% of the clonal diversity of
TILs in a tumor sample.
[0030] In some aspects, the heterogeneous population of TILs
after being placed in the
MR_M has a clonal diversity score of less than about 0.5, less than about
0.45, less than about 0.4,
less than about 0.35, less than about 0.3, less than about 0.275, less than
about 0.25, less than about
0.225, less than about 0.2, less than about 0.175, less than about 0.15, less
than about 0.125, less
than about 0.1, less than about 0_075, less than about 0.07, less than about
0.06, or less than about
0.05 as measured by Simpsons clonality.
[0031] In some aspects, the heterogeneous population of TILs
after being placed in the
MRIV1 has a clonal diversity score of less than about 0.3 as measured by
Simpsons clonality.
[0032] In some aspects, the heterogeneous population of TILs
after being placed in the
MRM has a clonal diversity score of less than about 0.25 as measured by
Simpsons clonality.
[0033] In some aspects, the heterogeneous population of TILs
after being placed in the
MRIV1 has a clonal diversity score of less than about 0.2 as measured by
Simpsons clonality.
[0034] In some aspects, the heterogeneous population of TILs
after being placed in the
MRN1 has a clonal diversity score of less than about 0.1 as measured by
Simpsons clonality.
[0035] Some aspects of the present disclosure are directed to a
composition of immune
cells, comprising one or more CD8+ TIL cultured according to any method
disclosed herein. In
some aspects, at least about 30%, at least about 35%, at least about 40%, at
least about 45%, at
least about 50%, at least about 55%, at least about 60%, at least about 65%,
at least about 70%, at
least about 75%, or at least about 80% of the immune cells are CD8+ TILs.
[0036] Some aspects of the present disclosure are directed to a
composition comprising a
population of immune cells, wherein at least about 30%, at least about 35%, at
least about 40%, at
least about 45%, at least about 50%, at least about 55%, at least about 60%,
at least about 65%, at
least about 70%, at least about 75%, or at least about 80% of the immune cells
are CD8+ TILs. In
some aspects, at least about 50% of the cells are CD8 TILs.
[0037] In some aspects, the cells exhibit increased expression
of TCF7 following culture
in the MRIVI, relative to TCF7 expression in a population of immune cells
following culture in a
control medium that is not hyperkalemic. In some aspects, at least about 5%,
at least about 10%,
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at least about 15%, at least about 20%, at least about 25%, at least about
30%, at least about 35%,
at least about 40%, at least about 45%, at least about 50%, at least about
55%, at least about 60%,
at least about 65%, at least about 70%, or at least about 75% of the immune
cells are CD8+/CD62L+
TILs. In some aspects, at least about 10%, at least about 15%, at least about
20%, at least about
25%, at least about 30%, at least about 35%, at least about 40%, or at least
about 50% of the CD8+
TILs obtained at the end of the initial T1L culture are PDF'. In some aspects,
at least about 10%,
at least about 15%, at least about 20%, at least about 25%, at least about
30%, at least about 35%,
at least about 40%, or at least about 50% of the CD8+ Tits obtained at the end
of the initial TlL
culture are CD39- In some aspects, at least about 10%, at least about 15%, at
least about 20%, at
least about 25%, at least about 30%, at least about 35%, at least about 40%,
or at least about 50%
of the CD8+ TILs are CD27+ In some aspects, at least about 10%, at least about
15%, at least about
20%, at least about 25%, at least about 30%, at least about 35%, at least
about 40%, or at least
about 50% of the CD8+ TILs are CD28+. In some aspects, at least about 10%, at
least about 15%,
at least about 20%, at least about 25%, at least about 30%, at least about
35%, at least about 40%,
or at least about 50% of the CD8+ TILs obtained at the end of the initial TIL
culture are PD1+
CD39+. In some aspects, at least about 10%, at least about 15%, at least about
20%, at least about
25%, at least about 30%, at least about 35%, at least about 40%, or at least
about 50% of the CD8+
TILs obtained at the end of the initial TIL culture are PDF' CD27+. In some
aspects, at least about
10%, at least about 15%, at least about 20%, at least about 25%, at least
about 30%, at least about
35%, at least about 40%, or at least about 50% of the CD8+ TILs are CD27+
CD62L+. In some
aspects, at least about 10%, at least about 15%, at least about 20%, at least
about 25%, at least
about 30%, at least about 35%, at least about 40%, or at least about 50% of
the CD8+ TILs obtained
at the end of the initial TIL culture are CD27 CD28' CD103' PD1' TCF7+.
[0038] In some aspects, the population of immune cells comprises
at least about 2 x 106, at
least about 3 x 106, at least about 4 x 106, at least about 5 x 106, at least
about 6 x 106, at least about
7 x 106, at least about 8 x 106, at least about 9 x 106, or at least about 1 x
107 cells. In some aspects,
the population of immune cells comprises at least about 1 x 106, at least
about 3 x 106, at least
about 4 x 106, at least about 5 x 106, at least about 6 x 106, at least about
7 x 106, at least about 8 x
106, at least about 9 x 106, or at least about 1 x 107 CD8+ cells.
[0039] In some aspects, the CD8 TILs have a clonal diversity
that is at least about 99% to
about 100%, at least about 98% to about 100%, at least about 97% to about
100%, at least about
96% to about 100%, at least about 95% to about 100%, at least about 94% to
about 100%, at least
about 93% to about 100%, at least about 92% to about 100%, at least about 91%
to about 100%,
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at least about 90% to about 100%, at least about 85% to about 100%, at least
about 80% to about
100%, at least about 75% to about 100%, at least about 70% to about 100%, at
least about 65% to
about 100%, at least about 60% to about 100%, at least about 55% to about
100%, at least about
50% to about 100%, at least about 45% to about 100%, or at least about 40% to
about 100% of the
clonal diversity of TILs in a tumor sample.
[0040] In some aspects, the CD8+ TILs have a clonal diversity
score (e.g., Simpson
clonality index) of less than about 0.5, less than about 0.45, less than about
0.4, less than about
0.35, less than about 0.3, less than about 0.275, less than about 0.25, less
than about 0.225, less
than about 0.2, less than about 0.175, less than about 0.15, less than about
0.125, less than about
0.1, less than about 0.075, less than about 0.07, less than about 0.06, or
less than about 0.05 as
measured by Simpsons clonality. In some aspects, the CD8+ TILs have a clonal
diversity score of
less than about 0.3 as measured by Simpsons clonality. In some aspects, the
CDS+ TILs have a
clonal diversity score of less than about 0.25 as measured by Simpsons
clonality. In some aspects,
the CD8+ TILs have a clonal diversity score of less than about 0.2 as measured
by Simpsons
clonality. In some aspects, the CDS+ TILs have a clonal diversity score of
less than about 0.1 as
measured by Simpsons clonality.
[0041] Some aspects of the present disclosure are directed to a
method of treating a cancer
(tumor) in a subject in need thereof, comprising administering a population of
TILs to the subject,
wherein the population of TILs are cultured according to any method disclosed
here. In some
aspects, the population of TILs is enriched for CDS+ TILs. In some aspects, at
least about 30%, at
least about 35%, at least about 40%, at least about 45%, at least about 50%,
at least about 55%, at
least about 60%, at least about 65%, at least about 70%, at least about 75%,
or at least about 80%
of the TILs in the population of TILs are CD8 TILs. In some aspects, at least
about 50% of the
TILs in the population of TILs are CD8+ TILs. In some aspects, the tumor is
refractory to a
checkpoint inhibitor. In some aspects, the checkpoint inhibitor comprises a PD-
1 antagonist, a
CTLA-4 antagonist, a TIM3 antagonist, a GITR antagonist, a KIR antagonist, a
LAG3 antagonist,
or any combination thereof In some aspects, the checkpoint inhibitor comprises
an anti-PD1
antibody, an anti-CTLA-4 antibody, an anti-TIM3 antibody, an anti-GITR
antibody, an anti-KIR
antibody, an anti-LAG3 antibody, or any combination thereof. In some aspects,
the tumor is
relapsed. In some aspects, the tumor is metastatic.
[0042] Some aspects of the present disclosure are directed to a
method treating a cancer
(tumor) in a subject in need thereof, comprising administering to a subject a
composition disclosed
herein. In some aspects, the cancer comprises a solid tumor. In some aspects,
the cancer comprises
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a solid tumor derived from a melanoma, a colon cancer, a lung cancer, a
cervical cancer, a
gastrointestinal cancer, a breast cancer, a prostate cancer, a liver cancer,
bone cancer, a pancreatic
cancer, a small cell carcinoma of the head and neck, lung squamous cell
carcinoma, lung
adenocarcinoma, pancreatic adenocarcinoma, head and neck squamous cell
carcinoma, testicular
germ cell tumors, stomach adenocarcinoma, skin cutaneous melanoma,
mesothelioma, kidney
renal clear cell carcinoma, cervical squamous cell carcinoma and endocervical
adenocarcinoma,
esophageal carcinoma, bladder urothelial carcinoma, breast invasive carcinoma,
kidney renal
papillary cell carcinoma, colon adenocarcinoma, non-small cell lung cancer, or
any combination
thereof, In some aspects, the tumor is refractory to a checkpoint inhibitor.
In some aspects, the
checkpoint inhibitor comprises a PD-1 antagonist, a CTLA-4 antagonist, a TIM3
antagonist, a
GITR antagonist, a KIR antagonist, a LAG3 antagonist, or any combination
thereof. In some
aspects, the checkpoint inhibitor comprises an anti-PD1 antibody, an anti-CTLA-
4 antibody, an
anti-TIM3 antibody, an anti-GITR antibody, an anti-KIR antibody, an anti-LAG3
antibody, or any
combination thereof. In some aspects, the tumor is relapsed. In some aspects,
the tumor is
metastatic.
[0043] In some aspects, the method comprises administering at
least about 2 x 109, at least
about 3 x 109, at least about 4 x 109, at least about 5 x 109, at least about
6 x 109, at least about 7 x
109, at least about 8 x 109, at least about 9 x 109, or at least about 1 x
1010, or at least about 10 x
1010, or at least about 15 x 1010, or at least about 20 x 1010, or at least
about 25 x 1010, or at least
about 30 x 1010 cells to the subject. In some aspects, the method comprises
administering at least
about 1 x 109, at least about 3 x 109, at least about 4 x 109, at least about
5 x 109, at least about 6 x
109, at least about 7 x 109, at least about 8 x 109, at least about 9 x 109,
or at least about 1 x 109
CD8 cells to the subject. In some aspects, the method comprises administering
about 1x109 to
about 4 x 109, about 5 x 109 to about 7 x 109, about 10 x 109 to about 30 x
109, about 40 x 109 to
about 60 x 109, about 70 x 109 to about 90 x 109 cells to the subject. In some
aspects, the method
comprises administering more than 90 x 109 cell to the subject. In some
aspects, the method
comprises administering about 5 x 109 to about 8 x 109, about 10 x 109 to
about 40 x 109, about 50
x 109 to about 80 x 109 cells to the subject.
[0044] In some aspects, the method further comprises
administering a checkpoint inhibitor.
In some aspects, the checkpoint inhibitor is administered to the subject after
administering the
population of cells. In some aspects, the checkpoint inhibitor comprises a
CTLA-4 antagonist, a
PD1 antagonist, a TIM-3 antagonist, or a combination thereof. In some aspects,
the checkpoint
inhibitor comprises an anti-CTLA-4 antibody, an anti-PD1 antibody, an anti-PD-
Li antibody, an
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anti-TIM-3 antibody, or a combination thereof. In some aspects, the method
further comprises
administering a checkpoint activator. In some aspects, the checkpoint
inhibitor is administered to
the subject after administering the population of TILs. In some aspects, the
checkpoint activator
comprises an 0X40 agonist, a LAG-3 agonist, a 4-1BB (CD137) agonist, a GITR
agonist, a TIM3
agonist, or a combination thereof. In some aspects, the checkpoint activator
comprises an anti-
0X40 antibody, an anti-LAG-3 antibody, an anti-CD137 antibody, an anti-GITR
antibody, an anti-
TIM3 antibody, or a combination thereof.
[0045] In some aspects, the method further comprises
administering a cytokine. In some
aspects, the cytokine is administered to the subject after administering the
population of TILs. In
some aspects, the cytokine is IL-2.
[0046] In some aspects, the method further comprises
administering a lymphodepleting
therapy to the subject prior to administering the population of cells. In some
aspects, the
lymphodepleting therapy comprises cyclophosphamide, fludarabine, or both
cyclophosphamide
and fludarabine.
[0047] Some aspects of the present disclosure are directed to a
population of expanded
TILs having a clonal diversity that is at least about 99% to about 100%, at
least about 98% to about
100%, at least about 97% to about 100%, at least about 96% to about 100%, at
least about 95% to
about 100%, at least about 94% to about 100%, at least about 93% to about
100%, at least about
92% to about 100%, at least about 91% to about 100%, at least about 90% to
about 100%, at least
about 85% to about 100%, at least about 80% to about 100%, at least about 75%
to about 100%,
at least about 70% to about 100%, at least about 65% to about 100%, at least
about 60% to about
100%, at least about 55% to about 100%, at least about 50% to about 100%, at
least about 45% to
about 100%, or at least about 40% to about 100% of the clonal diversity of
TILs in a tumor sample.
[0048] Some aspects of the present disclosure are directed to a
population of expanded
TILs having a clonal diversity score of less than about 0.5, less than about
0.45, less than about
0.4, less than about 0.35, less than about 0.3, less than about 0.275, less
than about 0.25, less than
about 0.225, less than about 0.2, less than about 0.175, less than about 0.15,
less than about 0.125,
less than about 0.1, less than about 0.075, less than about 0.07, less than
about 0.06, or less than
about 0.05 as measured by Simpsons clonality. In some aspects, the clonal
diversity score is less
than about 0.3 as measured by Simpsons clonality. In some aspects, the clonal
diversity score is
less than about 0.25 as measured by Simpsons clonality. In some aspects, the
clonal diversity score
is less than about 0.2 as measured by Simpsons clonality. In some aspects, the
clonal diversity
score is less than about 0.1 as measured by Simpsons clonality.
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[0049] In some aspects, the population of expanded TILs comprise
at least about at least
about 2 x 106, at least about 3 x 106, at least about 4 x 106, at least about
5 x 106, at least about 6 x
106, at least about 7 x 106, at least about 8 x 106, at least about 9 x 106,
or at least about 1 x 107
cells. In some aspects, at least at least about 1 x 106, at least about 3 x
106, at least about 4 x 106,
at least about 5 x 106, at least about 6 x 106, at least about 7 x 106, at
least about 8 x 106, at least
about 9 x 106, or at least about 1 x 107 cells in a population of expanded TIL
are CD8+ cells.
[0050] In some aspects, at least about 30%, at least about 35%,
at least about 40%, at least
about 45%, at least about 50%, at least about 55%, at least about 60%, at
least about 65%, at least
about 70%, at least about 75%, or at least about 80% of the expanded TILs are
CD8+ TILs. In some
aspects, at least about 50% of the expanded TILs are CD8+ TILs.
[0051] In some aspects, the expanded TILs exhibit increased
expression of TCF7 following
culture in the MR1VI, relative to TCF7 expression in a population of immune
cells following culture
in a control medium that is not hyperkalemic. In some aspects, at least about
5%, at least about
10%, at least about 15%, at least about 20%, at least about 25%, at least
about 30%, at least about
35%, at least about 40%, at least about 45%, at least about 50%, at least
about 55%, at least about
60%, at least about 65%, at least about 70%, or at least about 75% of the
expanded TILs are
CD8 /CD62L+ TILs. In some aspects, at least about 10%, at least about 15%, at
least about 20%,
at least about 25%, at least about 30%, at least about 35%, at least about
40%, or at least about
50% of the CD8+ TILs are PDF'. In some aspects, at least about 10%, at least
about 15%, at least
about 20%, at least about 25%, at least about 30%, at least about 35%, at
least about 40%, or at
least about 50% of the CD8+ TILs are CD39+. In some aspects, at least about
10%, at least about
15%, at least about 20%, at least about 25%, at least about 30%, at least
about 35%, at least about
40%, or at least about 50% of the CD8 TILs are CD27. In some aspects, at least
about 10%, at
least about 15%, at least about 20%, at least about 25%, at least about 30%,
at least about 35%, at
least about 40%, or at least about 50% of the CD8+ TILs are CD28+. In some
aspects, at least about
10%, at least about 15%, at least about 20%, at least about 25%, at least
about 30%, at least about
35%, at least about 40%, or at least about 50% of the CD8 TILs are PD1' CD39
. In some aspects,
at least about 10%, at least about 15%, at least about 20%, at least about
25%, at least about 30%,
at least about 35%, at least about 40%, or at least about 50% of the CD8+ TILs
are PDF' CD27 .
In some aspects, at least about 10%, at least about 15%, at least about 20%,
at least about 25%, at
least about 30%, at least about 35%, at least about 40%, or at least about 50%
of the CD8+ TILs
are CD27+ CD62L+. In some aspects, at least about 10%, at least about 15%, at
least about 20%,
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at least about 25%, at least about 30%, at least about 35%, at least about
40%, or at least about
50% of the CD8+ TILs are CD27+ CD28+ CD103+ PDF TCF7+.
[0052] In some aspects, the composition disclosed herein or the
population of expanded
TILs disclosed herein comprises at least one immune cell expression one or
more stem-like
markers and one or more effector-like markers. In some aspects, the stem-like
markers comprise
CD45RA I, CD62L I, CCR7 I, CD27 I, CD28 I, BACII2 I, LEF1 I, TCF7 I, or any
combination
thereof. In some aspects the stem-like markers comprise CD45RA+, CD62L+,
CCR7+, and
TCF7+, or any combination thereof. In some aspects, the effector-like markers
comprise
pSTAT5+, STAT5+, pSTAT3+, STAT3+, or any combination thereof. In some aspects,
at least
about 40%, at least about 50%, at least about 60%, at least about 70%, at
least about 80%, at least
about 85%, at least about 90%, at least about 95%, at least about 99%, or
about 100% of expanded
TILs in composition or population comprise at least one immune cell expression
one or more stem-
like markers and one or more effector-like markers.
[0053] Some aspects of the present disclosure are directed to a
TIL expressing one or more
stem-like markers and one or more effector-like markers. In some apects, the
stem-like markers
comprise CD45RA+, CD62L+, CCR7+, CD27+, CD28+, BACH2+, LEF I+, TCF7+, or any
combination thereof. In some aspects, the stem-like cells are CD39- CD69-. In
some aspects, the
stem-like markers comprise one or more of the following: CD45RA+, CD62L+,
CCR7+, CD27+,
CD28+, BACH2+, LEF1+, TCF7+, CD39-, and CD69-. In some aspects, the stem-cell
markers
comprise comprise CD45RA+, CD62L+, CCR7+, and TCF7+. In some aspects, the
effector-like
markers comprise pSTAT5+, STAT5+, pSTAT3+, STAT3+, or any combination thereof.
[0054] Some aspects of the present disclosure are directed to a
population of expanded
TILs comprising a TIL disclosed herein, e.g., a TIL comprising one or more
stem-like markers and
one or more effector-like markers. In some aspects, at least about 40%, at
least about 50%, at least
about 60%, at least about 70%, at least about 80%, at least about 85%, at
least about 90%, at least
about 95%, at least about 99%, or about 100% of the population of expanded
TILs comprises the
TILs comprising one or more stem-like markers and one or more effector-like
markers.
[0055] Some aspects of the present disclosure are directed to a
pharmaceutical composition
comprising a TIL comprising one or more stem-like markers and one or more
effector-like markers
and a pharmaceutically acceptable carrier.
[0056] Certain aspects of the present disclosure are directed to
a method of treating a
disease or condition in a subject in need thereof comprising administering a
TlL disclosed herein,
a population of expanded TILs disclosed herein, or a pharmaceutical
composition disclosed herein
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to the subject. In some aspects, the disease or condition is a cancer (tumor).
In some aspects, the
tumor is refractory to a checkpoint inhibitor. In some aspects, the checkpoint
inhibitor comprises
a PD-1 antagonist, a CTLA-4 antagonist, a TIN/13 antagonist, a GITR
antagonist, a KIR antagonist,
a LAG3 antagonist, or any combination thereof. In some aspects, the checkpoint
inhibitor
comprises an anti-PD1 antibody, an anti-CTLA-4 antibody, an anti-TIM3
antibody, an anti-GITR
antibody, an anti-KIR antibody, an anti-LAG3 antibody, or any combination
thereof In some
aspects, the tumor is relapsed. In some aspects, the tumor is metastatic
[0057] In some aspects, the population of TILs comprises an
increased proportion of CD39-
/CD69- Tits following culture in the MRM, relative to the proportion of
CD391CD69- TILs
following culture in a control medium.
[0058] In some aspects, the population of expanded TILs
comprises at least about 10%, at
least about 15%, at least about 20%, at least about 25%, at least about 30%,
at least about 35%, or
at least about 40% of the total number of TILs in the population of TILs are
CD391CD69-.
[0059] In some aspects, the culturing of the TILs in the
secondary TIL culture medium is
carried out in one or more gas-permeable containers. In some aspects, the
culturing of the TILs in
the third (or final) TIL culture medium is not carried out in one or more gas-
permeable containers.
In some aspects, the TILs are split between the culturing of the TILs in the
secondary TIL culture
medium and the culturing of the TILs in the third (or final) TIL culture
medium.
[0060] Some aspects of the present disclosure are directed to
methods of expanding TILs
obtained from a human subject comprising: (a) culturing the TILs in a medium
comprising greater
than 4 mM potassium ion, a CD3 agonist, and antigen-presenting cells (a
"static-REP step"); and
(b) adding to the TILs from the static-REP step a medium comprising greater
than 4 mM potassium
ion, wherein agitation is applied to the culture (a "dynamic-REP step").
[0061] In some aspects, no CD3 agonist and no antigen-presenting
cells are added during
the dynamic-REP step.
[0062] In some aspects, the agitation comprises rocking.
[0063] In some aspects, the dynamic-REP step comprises perfusion
In some aspects, the
perfusion comprises continuous medium exchange at a rate of about 10%, about
15%, about 20%,
about 25%, about 30%, about 35%, about 40%, about 45%, about 50%, about 55%,
or about 60%
of the working volume of the culture every 24 hours. In some aspects, the
perfusion comprises
continuous medium exchange at a rate of about 25% of the working volume of the
culture every
24 hours. In some aspects, the perfusion comprises continuous medium exchange
at a rate of about
50% of the working volume of the culture every 24 hours.
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[0064] In some aspects, the perfusion comprises continuous
medium exchange at a rate of
about 25% of the working volume of the culture every 24 hours for the first 48
hours of the
dynamic-REP culture, and wherein the perfusion comprises continuous medium
exchange at a rate
of about 50% of the working volume of the culture every 24 hours for the
remainder of the
dynamic-REP culture.
[0065] In some aspects, the continuous medium exchange comprises
addition to the culture
of fresh MRM, wherein the fresh MRM does not comprise an anti-CD-3 antibody.
In some aspects,
the continuous medium exchange comprises addition to the culture of fresh
1V11RM, wherein the
fresh MRM does not comprise antigen-presenting cells.
[0066] In some aspects, the concentration of CD3 agonist in the
dynamic-REP culture
decreases at a rate of about of about 10%, about 15%, about 20%, about 25%,
about 30%, about
35%, about 40%, about 45%, about 50%, about 55%, or about 60% every 24 hours.
In some
aspects, the concentration of CD3 agonist in the dynamic-REP culture is less
than about 75% that
of the concentration of CD3 agonist in the static-REP culture at least about
24 hours after initiation
of the dynamic-REP. In some aspects, the concentration of CD3 agonist in the
dynamic-REP
culture is less than about 45% that of the concentration of CD3 agonist in the
static-REP culture at
least about 48 hours after initiation of the dynamic-REP. In some aspects, the
concentration of CD3
agonist in the dynamic-REP culture is less than about 35% that of the
concentration of CD3 agonist
in the static-REP culture at least about about 72 hours after initiation of
the dynamic-REP. In some
aspects, the concentration of CD3 agonist in the dynamic-REP culture is less
than about 27% that
of the concentration of CD3 agonist in the static-REP culture at least about
96 hours after initiation
of the dynamic-REP. In some aspects, the concentration of CD3 agonist in the
dynamic-REP
culture is less than about 21% that of the concentration of CD3 agonist in the
static-REP culture at
least 120 hours after initiation of the dynamic-REP. In some aspects, the
number of viable antigen-
presenting cells in the dynamic-REP culture is less than about 90%, less than
about 80%, less than
about 70%, less than about 60%, less than about 50%, less than about 45%, less
than about 40%,
less than about 35%, less than about 30%, less than about 25%, less than about
20%, less than
about 15%, less than about 10%, or less than about 5% the number of antigen-
presenting cells in
the static-REP at least about 24 hours after initiation of the dynamic-REP. In
some aspects, the
number of viable antigen-presenting cells in the dynamic-REP is culture less
than about 50%, less
than about 45%, less than about 40%, less than about 35%, less than about 30%,
less than about
25%, less than about 20%, less than about 15%, less than about 10%, or less
than about 5% the
number of antigen-presenting cells in the static-REP at least about 48 hours
after initiation of the
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dynamic-REP. In some aspects, the number of viable antigen-presenting cells in
the dynamic-REP
culture is less than about 25%, less than about 20%, less than about 15%, less
than about 10%, or
less than about 5% the number of antigen-presenting cells in the static-REP at
least about 72 hours
after initiation of the dynamic-REP. In some aspects, the number of viable
antigen-presenting cells
in the dynamic-REP culture is less than about 10% the number of antigen-
presenting cells in the
static-REP at least about 96 hours after initiation of the dynamic-REP.
[0067] In some aspects, the anti-CD3 antibody comprises OKT-3.
In some aspects, the
antigen-presenting cells comprise irradiated PBMCs.
[0068] In some aspects, the TILs are cryopreserved following
disoccociation of tumor
fragements. In some aspects, the Tits are cryopreserved following the pre-REP.
In some aspects,
the TILs are cryopreserved following the static-REP and prior to the dynamic-
REP. In some
aspects, the TILs are cryopreserved following the dynamic-REP.
[0069] In some aspects, the TILs are subjected to the static-REP
for about 5 days. In some
aspects, the TILs are subjected to the static-REP for about 6 days. In some
aspects, the TILs are
subjected to the static-REP for about 7 days. In some aspects, the TILs are
subjected to the
dynamic-REP for about 9 days to about 12 days. In some aspects, the TILs are
subjected to the
dynamic-REP for about 9 days to about 13 days. In some aspects, the TILs are
subjected to the
static-REP for about 5 days followed by the dynamic-REP for about 9 days. In
some aspects, the
TILs are subjected to the static-REP for about 5 days followed by the dynamic-
REP continues for
about 12 days. In some aspects, the TILs are subjected to the static-REP for
about 5 days followed
by the dynamic-REP continues for about 13 days.
[0070] In some aspects, the medium of the static-REP culture
comprises IL-2. In some
aspects, the medium of the static-REP culture comprises at least about 1000
IU, at least about 1100
IU, at least about 1200 IU, at least about 1300 IU, at least about 1400 IU, at
least about 1500 IU,
at least about 1600 IU, at least about 1700 IU, at least about 1800 IU, at
least about 1900 IU, or at
least about 2000 IU IL-2. In some aspects, the medium of the static-REP
culture comprises about
1500 IU 1L-2.
[0071] In some aspects, the medium of the static-REP culture
comprises IL-21. In some
aspects, the medium of the static-REP culture comprises at least about 5
ng/mL, at least about 6
ng/mL, at least about 7 ng/mL, at least about 8 ng/mL, at least about 9 ng/mL,
at least about 10
ng/mL, at least about 11 ng/mL, at least about 12 ng/mL, at least about 13
ng/mL, at least about 14
ng/mL, or at least about 15 ng/mL IL-21. In some aspects, the medium of the
static-REP culture
comprises about 10 ng/mL IL-21.
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[0072] In some aspects, the medium of the static-REP culture
comprises IL-15. In some
aspects, the medium of the static-REP culture comprises at least about 0.1
ng/mL, at least about
0.2 ng/mL, at least about 0.3 ng/mL, at least about 0.4 ng/mL, at least about
0.5 ng/mL, at least
about 0.6 ng/mL, at least about 0.7 ng/mL, at least about 0.8 ng/mL, at least
about 0.9 ng/mL, or
at least about 1 ng/mL IL-15. In some aspects, the medium of the static-REP
culture comprises
about 0.4 ng/mL IL-15.
[0073] In some aspects, the medium of the static-REP culture
comprises about 1500 IU IL-
2, about 10 ng/mL IL-21, and about 0.4 ng/mL IL-15.
[0074] In some aspects, the medium of the perfused fresh medium
of the dynamic-REP
culture comprises IL-2. In some aspects, the perfused fresh medium of the
dynamic-REP culture
comprises at least about 1000 IU, at least about 1100 IU, at least about 1200
IU, at least about 1300
IU, at least about 1400 IU, at least about 1500 IU, at least about 1600 IU, at
least about 1700 IU,
at least about 1800 IU, at least about 1900 IU, or at least about 2000 IU IL-
2. In some aspects, the
perfused fresh medium of the dynamic-REP culture comprises about 1500 IU IL-2.
[0075] In some aspects, the perfused fresh medium of the dynamic-
REP culture comprises
IL-21. In some aspects, the perfused fresh medium of the dynamic-REP culture
comprises at least
about 5 ng/mL, at least about 6 ng/mL, at least about 7 ng/mL, at least about
8 ng/mL, at least
about 9 ng/mL, at least about 10 ng/mL, at least about 11 ng/mL, at least
about 12 ng/mL, at least
about 13 ng/mL, at least about 14 ng/mL, or at least about 15 ng/mL IL-21. In
some aspects, the
perfused fresh medium of the dynamic-REP culture comprises about 10 ng/mL IL-
21.
[0076] In some aspects, the perfused fresh medium of the dynamic-
REP culture comprises
IL-15. In some aspects, the perfused fresh medium of the dynamic-REP culture
comprises at least
about 0.1 ng/mL, at least about 0.2 ng/mL, at least about 0.3 ng/mL, at least
about 0.4 ng/mL, at
least about 0.5 ng/mL, at least about 0.6 ng/mL, at least about 0.7 ng/mL, at
least about 0.8 ng/mL,
at least about 0.9 ng/mL, or at least about 1 ng/mL IL-15. In some aspects,
the perfused fresh
medium of the dynamic-REP culture comprises about 0.4 ng/mL IL-15. In some
aspects, the
perfused fresh medium of the dynamic-REP culture comprises about 1500 IU IL-2,
about 10 ng/mL
IL-21, and about 0.4 ng/mL IL-15.
[0077] In some aspects, prior to the static-REP, the TILs are
subjected to an initial TIL
expansion. In some aspects, the initial TIL expansion comprises culturing a
tumor sample or a
portion thereof in MRM, wherein the MRM further comprises IL-2. In some
aspects, the MRM
further comprises IL-7, IL-15, IL-21, or any combination thereof during the
initial TIL expansion.
In some aspects, the MR1\4 comprises IL-2 and IL-21 during the initial TIL
culture. In some
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aspects, the MRIVI comprises about 6000 IU/mL IL-2 and about 30 ng/mL IL-21
during the initial
TM culture.
[0078] In some aspects, the initial TM culture lasts about 11
days to about 15 days. In some
aspects, the initial TIL culture lasts at least about 11 days. In some
aspects, the initial TIL culture
lasts at least about 13 days. In some aspects, the initial TIL culture lasts
at least about 15 days.
[0079] In some aspects, the initial TIL culture lasts until cell
yield in the initial TIL culture
reaches at least about 10x106-50x106 cells. In some aspects, the initial TIL
culture lasts until cell
yield in the initial culture reaches at least about 10x106, at least about
15x106, at least about 20x106,
at least about 25x106, at least about 30x106, at least about 35x106, at least
about 40x106, at least
about 45x106, or at least about 50 x106 cells. In some aspects, the initial
TIL culture lasts until cell
yield in the initial culture reaches at least about 30x106 cells.
[0080] In some aspects, the initial TIL expansion further
comprises contacting the TILs
with TRANSACTTm. In some aspects, the TILs are contacted with TRANSACTTm on
about day
3, about day 4, about day 5, about day 6, or about day 7 of the initial TIL
culture. In some aspects,
the TILs are contacted with TRANSACTTm on about day 4 of the initial TIL
culture. In some
aspects, the initial TIL expansion further comprises contacting the TILs with
4-1BB ligand. In
some aspects, the TILs are contacted with 4-1BB ligand on about day 3, about
day 4, about day 5,
about day 6, or about day 7 of the initial TIL culture. In some aspects, the
TILs are contacted with
4-1BB ligand on about day 4 of the initial TIL culture. In some aspects, the
initial TIL expansion
comprises contacting the TILs with TRANSACTTm and 4-1BB ligand on about day 4
of the initial
TIL culture.
[0081] Some aspects of the present disclosure are directed to a
population of TILs obtained
by a method disclosed herein. In some aspects, about 20% to about 80% of the
CD8+ TILs are
stemlike TILs. In some aspects, at least 50% to 90% of the CD8+ cells are
putative tumor reactive
TILs. In some aspects, at least 35% to 90% of the TILs are tumor reactive
TILs.
[0082] Some aspects of the present disclosure are directed to a
method of treating a subject
in need thereof comprising administering to the subject a population of TILs
disclosed herein. In
some aspects, the subject is afflicted with a cancer. In some aspects, the
cancer comprises a solid
tumor. In some aspects, the cancer comprises a solid tumor derived from a
melanoma, a colon
cancer, a lung cancer, a cervical cancer, a gastrointestinal cancer, a breast
cancer, a prostate cancer,
a liver cancer, bone cancer, a pancreatic cancer, a small cell carcinoma of
the head and neck, lung
squamous cell carcinoma, lung adenocarcinoma, pancreatic adenocarcinoma, head
and neck
squamous cell carcinoma, testicular germ cell tumors, stomach adenocarcinoma,
skin cutaneous
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melanoma, mesothelioma, kidney renal clear cell carcinoma, cervical squamous
cell carcinoma
and endocervical adenocarcinoma, esophageal carcinoma, bladder urothelial
carcinoma, breast
invasive carcinoma, kidney renal papillary cell carcinoma, colon
adenocarcinoma, or any
combination thereof.
[0083] In some aspects, the tumor is refractory to a checkpoint
inhibitor. In some aspects,
the checkpoint inhibitor comprises a PD-1 antagonist, a CTLA-4 antagonist, a
TIM3 antagonist, a
GITR antagonist, a KIR antagonist, a LAG3 antagonist, or any combination
thereof In some
aspects, the checkpoint inhibitor comprises an anti-PD1 antibody, an anti-CTLA-
4 antibody, an
anti-TIM3 antibody, an anti-GITR antibody, an anti-K1R antibody, an anti-LAG3
antibody, or any
combination thereof. In some aspects, the tumor is relapsed. In some aspects,
the tumor is
metastatic_
BRIEF DESCRIPTION OF THE DRAWINGS/FIGURES
[0084] FIGs. 1A-1F are schematics showing exemplary processes of
culturing and
expanding TILs from tumor fragments. FIGs. 1A-1B show exemplary processes
comprising an
initial expansion and a secondary expansion, wherein the TILs are optionally
stimulated (e.g.,
according to the methods disclosed herein, e.g., by contacting the cells with
4-1BBL,
TRANSACTTh, anti-CD3 antibody, an antigen presenting cell, or any combination
thereof) at the
transition from the initial TIL culture to the secondary TIL expansion (FIGs.
1A-1B) and during
the initial TIL culture (FIG. 1B). FIGs. 1C-1D show exemplary processes
comprising an initial
expansion, a secondary expansion, and a final expansion, wherein the TILs are
optionally
stimulated (i) at the transition from the initial TIL culture to the secondary
TIL expansion (FIGs.
1C-1D); (ii) at the transition from the secondary TIL expansion to the final
TIL expansion (FIGs.
1C-1D); and (iii) during the initial TIL culture (FIG. 1D). FIGs. 1E-1F show
exemplary processes
for generating young TILs, wherein the initial expansion and the secondary
expansion are shorter
in duration, e.g., 11 days (or less) for each expansion, and wherein the TILs
are optionally
stimulated at the transition from the initial TIL culture to the secondary TIL
expansion (FIGs. 1E-
1F) and during the initial TIL culture (FIG. 1F).
[0085] FIGs. 2A-2B are graphical representations of FACS cell
phenotyping of TILs after
initial culture (day 14) in T cell conditioned media (e.g., CTSTm OPTIMIZERTm;
also referred to
herein as "control media"; FIG. 2A) or metabolic reprogramming media (also
referred to herein as
"MRM"; FIG. 2B). FIGs. 2A and 2B show that culture in MRM produced TILs with
enhanced
expression of CD39 and PD1 (greater than 20%) as compared to TILs cultured in
control media.
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FIG. 2C is a scatter plot showing the individual differences in the percentage
of CD8 + cells
obtained by culturing TILs from various tumor types in either control or MRM.
Each of the linked
points represent TILs obtained from the same sample such that the figure
summarizes data from
13 patients. Asterisks indicate that the average percentage of CD8 + TILs
following culture in
control media is significantly different than the average percentage of CD8 +
TILs following culture
in MRM. These data show that culturing TILs in MRM results in enrichment of
CD8 + T cells as
compared to culturing TILs in control media.
[0086] FIGs. 3A-3E are graphical representations of FACS cell
phenotyping based on
expression of PD1 and CD27 of cultured CD4 + (FIGs. 3A-3B) and CD8 + (FIGs. 3C-
3D) Tits
following 14-day culture in control media (FIG. 3A and 3C) or MRM (FIGs. 3B
and 3D). FIG. 3C
is a scatter plot showing the individual differences in the percentage of
CD27+13D1+ cells obtained
by culturing TILs from various tumor types in either control or MR1VI. Each of
the linked points
represent TILs obtained from the same sample such that FIG. 3C summarizes data
from 9 patients.
Asterisks indicate that the average percentage of CD27 13D1+ TILs following
culture in control
media is significantly different than the average percentage of CD27 PD1+ TILs
following culture
in MRM. These data show that culturing TILs in MR1\4 results in enrichment of
CD27+131)1+ T
cells as compared to culturing TILs in control media.
[0087] FIG. 4 is a graphical representation illustrating the
statistically significant
difference in the percentages of CD27+CD28+ cells obtained by culturing TILs
from various tumor
types in either control media or MRIVI after the initial culture (day 14).
Each of the linked points
represent TILs obtained from the same sample such that FIG. 4 summarizes data
from 9 patients.
These data show that culturing TILs in MRNI results in enrichment of CD27
CD28+ T cells as
compared to TILs cultured in control media.
[0088] FIGs. 5A-5B are graphical representations of FACS cell
phenotyping of TILs
cultured (day 14) in control media (FIG. 5A) or MRIVI (FIG. 5B), gated first
by CD8 or CD4
expression, followed by CD28 and CD27 expression, followed by CD103 and CD27
expression,
followed by PD1 and CD103 expression, and finally by TCF7 and CD27 expression.
FIG. 5C is a
graphical representation illustrating the mean fluorescence intensity (MFI) of
TCF7+ TILs
following initial culture in control media (1) or MRM (2) (about day 14).
[0089] FIGs. 6A-6H are graphical representations of FACS cell
phenotyping of TILs
expanded in control media (FIGs. 6A-6D) or MRM (FIGs. 6E-6H) after the
secondary expansion
(about day 21-26), gated first by CD8 or CD4 expression (FIGs 6A and 6E), CD28
and CD27
expression gated on CD8 cells (FIGs. 6B and 6F), PD1 and CD27 expression
gated on CD8 + cells
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(FIGs. 6C and 6G), and finally by TCF7 and CD39 expression gated on CD8 +
cells (FIGs. 6D and
6H). FIGs. 6B-6D and 6F-6H are CD8 + cells.
[0090] FIGs. 7A-7H are graphical representations of FACS cell
phenotyping of CD8 + TILs
expanded by co-culture with mutant KRAS-pulsed dendritic cells in control
media (FIGs. 7A-7D)
or MRNI (FIGs. 7E-7H) after the secondary expansion (about day 21), gated
first by CD8 or CD4
expression (FIGs. 7A and 7E), followed by CD28 and CD27 expression gated on
CD8 + cells (FIGs.
7B and 7F), followed by PD1 and CD27 expression gated on CD8 + cells (FIGs. 7C
and 7G), and
finally by TCF7 and CD8 expression gated on PD1 + only and CD27, PD1+ cells
(FIGs. 7D and
7H). FIGs. 7B-7D and 7F-7H are CD8 + cells.
[0091] FIGs. 8A-8H are graphical representations of FACS cell
phenotyping of TILs
expanded by co-culture with wild-type KRAS-pulsed dendritic cells in control
media (FIGs. 8A-
8D) or MRM (FIGs. 8E-8H) after the secondary expansion (about day 21), gated
first by CD8 or
CD4 expression (FIGs. 8A and 8E), followed by CD28 and CD27 expression gated
on CD8 + cells
(FIGs. 8B and 8F), followed by PD1 and CD27 expression gated on CD8 + cells
(FIGs. 8C and 8G),
and finally by TCF7 and CD8 expression gated on PD1 + only and CD27, PD1 +
cells (FIGs. 8D
and 8H). FIGs. 8B-8D and 8F-8H are CD8 cells.
[0092] FIGs. 9A-9B are graphical representations of FACS cell
phenotyping of cultured
TILs following secondary expansion (about day 21-26) in control media (FIG.
9A) or MR1VI (FIG.
9B).
[0093] FIG. 10 is a bar graph showing the fold-change (FC) in
gene expression of IL-2,
B2M, GZMB, IFNy, and TCF7 in TILs cultured in control media or MEM after the
secondary
expansion (about day 21). Expression of each gene is normalized to the
expression in TILs cultured
in control media.
[0094] FIGs. 11A-11L are graphical representations of FACS cell
sorting of CD4 + or CD8+
TILs cultured in control media (FIGs. 11A, 11B, 11E, and 11F) or MRM (FIGs.
11C, 11D, and
11G-11L) after secondary expansion (about day 21-26), gated by PD1 expression
(FIGs. 11A-11D)
or CD103 expression (FIGs. 11E-11H) and CD39 expression (FIGs. 11A-11H). FIGs.
11I-11L
show gating of CD4 + TILs (FIGs. 111 and 11K) and CD8 + TILs (FIGs. 11J and
11L) gated on PD1
and CD39 expression (FIGs. 11I-11J) followed by CD45R0 and CD103 expression
(FIGs. 11K-
11L).
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[0095] FIG. 12 is a bar graph illustrating the Simpsons
clonality values for immune cells
in tumor fragments ("tumor"), TILs expanded using control media ("control"),
and TILs expanded
using metabolic reprogramming media ("MRM").
[0096] FIGs. 13A-13B are differential abundance (DA) plots
generated using the the data
presented in FIG. 12 for TILs expanded in control media (FIG. 13A) and TILs
expanded in MRM
(FIG. 13B). FIGs. 13C-13D are graphical representations of tumor antigen
recognition of the top
50 dominant tumor TCRs in a TIL population cultured in control media (FIG.
13C) or in MRM
(FIG. 13D).
[0097] FIG. 14 is a diagram showing KRAS mutant activity of TIT,
cultured in MRM. 1SEQ
ID NO: 6; 2SEQ ID NO: 1; 3 SEQ ID NO: 7; 4SEQ ID NO: 8; 5SEQ ID NO: 9; 6SEQ ID
NO: 10;
7SEQ ID NO: 11.
[0098] FIGs. 15A-15D are bar graphs illustrating the tumor
recognition and tumor killing
activity of TILs generated using control media or MRM, as evidenced by
secreted IFN-gamma
(FIGs. 15A and 15D), secreted IL-2 (FIG. 15A), secreted TNF-alpha (FIG. 15B),
percent tumor
cell killing (FIG. 15C). A= TILs generated using control media, and B=TILs
generated using MRM
(FIGs. 15A, 15B, and 15D); "Control TIL" = Tits generated using control media
and "MRM TIL"
= TILs generated using MRM (FIG. 15C); and "TC line" = tumor cell line (FIG.
15D).
[0099] FIG. 16 is a graphical representation of the percent of
cell lysis of autologous
melanoma tumor cells culured ex vivo over time, following contact and co-
culture with TILs (at
the time indicated by the arrow). TILs were cultured in either control media
or MRM and added to
the cultuted tumor cells at a ratio of 1:1 effector T cell (E) to tumor cell
(T), 2:1 E:T, and 4:1 E:T,
as indicated.
[0100] FIGs. 17A-17H are graphical represenations, illustrating
the expression of marker
genes in NSCLC TILs expanded using a control process (FIGs. 17A-17D) or MRM
(17E-17H).
TILs expanded in MRM exhibited superior phenotypic characteristics as measured
by CD8+ T cell
fraction, low CD39/CD69 expression (FIGs. 17B and 17D), central memory
(CD45RO+CD6L+;
FIGs. 17C and 17G) and high CD27 expression (FIGs. 17D and 17H). Dashed line
highlighted box
indicates unfavorable phenotype and solid line highlighted box indicates
favorable phenotype.
[0101] FIGs. 18A-18C are graphical represenations, illustrating
negative expression by
CD8+ T cells of both CD39 and CD69 within the T cell compartment in TILs
obtained from a
melanoma (FIG. 8A), a NSCLC (FIG. 18B), or a colorectal cancer (FIG. 18C).
Cultures were
initiated from freshly supplied human tumor samples and cells were expanded
under control or
MRIVI conditions. After final rapid expansion process (REP), TILs were
analyzed for negative
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expression by CD8+ T cells of both CD39 and CD69 within the T cell
compartment. For each
analysis, TILs expanded from melanoma (n=6 independent tumors), NSCLC (n=5
independent
tumors) and colorectal cancer (n=11 independent tumors) were assessed.
Statistical significance
was measured by paired t test. 'p<.001, *p<.05.
[0102] FIGs. 19A and 19B are bar graphs illustrating the
Simpsons clonality values for
immune cells in tumor fragments ("tumor"), TILs expanded using control media
("control"), and
TILs expanded using metabolic reprogramming media ("MRI\4") for non-small cell
lung cancer
(NSCLC) (FIG. 19A) and melanoma (FIG. 19B).
[0103] FIGs. 20A-20J present data showing stem-like (FIGs. 20A-
20C and 20G-201) and
exhaustion (FIGs. 20D-20F, 20G, 20H, and 20J) phenotypes for CD8 (FIGs. 20A-
20F) and CD4
(FIGs. 20G-201) T1L products cultured by the standard (TILs cultured in AIM-
V), control (TILs
cultured in control media), or MR1VI (TILs cultured in MRM) process. FIGs.
20A, 20D, and 20G
are heat-maps illustrating the expression of stem-like markers (FIGs. 20A and
20G) and exhaustion
markers (FIGs. 20D and 20G) as measured by bulk RNAseq of CD8 (FIGs. 20A and
20D) and
CD4 (FIG. 20G) TILs. FIGs. 20B (stem-like markers), 20E (exhaustion markers),
and 20H (stem-
like and exhaustion markers) summarize the results of interrogation of various
genesets. FIG. 20C
is a graphical representation of the proportion of stem-like CD8 cells (each
data set represents a
single donor). FIG. 20F is a graphical representation of the proportion of
exhausted CD8 cells
(each data set represents a single donor). FIG. 201 is a graphical
representation of the proportion
of stem-like CD4 cells (each data set represents a single donor). FIG. 20J is
a graphical
representation of the proportion of exhausted CD4 cells (each data set
represents a single donor).
[0104] FIGs. 21A-21B present data illustrating the expression of
various genes associated
with a metabolic phenotype and cell cycle in TIL products cultured by the
standard, control, or
MRIVI process. FIG. 21A is a heat-map illustrating the expression of metabolic
phenotype and cell
cycle markers as measured by bulk RNAseq of CD8 TILs. FIG. 21B summarizes the
results of
interrogation of various genesets.
[0105] FIGs. 22A-22H are graphical representations of the
results of CITEseq + scTCRseq
was conducted on Day0 gMACS T cells from 6 donors. FIG. 22A shows an overlay
of the results
of each of the individual 6 donor samples (FIGs. 22B-22G).
[0106] FIGs. 23A-23D show the number of putative tumor reactive
clones identified in
each sample based on the exhausted phenotype in the day0 5' CITEseq data and
bulkTCR
frequency (top 100 clones) for CD8 (FIGs. 23A-23B) and CD4 (FIGs. 23C-23D)
components of
T1L products cultured by the standard, control, or MR_M process. FIG. 23E is a
bar graph
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illustrating the number of tumor reactive clones in stem-like product clusters
for various donors
for each of the three processes.
[0107] FIGs. 24A-24F provide data illustrating that MRM putative
tumor reactive cells
have differentially expressed genes compared to AIM-V and control putative
tumor reactive cells.
FIG. 24A is a heat-map showing differential expression of the Zhang TEX
3Tumors gene set (see
Table 3). FIG. 24B provides the results of interrogation of various gene sets.
FIGs. 24C-24F are
graphical representations showing differential expression of SEL and IL7R RNA
(FIG. 24C) and
CD39 and CD69 protein (FIGs. 24C-24F) in TIL products cultured by the standard
(FIGs. 24C and
24D), control (FIGs. 24C and 24E), or MRM (FIGs. 24C and 24F) process.
[0108] FIGs. 25A and 25B are graphical representations of
percent cytolysis by TIL
products cultured by the MRM protocol (at E:T ratios of 1:1, 5:1, and 10:1;
FIG. 25A) or standard,
control, MR1VI, and MRM without a 4-1BB/TRANSACTTm boost processes in co-
culture with
autologous tumor cells at an E:T ratio of 10:1 (FIG. 25B).
[0109] FIGs. 26A-26C are graphical representations of the
proportion of tumor-reactive
clones expressing 4-1BB. FIG. 26D is a heat map illustrating the differential
expression of various
differentiation and exhaustion marker genes. The numbers of unique clones in
each process group
are shown in FIG. 26E.
[0110] FIGs. 27A-27C are pie charts showing the likelihood of
reaching pre-REP TIL
expansion greater than 1.5-fold for each of the processes. FIGs. 27D-27F show
the pre-REP fold
expansion achieved for each process using metastatic colon (FIG. 27D),
metastatic melanoma
(FIG. 27E), and NSCLC (FIG. 27F) tumors as the starting material. Pre-REP fold
expansion was
calculated based on the number of cells seeded compared to the number of cells
at the end of pre-
REP. FIG. 27G shows the fold expansion of TILs following REP for each of the
processes. REP
fold expansion was calculated based on the number of cells seeded compared to
the number of
cells at the end of REP. FIGs. 27H and 271 show CTV dilution assay results
illustrating
proliferation of healthy PBMCs and irradiated PBMCs, respectively, following
REP.
[0111] FIG. 28 shows the gating strategy for phenotypic analysis
of the TfL products. TIL
products were gated on lymphocytes by FSCA vs SSCA, then singlets by FSCA vs
FSCH, then
live cells by exclusion dye against FSCA, then CD3+ T cells by CD3 vs FSCA.
For product
characterization, CD4+ and CD8+ T cells were gated separately by comparing CD4
against CD8.
The CD8+ T cells were further analyzed for sternness characteristics by
plotting CD39 against
CD69 and gating on the double positive population (CD39+CD69+) and double
negative
population (CC39-CD69-). For stem-like double positive populations in the CD8+
T cell subset,
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CD62L and CD27 or CD27 and CD127 were plotted against each other and gated on
the double
positive population (CD62L+CD27+ or CD27+CD127+).
[0112] FIGs. 29A-29B are box-plots illustrating the percent of
CD8+ (FIG. 29A) and
CD4+ (FIG. 29B) in the standard, control, and MRNI products. CD3+ T cells were
gated on CD8
versus CD4 in both control and Epi-R TIL cell product. Statistical
significance calculated by paired
T test. P-values were defined as: NS, *P<0.05, **P<0.01.
[0113] FIGs. 30A-30D are box-plots illustrating the levels of
various surface proteins on
CD8+ TILs for each process. Based on flow cytometric surface protein
expression, CD8+ TIL
were plotted on CD8 versus CD27, and the percentage frequency of parent
(P<0.0001) and
percentage frequency of grandparent (P<0.0001) of the CD27+ population were
calculated. CD8+
TIL were plotted on CD62L versus CD27 (P<0.0001) or CD127 versus CD27
(P=0.0011) and the
percentage frequency of parent for both double positive populations were
calculated. Statistical
significance calculated by paired T test. P-values were defined as: NS,
*P<0.05, **P<0.01,
***P<0.001, ****P<0.0001.
[0114] FIGs. 31A-31C are box-plots illustrating levels of CD39-
CD69- stem-like (FIGs.
31A-31B) and CD39+CD69+ (FIG. 31C) CD8+ TIL for standard, control, and MRNI
products,
based on flow cytometric surface protein expression. Statistical significance
calculated by paired
T test. P-values were defined as: NS, *P<0.05, **P<0.01, ***P<0.001,
****P<0.0001.
[0115] FIGs. 32A-32B are box-plots illustrating levels of CD39-
CD69- stem-like (FIG.
32A) and CD39+CD69+ (FIG. 32B) CD4+ TILs for standard, control, and MIRM
products, based
on flow cytometric surface protein expression. Statistical significance
calculated by paired T test.
P-values were defined as: NS, *P<0.05, **P<0.01, ***P<0.001, ****P<0.0001.
[0116] FIG. 33 is a box-plot illustrating the polyclonality of
raw tumor cells and TIL
products generated using the standard, control, and MRM processes, as measured
by simpson
clonality.
[0117] FIG. 34 provides a schematic of the experimental design
for identifying, tracking,
and analyzing putative tumor reactive clones as further described in Example
18.
[0118] FIG. 35A provides box plots illustrating the
polyclonality of the following TIL
products using Simpson clonality index: (i) initial tumor cells ("tumor"), and
(ii) generated using
the MRM process ("MRM") at research scale.
[0119] FIG. 35B provides a box-plots illustrating the
polyclonality of TIL products
generated using either the research scale MRM process or the large scale MRM
process. The
polyclonality of the TM products are shown using the Simpson clonality index.
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[0120] FIG. 36A shows autologous tumor cell killing by TILs
produced using the MRM
process at different effector to target cell ratios: (i) 10:1, (ii) 5:1, (iii)
1:1, and (iv) 0:1.
[0121] FIGs. 36B-36C show the ability of TILs produced using the
MRM process to
produce inflammatory cytokines (FIG. 36B) and immunosuppressive cytokines
(FIG. 36C) after
in vitro stimulation with anti-CD3/CD28 antibodies. Unstimulated cells were
used as control. In
FIG. 36B, the proinflammatory cytokines shown include: IFN-7 (left graph), TNF-
a, (middle
graph), and IL-2 (right graph). In FIG. 36C, the immunosuppressive cytokines
shown include: IL-
4 (left graph), IL-5 (middle graph), and IL-13 (right graph).
[0122] FIGs. 37A-37D show the UMAP representations for
identification of putative
tumor-reactive clones from day 0 tumor samples (11 melanoma, 3 lung, and 2
colon cancer) after
single-cell RNA/TCR and bulkTCR sequencing analysis_ FIG. 37A shows the bias
of the identified
putative tumor-reactive clones towards CD8+ T cells as opposed to CD4+ T
cells. FIGs. 37B and
37C show the identification of tumor reactive phenotype based on CXCL13, 4-
1BB, PD-1, and
TIGIT expression. FIG. 37D shows cells identified by tumor reactive phenotype
and high
frequency in bulkTCR-seq.
[0123] FIGs. 38A and 38B provide comparison of anti-tumor
activity of T cells from
healthy donors modified to express one of the top three putative tumor-
reactive TCRs identified
(i.e., TCR 1, TCR 2, or TCR 3). FIG. 38A shows the ability of the different
modified T cells to kill
autologous tumor cells (at 5:1 effector:target ration) using an in vitro
killing assay. Tumor only
group was used as a control. FIG. 38B shows IFN-7 production by the different
modified T cells
after in vitro stimulation with the autologous tumor cells (i.e., "TR TCR 1,"
"TR TCR 2," and "TR
TCR 3''). Tumor only and unstimulated cells (i.e., "TIL only") were used as
control.
[0124] FIG. 39 provides validation of the identified putative
tumor-reactive clones as being
tumor reactive when tested at high frequencies. TILs produced using the MRM
process were co-
cultured with a target (autologous tumor cell line or dissociated tumor
suspension, then, TIL
activation (41BB+/IFN-7+) was measured to determine tumor reactivity.
[0125] FIGs. 40A-40D show the UM AP representation of the
phenotypic characterization
of putative tumor-reactive clones after expansion using the control process or
MRM (research scale
or large scale) based on scRNAseq/scTCRseq analysis. In total, 23 different
TIL products were
analyzed (9 control, 9 MRM ¨ research scale, and 6 MRM ¨ large scale). For
each of FIGs. 40A-
40D, the control group is shown on the left, the MRM (research scale) is shown
in the middle, and
the MRM (large scale) is shown on the right. FIG. 40A shows the UMAP
representation of the
cells in the TIL products expanded under control, MRM research scale and MRM
large scale
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conditions. FIG. 40B shows the UMAP representation stem-like clusters
identified by expression
of SELL (left), CD39 (center), or CD69 (right) and gene sets listed in Table
3. Stem-like clusters
were identified as SELL+, CD39-, and CD69-. FIG. 40C shows the UMAP
reprentation of all
stemlike cells within the different T1L groups. FIG. 40D provides a UMAP
representation of the
putative tumor-reactive cells present within the different TIL groups.
[0126] FIGs. 41A and 4111 provide heatmap analysis showing the
relative expression of
different genes associated with stemness (FIG. 41A) or exhaustion (FIG. 41B)
in putative tumor-
reactive cells after culturing using: (i) the control process, (ii) the
research scale 1V1RM process, or
(iii) the large scale MRM process for different tumor types
[0127] FIGs. 41C and 41D provides Gene Set Enrichment Analysis
(GSEA) showing the
relative expression of stemness-associated or exhausted-associated gene sets
in putative tumor-
reactive cells after culturing using the control process or the MRM process.
FIG. 41C provides the
results for MR1VI (research scale) as compared to the control. FIG. 41D
provides the results for
MR1V1 (large scale) as compared to the control.
[0128] FIG. 42 provides a graphical representation of the single
cell data presented in FIG.
40C and shows the proportion of CD8+ stemlike cells for TIL expanded using the
(i) control
process, (ii) MRM research scale process, and (iii) MRI\4 large scale process
for colorectal tumor
(CRC; left), metastatic melanoma (middle) and non-small cell lung cancer
(NSCLC; right). Each
dot represents single cell data from one donor.
[0129] FIGs. 43A-43C show the effect of thelVIR1VI process on
TILs from tumors that were
not previously treated with an immune checkpoint blockade ("Naïve") or from
tumors that were
previously treated with an immune checkpoint blockade ("ICB"). TILs (both from
naïve and ICB
groups) were expanded and cultured using the control process are also provided
for comparison
purposes ("control"). FIG. 43A provides a comparison of the pre-REP fold
expansion for the
control and ICB. FIG. 43B provides a comparison of the CD8+ T cell percentage.
FIG. 43C
provides the percentage of stem-like cells as evidenced by CD39- CD69-
expression.
DETAILED DESCRIPTION
[0130] The present disclosure is directed to methods of
culturing immune cells (e.g., TILs),
cells prepared by the methods (e.g., compositions comprising enrichment of
oligoclonal or
polyclonal tumor reactive, e.g., tumor specific, stem-like T-cells and/or CD8+
TILs), and/or
methods of treating a subject using the immune cells described herein. The
cell culturing methods
of the present disclosure are capable of enhancing the expansion of CDS+ TILs
and/or increasing
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multipotency and/or pluripotency of the cultured TILs. In some aspects, the
culturing methods are
capable of reducing and/or preventing immune cell exhaustion, e.g., TIL
exhaustion, when the
immune cells are cultured and/or the immune cells are used in therapy in vivo.
In some aspects the
culturing methods of the present disclosure are capable of preserving clonal
diversity of the TILs
derived from cancer patients.
[0131] In some aspects, the disclosure is directed to methods of
culturing TILs ex vivo or
in vitro comprising culturing a heterogeneous population of TILs in a
metabolic reprogramming
medium, e.g., a hyperkalemic medium comprising potassium ion at a
concentration higher than 40
mM, wherein the hyperkalemic medium is not hypertonic. In some aspects, the
disclosure is
directed to methods of increasing the number or percentage of CD8+ TILs ex
vivo or in vitro
comprising culturing a heterogeneous population of TILs in a metabolic
reprogramming medium,
e.g., a hyperkalemic medium comprising potassium ion at a concentration of at
least 5 mM. In
other aspects, the disclosure is directed to methods of preparing a CD8+-
enriched population of
tumor infiltrating lymphocytes (TILs), comprising culturing a heterogeneous
population of TILs
ex vivo or in vitro in a metabolic reprogramming medium, e.g., a hyperkalemic
medium comprising
potassium ion at a concentration of at least 5 mM. In some aspects, the
disclosure is directed to
methods of preparing a CD8 -enriched population of tumor infiltrating
lymphocytes (TILs),
comprising culturing a heterogeneous population of TILs ex vivo or in vitro in
a metabolic
reprogramming medium, e.g., a medium comprising potassium ion at a
concentration between 40
mM and 80 mM and NaCl at a concentration between 100 mM and 30 mM, wherein the
total
concentration of potassium ion and NaCl is between 110 and 140 mM.
[0132] In some aspects, the hyperkalemic medium is not
hypertonic. In some aspects, the
hyperkalemic medium is hypotonic. In some aspects, the hyperkalemic medium is
isotonic. In
some aspects, the hyperkalemic medium further comprises interleukin (IL)-2, IL-
21, IL-7, IL-15,
or any combination thereof. In some aspects, the hyperkalemic medium further
comprises sodium
ion, calcium ion, glucose, or any combination thereof
[0133] Before the present disclosure is described in greater
detail, it is to be understood
that this disclosure is not limited to the particular compositions or process
steps described, which,
of course, vary. As will be apparent to those of skill in the art upon reading
this disclosure, each of
the individual aspects described and illustrated herein has discrete
components and features which
can be readily separated from or combined with the features of any of the
other several aspects
without departing from the scope or spirit of the present disclosure. Any
recited method can be
carried out in the order of events recited or in any other order that is
logically possible.
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[0134] The headings provided herein are not limitations of the
various aspects of the
disclosure, which can be defined by reference to the specification as a whole.
It is also to be
understood that the terminology used herein is for the purpose of describing
particular aspects only,
and is not intended to be limiting.
I. Terms
[0135] In order that the present disclosure can be more readily
understood, certain terms
are first defined. As used in this application, except as otherwise expressly
provided herein, each
of the following terms shall have the meaning set forth below. Additional
definitions are set forth
throughout the application.
[0136] Throughout this disclosure, the term "a" or "an" entity
refers to one or more of that
entity; for example, "a chimeric polypeptide," is understood to represent one
or more chimeric
polypeptides. As such, the terms "a" (or "an"), "one or more," and "at least
one" can be used
interchangeably herein.
[0137] Furthermore, "and/or" where used herein is to be taken as
specific disclosure of
each of the two specified features or components with or without the other.
Thus, the term "and/or"
as used in a phrase such as "A and/or B" herein is intended to include "A and
B," "A or B," "A"
(alone), and "B" (alone). Likewise, the term "and/or" as used in a phrase such
as "A, B, and/or C"
is intended to encompass each of the following aspects: A, B, and C; A, B, or
C; A or C; A or B;
B or C; A and C; A and B; B and C; A (alone); B (alone); and C (alone). In
addition, "or" is used
mean an open list of the components in the list. For example, "wherein X
comprises A or B- means
X comprises A, X comprises B, X comprises A and B, or X comprises A or B and
any other
components.
[0138] It is understood that wherever aspects are described
herein with the language
"comprising," otherwise analogous aspects described in terms of "consisting
of' and/or "consisting
essentially of' are also provided.
[0139] Unless defined otherwise, all technical and scientific
terms used herein have the
same meaning as commonly understood by one of ordinary skill in the art to
which this disclosure
is related. For example, the Concise Dictionary of Biomedicine and Molecular
Biology, Juo, Pei-
Show, 2nd ed., 2002, CRC Press, The Dictionary of Cell and Molecular Biology,
3rd ed., 1999,
Academic Press; and the Oxford Dictionary of Biochemistry and Molecular
Biology, Revised,
2000, Oxford University Press, provide one of skill with a general dictionary
of many of the terms
used in this disclosure.
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[0140] Units, prefixes, and symbols are denoted in their Systeme
International de Unites
(SI) accepted form. Numeric ranges are inclusive of the numbers defining the
range.
[0141] Abbreviations used herein are defined throughout the
present disclosure. Various
aspects of the disclosure are described in further detail in the following
subsections.
[0142] The terms "about" or "comprising essentially of' refer to
a value or composition
that is within an acceptable error range for the particular value or
composition as determined by
one of ordinary skill in the art, which will depend in part on how the value
or composition is
measured or determined, i.e., the limitations of the measurement system. For
example, "about" or
"comprising essentially of' can mean within 1 or more than 1 standard
deviation per the practice
in the art. Alternatively, "about" or "comprising essentially of' can mean a
range of up to 10%.
Furthermore, particularly with respect to biological systems or processes, the
terms can mean up
to an order of magnitude or up to 5-fold of a value. When particular values or
compositions are
provided in the application and claims, unless otherwise stated, the meaning
of "about" or
"comprising essentially of' should be assumed to be within an acceptable error
range for that
particular value or composition.
[0143] As used herein, the term "approximately," as applied to
one or more values of
interest, refers to a value that is similar to a stated reference value. In
some aspects, the term
"approximately" refers to a range of values that fall within 10%, 9%, 8%, 7%,
6%, 5%, 4%, 3%,
2%, 1%, or less in either direction (greater than or less than) of the stated
reference value unless
otherwise stated or otherwise evident from the context (except where such
number would exceed
100% of a possible value).
[0144] As described herein, any concentration range, percentage
range, ratio range or
integer range is to be understood to include the value of any integer within
the recited range and,
when appropriate, fractions thereof (such as one tenth and one hundredth of an
integer), unless
otherwise indicated.
[0145] The term "control media" as used herein refers to any
media in comparison to the
metabolic reprogramming media ("MRM") disclosed herein. Control media can
comprise the same
components as the metabolic reprogramming media except certain ion
concentrations, e.g,
potassium ion. In some aspects, metabolic reprogramming media described herein
are prepared
from control media by adjusting one or more ion concentrations, e.g.,
potassium ion concentration,
as described herein. In some aspects, control media comprise basal media,
e.g., CTSTm
OPTIMIZERTm. In some aspects, control media comprise AIM V, RPMI, or a mixture
comprising
AIM V and RPMI. In some aspects, control media comprise (i) 50% AIM V, (ii)
50% RPMI1640,
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(iii) 5% or 10% human serum, and (iv) IL-2. In some aspects, control media
thus comprises one or
more additional components, including, but not limited to, amino acids,
glucose, glutamine, T cell
stimulators, antibodies, substituents, etc. that are also being added in the
metabolic reprogramming
media, but control media have certain ion concentrations different from the
metabolic
reprogramming media. Unless indicated otherwise, the terms "media" and
"medium" can be used
interchangeably.
[0146] As used herein, a "control process" or an "MRM process"
refers to a method
disclosed herein, wherein the TIL culturing and expansion process includes a
control medium, or
an MR1\4 medium, respectively. As such, a "control process" refers to the
culturing of Tits in a
control medium disclosed herein, such as AIIVI V or TCM without a modified
concentration of
potassium (i.e., less than 40 mM, e.g., 5 mM, potassium ion), and an "MR_I\4
process" refers to the
culturing of TILs in an MRI\4 medium disclosed herein.
[0147] As used herein, the term "immune cell" refers to a cell
of the immune system. In
some aspects, the immune cell is selected from a T lymphocyte ("T cell"), B
lymphocyte ("B cell"),
natural killer (INK) cell, macrophage, eosinophil, mast cell, dendritic cell
or neutrophil). In some
aspects, the immune cell is a tumor-infiltrating cell (TIL). As used herein, a
"TIL" refers to T cell
that has at least once entered into a tumor or is capable of entering a tumor,
e.g., within the
parenchyma of a tumor. In some aspects, the tumor is a solid tumor. In some
aspects, the tumor is
a liquid tumor, e.g., a hematopoietic cancer. TILs prepared by the present
methods can have one
or more properties that are the same as the naturally occurring TILs. In some
aspects, TILs prepared
by the present methods have one or more properties that are not present in the
naturally occurring
TILs. TILs can be obtained using any methods. In some aspects, the TILs are
obtained from a
tumor sample from a subject. In some aspects, the tumor sample, or a portion
thereof, is cultured
under conditions that promote evasion of the TILs from the tumor tissue,
proliferation of the TILs,
and/or expansion of the TILs. In some aspects, the medium used to promote
evasion, proliferation,
and/or expansion of the TILs is any metabolic reprogramming medium, e.g.,
hyperkalemic
medium, disclosed herein.
[0148] As used herein, a "population" of cells refers to a
collection of more than one cell,
e.g., a plurality of cells. In some aspects, the population of cells comprises
more than one TILs,
e.g., a plurality of TILs In some aspects, the population of cells is
comprises a heterogeneous
mixture of cells, comprising multiple types of cells, e.g., a heterogeneous
mixture of TILs and cells
other than TILs.
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[0149] TILs include, but are not limited to, CD8+ T cells (i.e.
cytotoxic T cells), CD4+ T
cells, B cells, and natural killer cells. TILs include both primary (e.g.,
obtained from a patient tissue
sample) and secondary TILs (e.g., TIL cell populations that have been
cultured, expanded or
proliferated from primary TILs. In some aspects the TILs are genetically
modified. In some
aspects, the TIL is a CD8+ T cell. CD8+ TILs are generally considered to be
the subpopulation of
TILs responsible for destroying cancer cells. Conversely, CD4+ TILs are
generally considered to
act as suppressors of the immune response, which can limit the immune response
against the tumor.
[0150] In some aspects, TILs can be defined biochemically using
cell surface markers.
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. In some
aspects,
TILs can be defined functionally by their ability to infiltrate tumors and
selectively kill the cancer
cells.
[0151] As used herein, the terms "T cell" and "T lymphocyte" are
interchangeable and refer
to any lymphocytes produced or processed by the thymus gland. Non-limiting
classes of T cells
include effector T cells (such as CD8+ T cell) and Th cells (such as CD4+ T
cells). In some aspects,
the immune cell is a Thl cell. In some aspects, the immune cell is a Th2 cell.
In some aspects, the
immune cell is a Tc17 cell. In some aspects, the immune cell is a Th17 cell.
In some aspects, the
immune cell is a Trcg cell.
[0152] As used herein, the term "memory" T cells refers to T
cells that have previously
encountered and responded to their cognate antigen (e.g., in vivo, in vitro,
or ex vivo) or which
have been stimulated with, e.g., an anti-CD3 antibody (e.g., in vitro or ex
vivo). Immune cells, e.g.,
TILs, having a "memory-like" phenotype, upon secondary exposure to antigen or
stimulation,
reproduce or proliferate to mount a faster and strong immune response than
during the primary
exposure. In some aspects, memory T cells comprise central memory T cells (Tcm
cells), effector
memory T cells (TEm cells), tissue resident memory T cells (TRm cells), stem
cell-like memory T
cells (Tscm cells), or any combination thereof
[0153] As used herein, the term "stem-like" or "stem cell-like"
refers to a property or an
ability of a cell to self-renew and has the multipotent capacity to generate
and reconstitute the entire
spectrum of memory and effector T cell subsets. In some aspects, a stem-like
cell can be measured
by specific markers expressed by the cell. In some aspects, those stem-like
markers can be one or
more of CD45RA+, CD62L+, CCR7-1, CD27-1, CD28+, BACH2+, LEF1+, and TCF7+. In
some
aspects, stem-like cells can be identified based on CD39- and CD69-
expression. As will be evident
to those skilled in the arts, the symbol "-" adjacent to a marker (e.g., CD39-
) does not necessarily
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indicate that a cell completely lacks the expression of the marker. For
instance, in some aspects,
CD39- cells can have some CD39 expression but the expression is much reduced
as compared to
a reference cell, e.g., CD39+ cell. Therefore, unless indicated otherwise,
"CD39- cells" can
comprise cells with no expression of CD39 as well as cells with reduced CD39
expression as
compared to a reference cell, e.g., CD39+ cells. As shown in FIG. 55, CD39-
CD69- cells (i.e.,
stem-like cells) can be identified using flow cytometry. For instance, the
CD39- CD69- cells can
be identified by first gating on the CD8+ or CD4+ T cells (see left flow
plot). Then, the CD39+
CD69+ cells are identified within the CDS+ or CD4+ T cell population (see
upper right quadrant
in the right flow plot). The CD39- CD69- cells correspond to the bottom left
two boxes (see the
two arrows). (Krishna et al., Science 370:1328-34 (Dec. 11, 2020); and/or
Galletti et al., Nature
Immunology (October 2018). In some aspects, CD39 dim/- CD69- stem-like TIL can
be gated with
the use of fluorescent minus one (FMO) controls to determine upper boundary
for background
signal for both CD39 and CD69. Populations that fall within the FMO boundary
are considered as
negative expression for the associated marker. Using FMO boundaries,
populations are gated for
CD39 dim/- CD69- expression. CD39 gates are extended slightly past the FMO
boundary to
include CD39 dim cells that remained CD69-. In some aspects, stem-like cells
can be identified
based on one or more of the following: CD45RA+, CD62L+, CCR7+, CD27+, CD28+,
BACH2+,
LEF1+, TCF7+, CD39-, and CD69-. In some aspects, the stem-like cells can be
identified by a
transcriptome analysis (alone or in combination with the phenotypic markers
described above),
e.g., using sternness gene signatures disclosed herein. In some aspects, the
effector-like marker
comprises a marker disclosed in Krishna et al., Science 370:1328-34 (Dec. 11,
2020); and/or
Galletti et al., Nature Immunology (October 2018), each of which is
incorporated by reference
herein in its entirety.
[0154] As used herein, the term "stem cell-like memory T cells,"
"T memory stem cells,"
or "Tscm cells" refer to memory T cells that express CD95, CD45RA, CCR7, and
CD62L and are
endowed with the stem cell-like ability to self-renew and the multipotent
capacity to reconstitute
the entire spectrum of memory and effector T cell subsets.
[0155] As used herein, the term "central memory T cells" or "Tcm
cells" refer to memory
T cells that express CD45RO, CCR7, and CD62L. Central memory T cells are
generally found
within the lymph nodes and in peripheral circulation.
[0156] As used herein, the term "effector-like" or "effector
cell-like" refers to tumor cell
killing capacity and cytokine polyfunctionality, e.g., ability of a cell to
produce inflammatory
cytokines and/or cytotoxic molecules. In some aspects, an effector-like cell
can be measured by
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specific markers expressed by the cell. In some aspects, those effector-like
markers can be one or
more of pSTAT5+, STAT5+, pSTAT3+, and STAT3+. In some aspects, the effector-
like marker
comprises a STAT target selected from the group consisting of AKT1, AKT2,
AKT3, BCL2L1,
CBL, CBLB, CBLC, CCND1, CCND2, CCND3, CISH, CLCF1, CNTF, CNTFR, CREBBP,
CRLF2, CSF2, CSF2RA, CSF2RB, CSF3, CSF3R, CSH1, CTF1, EP300, EPO, EPOR, GH1,
GH2,
GIIR, GRB2, IFNA1, IFNA10, IFNA13, IFNA14, IFNA16, IFNA17, IFNA2, IFNA21,
IFNA4,
IFNA5, IFNA6, IFNA7, IFNA8, IFNAR1, IFNAR2, IFNB1, IFNE, IFNG, IFNGR1, IFNGR2,
IFNK, IFNL1, IFNL 2, IFNL3, IFNLR1, IFNVV1, IL10, ILlORA, IL 1 ORB, IL11,
IL11RA, IL12A,
IL12B, IL12RB1, IL12RB2, IL13, IL13RA1, 1L13RA2, 1L15, IL15RA, IL19, IL2,
IL20, IL20RA,
IL20RB, IL21, IL21R, IL22, IL22RA1, IL22RA2, IL23A, IL23R, 1L24, IL26, IL2RA,
IL2RB,
IL2RG, IL3, IL3RA, IL4, IL4R, IL5, IL5RA, 11,6, IL6R, IL6ST, IL7, IL7R, IL9,
IL9R, IRF9,
JAK1, JAK2, JAK3, LEP, LEPR, LIF, LIFR, MPL, MYC, OSM, OSMR, PIAS1, PIAS2,
PIAS3,
PIAS4, PIK3CA, PIK3CB, PIK3CD, PIK3CG, PIK3R1, P11K3R2, PIK3R3, P1K3R5, PIM1,
PRL,
PRLR, PTPN11, PTPN6, SOCS1, SOCS2, SOCS3, SOCS4, SOCS5, SOCS7, SOS1, SOS2,
SPRED1, SPRED2, SPRY1, SPRY2, SPRY3, SPRY4, STAM, STAM2, STAT1, STAT2, STAT3,
STAT4, STAT5A, STAT5B, STAT6, TPO, TSLP, TYK2, and any combination thereof. In
some
aspects, the effector-like cells can be identified by a transcriptome
analysis. In some aspects, the
effector-like marker comprises a marker disclosed in Kaech et al., Cell
111:837-51(2002); Tripathi
et al., J. Immunology 185:2116-24 (2010); and/or Johnnidis et al., Science
Immunology 6: eabe3702
(Jan. 15, 2021), each of which is incorporated by reference herein in its
entirety.
[0157] In some aspects, the effector-like cells are
characterized using an effector-
associated gene set described in Gattinoni, L., et al., Nat Med 17(10):1290-97
(2011). In some
aspects, the gene signature for effector-like cells comprises one or more
genes selected from
MTCH2, RAB6C, KIAA0195, SETD2, C2orf24, NRD1, GNA13, COPA, SELT, TNIP1,
CBFA2T2, LRPIO, PRKCI, BRE, ANKS 1A, PNPLA6, ARL61P1, WDFY1, MAPK1, GPR153,
SHKBP1, MAP1LC3B2, PIP4K2A, HCN3, GTPBP1, TLN1, C4orf34, KIF3B, TC1RG1,
PPP3CA, ATG4D, TYMP, TRAF6, Cl 7orf76, WIPF1, FAM108A1, MYL6, NRM, SPCS2,
GGT3P, GALK1, CLIP4, ARL4C, YWHAQ, LPCAT4, ATG2A, IDS, TBC1D5, DMPK,
ST6GALNAC6, REEP5, ABHD6, KIAA0247, EMB, TSEN54, SPIRE2, PIWIL4, ZSCAN22,
ICAM1, CHD9, LPIN2, SETD8, ZC3H12A, ULBP3, IL15RA, HLA-DQB2, LCP1, CHP,
RUNX3, TMEM43, REEP4, MEF2D, ABL1, TMEM39A, PCBP4, PLCD1, CHST12, RA SGRP1,
C1orf58, Cl lorf63, C6orf129, FHOD1, DKFZp434F142, PIK3CG, ITPR3, BTG3,
C4orf50,
CNNM3, IFI16, AK1, CDK2AP1, REL, BCL2L1, MVD, TTC39C, PLEKHA2, FKBP11, EML4,
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FANCA, CDCA4, FUCA2, MFSD10, TBCD, CAPN2, IQGAP1, CHST11, PIK3R1, MY05A,
KIR2DL3, DLG3, MXD4, RALGDS, SIPR5, WSB2, CCR3, TIPARP, SP140, CDI51, SOX13,
KRTAP5-2, NF1, PEA15, PARP8, RNF166, UEVLD, LIMK1, CACNB1, TMX4, SLC6A6,
LBA1, SV2A, LLGL2, IRF1, PPP2R5C, CD99, RAPGEF1, PPP4R1, OSBPL7, FOXP4, SLA2,
TBC1D2B, ST7, JAZFL GGA2, PI4K2A, CD68, LPGAT1, STX11, ZAK, FAM160B1, RORA,
C8orf80, APOBEC3F, TGFBI, DNAJC1, GPR114, LRP8, CD69, CMI, NAT13, TGFB1,
FLJ00049, ANTXR2, NR4A3, IL12RB1, NTNG2, RDX, MLLT4, GPRIN3õ ADCY9, CD300A,
SCD5, ABI3, PTPN22, LGALS1, SYTL3, BMPR1A, TBK1, PMAIP 1, RAS GEF 1Aõ GCNT1,
GABARAPL1, STOM, CALI-EVI2, ABCA2, PPP1R16B, SYNE2, PAM, C12orf75, CLCF1,
MXRA7, APOBEC3C, CLSTN3, ACOT9, HIP1, LAG3, TNFAIP3, DCBLDI, KLF6, CACNB3,
RNF19A, RAB27A, FADS3, DLG5, APOBEC3D, TNFRSF1B, ACTN4, TBKBP1, ATXN1,
ARAP2, ARHGEF12, FAM53B, MAN1A1, FAA/138A, PLXNC1, GRLFI, SRGN, HLA-DRB5,
B4GALT5, WIPI1, PTPRJ, SLFN11, DUSP2, ANXA5, AHNAK, NE01, CLIC1, EIF2C4,
MAP3K5, IL2RB, PLEKHG1, MY06, GTDC1, EDARADD, GALM, TARP, ADAN18, MSC,
HNRPLL, SYT11, ATP2B4, NHSL2, MATK, ARFIGAP18, SLFN12L, SPATS2L, RAB27B,
PIK3R3, TP53INP1, MBOAT1, GYG1, KATNALI, FAM46C, ZC3HAV1L, ANXA2P2,
CTNNA1, NPC1, C3AR1, CRIM1, SH2D2A, ERNI, YPEL1, TBX21, SLC1A4, FASLG,
PHACTR2, GALNT3, ADRB2, PIK3AP1, TLR3, PLEKHA5, DUSP10, GNA01, PTGDR,
FRNID4B, ANXA2, EOMES, CADMI, MAF, TPRG1, NBEAL2, PPP2R2B, PELO, SLC4A4,
KLRF1, FOSL2, RGS2, TGFBR3, PRF1, MY0 IF, GAB3, C17orf66, MICAL2, CYTH3, TOX,
HLA-DRA, SYNE1, WEE1, PYHIN1, F2R, PLD1, THBS1, CD58, FAS, NET02, CXCR6,
ST6GALNAC2, DUSP4, AUT S2, C 1 orf21, KLRG1, TNIP3, GZMA, PRR5L, PRDM1,
ST8SIA6,
PLXND1, PTPRM, GFPT2, MYBL1, SLAMF7, FLJ16686õ GNLY, ZEB2, CST7, IL18RAP,
CCL5, KLRD1, KLRB1, and any combination thereof (see, e.g., Gattinoni, L., et
at., Nat Med
17(10):1290-97 (2011).
[0158] As used herein, the term "effector memory T cells" or
"TEm cells" refer to memory
T cells that express CD45R0 but lack expression of CCR7 and CD62L Because
effector memory
T cells lack lymph node-homing receptors (e.g., CCR7 and CD62L), these cells
are typically found
in peripheral circulation and in non-lymphoid tissues.
[0159] As used herein, the term "tissue resident memory T cells"
or "TRm cells" refer to
memory T cells that do not circulate and remain resident in peripheral
tissues, such as the skin,
lung, and the gastrointestinal tract. In some aspects, tissue resident memory
T cells are also effector
memory T cells
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[0160] As used herein, the term "naive T cells," "TN cells," or
"naive" TILs" refers to T
cells and/or TILs that express CD45RA, CCR7, and CD62L, but which do not
express CD95.
These cells represent the most undifferentiated cell in the T cell lineage.
The interaction between
a naive T cell and an antigen presenting cell (APC) induces differentiation of
the naive T cell
towards an actiaved TEFF cell and an immune response.
[0161] As used herein, the term "fragmenting," "fragment," and
"fragmented" describe
processes for disrupting a tumor, including mechanical fragmentation methods
such as crushing,
slicing, dividing, and morcellating tumor tissue as well as any other methods
for disrupting the
physical structure of tumor tissue.
[0162] The term "culturing" as used herein refers to the
controlled growth of cells ex vivo
and/or in vitro. As used herein, "culturing" includes the growth of cells,
e.g., TILs, during cell
expansion. In some aspects, the cultured cells are obtained from a subject,
e.g., a human subject.
In some aspects, the cultured cells comprise TILs obtained from a human
subject. In some aspects,
the culturing comprises placing a tumor sample or tumor fragmentinto a medium
disclosed herein,
wherein the medium promotes TIL evasion from the tumor sample and TIL
expansion. In some
aspects, the tumor sample or tumor fragement is a dissociated tumor sample
(e.g., single cell
suspension). In some aspects, the dissociated tumor sample (e.g., single cell
suspension) is
dissociated in a medium disclosed herein. In some aspects, the culturing
comprises placing a
dissocoiated tumor sample or tumor fragment into a medium disclosed herein. In
some aspects, the
TILs are isolated or purified prior to the culture. In some aspects, the cell
culturing is intended to
expand the number of cultured cells, e.g., to increase proliferation of the
cells.
[0163] "Expand" or "expansion," as used herein in reference to
TILs refers to the process
of stimulating or activating the cells and culturing the cells. The expansion
process can lead to an
increase in the proportion or the total number of desired cells, e.g., an
increase in the proportion or
total number of TILs, in a population of cultured cells, after the cells are
stimulated or activated
and cultured. Expansion does not require that all cell types in a population
of cultured cells are
increased in number. Rather, in some aspects, only a subset of cells in a
population of cultured
cells are increased in number during expansion, while the number of other cell
types may not
change or may decrease.
[0164] As used herein, the term "yield" refers to the total
number of cells following a
culture method or a portion thereof. In some aspects, the term "yield" refers
to a particular
population of cells, e.g., stem-like TILs in a population of Tits. The yield
can be determined using
any methods, including, but not limited to, estimating the yield based on a
representative sample.
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[0165] As used herein, the term "stem cell-like," "stem-like,"
or "less-differentiated" refers
to a cell, e.g., an immune cell (e.g., a TIL), that expresses markers
consistent with a more naive
phenotype. For example, a less differentiated TIL can express one or more
markers characteristic
of a TN or a Tscm cell. In some aspects, a "less-differentiated" or "stem-
like" TIL expresses
CD45RA, CCR7, and CD62L. In some aspects, a "less-differentiated" or "stem-
like" TIL expresses
CD45RA, CCR7, and CD62L, and is CD45R010w. In some aspects, a "less-
differentiated" or "stem-
like" immune cell (e.g., TIL) expresses CD45RA, CCR7, and CD62L, and does not
express
CD45RO. In some aspects, a "less-differentiated" or "stem-like" T cell
expresses CD45RA, CCR7,
CD62L, and TCF7. In some aspects, the methods disclosed herein promote the
growth and/or
proliferation of cells, e.g., TILs, having a less-differentiated phenotype.
Without being bound by
any particular mechanism, in some aspects, the methods disclosed herein block,
inhibit, or limit
differentiation of less-differentiated cells, e.g., TILs, resulting in an
increased number of stem-like
cells in culture. For example, it is generally thought that to effectively
control tumors, adoptive
transfer of less-differentiated TILs with a stem cell-like memory or central
memory phenotype are
preferred. See, e.g., Gattinoni, L., et al., J. Cl/n. Invest. 115:1616-
1626(2005); Gattinoni, L., et al.
Nat Med 15(7):808-814 (2009); Lynn, R.C., et al., Nature 576(7786): 293-300
(2019), Gattinoni,
L., et al., J. Cl/n. Invest. 115:1616-1626 (2005); Gattinoni, L., etal. Nat
Med 15(7):808-814
(2009); and Gattinoni, L., et al., Nat Med 17(10): 1290-1297 (2011).
[0166] Stemness is characterized by the capacity to self-renew,
the multipotency, and the
persistence of proliferative potential. In some aspects, sternness is
characterized by a particular
gene signature, e.g., a combined pattern of expression across a multitude of
genes. In some aspects,
the gene signature comprises one or more genes selected from ACTN1, DSCI,
TSHZ2, MYB,
LEF I, TIMD4, MAL, KRT73, SESN3, CDCA7L, L0C283174, TCF7, SLC16A10, LASS6,
UBE2E2, IL7R, GCNT4, TAF4B, SULT1B1, SELP, KRT72, STXBP1, TCEA3, FCGBP,
CXCR5, GPA33, NELL2, APBA2, SELL, VIPR1, FAM153B, PPFIBP2, FCERIG, GJB6,0CM2,
GCET2, LRRN1, IL6ST, LRRC16A, IGSF9B, EFHA2, L0C129293, APP, PKIA, ZC3H12D,
KIAA0748, SLC22A17, FLJ13197, NRCAM, C5orf13, GIPC3, WNT7A, FAM117B,
BENDS, LGMN, FAM63A, FAM153B, ARI-1GEF I I, RBM11, RIC3, LDLRAP1, PELI1, PTK2,
KCTD12, LM07, CEP68, SDK2, MCOLN3, ZNF238, EDAR, FAM153C, FAAH2, BCL9,
C17orf48, MAP1D, Z SWIM I , SORB S3, IL4R, SERPINF 1, C16orf45, SPTBN1, KCNQ
1, LDHB,
BZW2, NBEA, GAL3ST4, CRTC3, MAP3K1, HLA-D0A, RAB43, SGTB, CNN3, CWH43,
KLI-113, PIM2, RGMB, C16orf74, AEBP1, SNORD115-1 , SNORD115-11, GRAP, and any
combination thereof (see, e.g., Gattinoni (2011)). In some aspects, the gene
signature comprises
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one or more gene selected from NOG, TIMD4, MYB, UBE2E2, FCER1G, HAVCR1, FCGBP,
PPFIBP2, TPST1, ACTN1, IGF IR, KRT72, SLC16A10, GJB6, LRRN1, PRAGMIN, GIPC3,
FLNB, ARRB1, SLC7A8, NUCB2, LRRC7, MY015B, MAL, AEBP1, SDK2, BZW2, GAL3 ST4,
PITPNM2, ZNF496, FAM117B, C16orf74, TDRD6, TSPAN32, Ci8orf22, C3orf44,
L0C129293,
ZC3H12D, MLXIP, C7orf10, STXBP1, KCNQ1, FLJ13197, LDLRAP1, RAB43, RIN3,
SLC22A17, AGBL3, TCEA3, NCRNA00185, FAM153B, FAM153C, VIPR1, MNIP19, IIBS1L,
EEF2K, SNORA5C, UBASH3A, F1143390, RP6-213H19.1, INPP5A, PIM2, TNFRSF10D,
SNRK, L0C100128288, PIGV, L0C100129858, SPTBN1, PROS1, MMP28, HES1, CACHD1,
NSUN5C, LEF1, TTTY14, SNORA54, HSF2, C16orf67, NSUN5B, KIAA1257, NRG2, CAD,
TARBP1, STRADB, MTIF, TMEM41B, PDHX, KDM6B, L0C100288322, UXS1, LGMN,
NANOS2, PYGB, RASGRP2, C14orf80, XP06, SLC24A6, FAM113A, MRI\41, FBXW8,
NDUF S2, KCTD12, and any combination thereof (see, e.g., Gattinoni, L., et
al., Nat Med 17(10):
1290-1297 (2011) or Galletti et al. Nat Immunol 21, 1552-1562 (2020)). In some
aspects, the gene
signature comprises one or more gene selected from SELL, CCR7, S1PR1, KLF3,
TCF7, GPR183,
SC5D, FAAH2, LTB, SESN3, MAL, TSHZ2, LEFI, AP3M2, SLC2A3, ICAM2, PLAC8,
SCML1, IL7R, ABLIM1, RASGRP2, TRABD2A, SATB I, ALG13, ARID5A, BACH2, PABPC1,
GPCPDI, NELL2, TAF4B, FCMR, ARRDC2, Clorf162, FAM177A1, ANKRD12, TXK,
SORL1, AQP3, ADTRP, FXYD7, CD28, P2RY8, CRYBG1, TNFSF8, BEX2, PGAP1, PTGER4,
MAML2, BEX3, PC SKIN, INPP4B, AC119396.1, CXCR5, LINC00402, CCR4, IL6R,
ZBTB10,
ITGA6, ARMIII, RILPL2, FOXP1, TESPA1, YPEL5, LPAR6, CMSS1, RIPOR2, ZNF331,
EIVFP3, GIMAP7, WDR74, RIC3, CYSLTR1, ITGBI, CD5, SAMHD1, SERINC5, and any
combination thereof (see e.g., Caushi et al., Nature 596: 126-132 (2021)).
[0167] In the presence of prolonged antigen exposure, such as in
many cancers, more
differentiated immune cells, e.g., effector and effector memory T cells, often
become exhausted
and lose their anti-tumor function. Biomarkers, e.g., T cell markers, can be
measured using any
methods. In some aspects, T cells are identified using antibody-staining
following by gated flow
cytometry.
[0168] The term "clonotype," as used herein, refers to a
population of T cells with unique
DNA sequences that result from TCRa or TCRB rearrangements. A unique variable
a chain (VA)
sequence may pair up with more than one variable 13 chain (VB) sequence.
Conversely, a unique
VB sequence may pair up with more than one VA sequence.
[0169] As used herein, the term "tonicity" refers to the measure
of the effective osmotic
pressure gradient across a cell membrane. Tonicity can be measured or
calculated based on the
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level of potassium ion and sodium chloride (NaCl) in a solution. Herein,
tonicity is calculated as
the sum of the concentration of potassium ion (K+) and the concentration of
sodium chloride
(NaCl), multiplied by two. Tonicity can be expressed in terms of the
osmolality of the solution,
e.g., the media. As used herein, a solution, e.g., medium, is considered
"isotonic" when the
concentration of solutes in the media is equivalent to the concentration of
solutes inside the cell.
As used herein, an isotonic medium has an osmolality of about 280 mOsm/L
(e.g., ([K ] I [NaCl])
X 2 = 280).
[0170] As used herein, a solution, e.g., a medium, is considered
"hypotonic" if the
concentration of solutes in the solution is lower than the concentration of
solutes in the cell. As
used herein, a hypotonic solution has a tonicity of less than 280 mOsm/L
(e.g., ([K+] + [NaCl]) X
2 <280). In some aspects, a hypotonic medium described herein has an
osmolality of about 240
mOsm/L or about 250 mOsm/L. In some aspects, a hypotonic medium has a tonicity
from at least
about 220 mOsm/L to less than about 280 mOsm/L. In some aspects, a hypotonic
medium has a
tonicity from at least about 230 mOsm/L to less than about 280 mOsm/L. In some
aspects, a
hypotonic medium has a tonicity from at least about 240 mOsm/L to less than
about 280 mOsm/L.
In some aspects, a hypotonic medium described herein has a tonicity of about
250 mOsm/L (e.g.,
([K+] + [NaCl]) X 2 = 250).
[0171] As used herein, a solution, e.g., a medium, is considered
"hypertonic" if the
concentration of solutes in the solution is higher than the concentration of
solutes in the cell. As
used herein, a hypertonic solution has an osmolality of greater than 300
mOsm/L (e.g., ([K+] +
[NaCl]) X 2 > 280). In some aspects, a hypertonic medium described herein has
an osmolality of
about 320 mOsm/L. In certain aspects, the tonicity of the solution, e.g.,
medium is adjusted by
increasing or decreasing the concentration of one or more solute selected from
potassium ions,
sodium ions, glucose, and any combination thereof. In some aspects, the
tonicity of the solution,
e.g., medium is adjusted by increasing or decreasing the concentration of
potassium ions and NaCl.
In some aspects, the tonicity of a medium can be maintained by offsetting the
increase of one solute
with a decrease in a second solute. For example, increasing the concentration
of potassium ion in
a medium without changing the concentration of sodium ions can increase the
tonicity of the
medium. However, if the concentration of potassium ions is increased and the
concentration of
sodium ions is decreased, the tonicity of the original medium can be
maintained. As used herein,
the tonicity of a medium is defined by the sum of the potassium concentration
and the NaC1
concentration, multiplied by two. See, e.g., Table 2.
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[0172] As used herein, the terms "potassium," "potassium ion,"
"potassium cation," and
are used interchangeably to refer to elemental potassium. Elemental potassium
exists in
solution as a positive ion. However, it would be readily apparent to a person
of ordinary skill in
the art that standard means of preparing a solution comprising potassium ion
include diluting a
potassium containing salt (e.g., KC1) into a solution. As such, a solution,
e.g., a medium,
comprising a molar (M) concentration of potassium ion, can be described as
comprising an equal
molar (M) concentration of a salt comprising potassium.
[0173] As used herein, the terms "sodium ion" and "sodium
cation" are used
interchangeably to refer to elemental sodium. Elemental sodium exists in
solution as a monovalent
cation. However, it would be readily apparent to a person of ordinary skill in
the art that standard
means of preparing a solution comprising sodium ion include diluting a sodium-
containing salt
(e.g., NaCl) into a solution. As such, a solution, e.g., a medium, comprising
a molar (M)
concentration of sodium ion, can be described as comprising an equal molar (M)
concentration of
a salt comprising sodium.
[0174] As used herein, the terms "calcium ion" and "calcium
cation" are used
interchangeably to refer to elemental calcium. Elemental calcium exists in
solution as a divalent
cation. However, it would be readily apparent to a person of ordinary skill in
the art that standard
means of preparing a solution comprising calcium ion include diluting a
calcium-containing salt
(e.g., CaCl2) into a solution. As such, a solution, e.g., a medium, comprising
a molar (M)
concentration of calcium ion, can be described as comprising an equal molar
(M) concentration of
a salt comprising calcium.
[0175] As used herein, the term "hyperkalemic," e.g.,
"hyperkalemic medium," refers to a
medium that has an increased potassium concentration. In some aspects, the
hyperkalemic medium
comprises potassium ion at a concentration of greater than 5 mM. In some
aspects, the
hyperkalemic medium comprises potassium ion at a concentration higher than 40
mM. In some
aspects, the hyperkalemic medium a concentration of potassium ion of at least
about 10 mM, at
least about 15 mM, at least about 20 mM, at least about 25 mM, at least about
30 mM, at least
about 35 mM, at least about 40 mM, at least about 45 mM, at least about 50 mM,
at least about 55
mM, at least about 60 mM, at least about 65 mM, at least about 70 mM, about 75
mM, about 80
mM, about 85 mM, about 90 mM, about 95 mM, or about 100 mM The term "metabolic
reprogramming media," "metabolic reprogramming medium," or "MRM," as used
herein, refers to
a hyperkalemic medium of the present disclosure. In some aspects, the MRM
comprises potassium
ion at a concentration of about 50 mM. In some aspects, the 1\'IR_IV1
comprises potassium ion at a
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concentration of about 55 mM. In some aspects, the MRM comprises potassium ion
at a
concentration of about 60 mM. In some aspects, the MRM comprises potassium ion
at a
concentration of about 65 mM. In some aspects, the MRM comprises potassium ion
at a
concentration of about 70 mM. In certain aspects, the metabolic reprogramming
media comprises
about 40 mM to about 80 mM NaC1, about 40 mM to about 90 mM KC1, about 0.5 mM
to about
2.8 mM calcium, and about 10 mM to about 24 mM glucose. In some aspects, the
metabolic
reprograming media further comprises an osmolality of about 250 to about 340
mOsmol.
[0176] As used herein, the term "basal" media refers to any
starting media that is
supplemented with one or more of the additional elements disclosed herein,
e.g., potassium,
sodium, calcium, glucose, IL-2, IL-7, IL-15, IL-21, or any combination
thereof. The basal media
can be any media for culturing immune cells, e.g., TILs. In some aspects, the
basal media is
selected from a balanced salt solution (e.g., PBS, DPBS, HBSS, EBSS),
Dulbecco's Modified
Eagle's Medium (DMEM), Click's medium, Minimal Essential Medium (MEM), Basal
Medium
Eagle (BME), F-10, F-12, RPMI 1640, Glasgow Minimal Essential Medium (GMEM),
alpha
Minimal Essential Medium (alpha MEM), Iscove's Modified Dulbecco's Medium
(11\4DM), M199,
OPTMIZERTm CTSTm T-Cell Expansion Basal Medium (ThermoFisher), OPTMIZERTm
Complete, IM_MUNO CUL TTm XF ( S TEMCELLTm Technologies), IMMUNO CUL T Tm XF,
AIM
V, TEXMACSTm medium, TRANSACTTm TIL expansion medium, TIL rapid expansion
protocol
medium, and any combination thereof. In some aspects, the basal medium is
serum free. In some
aspects, the basal media comprises PRIME-XV T cell CDM. In some aspects, the
basal media
comprises OPTMIZER". In some aspects, the basal media comprises OPTMIZER" Pro.
In some
aspects, the basal media comprises XVIVOTM 15 (LONZA). In some aspects, the
basal media
comprises IMMUNOCULT". In some aspects, the basal media comprises Click's
medium. In
some aspects, the basal media comprises TRANSACT' TIL expansion medium. In
some aspects,
the basal media comprises TIL rapid expansion medium. In some aspects, the
basal medium further
comprises immune cell serum replacement (ICSR). For example, in some aspects,
the basal
medium comprises OPTMIZERTm Complete supplemented with ICSR, AIM V
supplemented with
ICSR, IMMUNOCULTTm XF supplemented with ICSR, RPMI supplemented with ICSR,
TEXMACSTm supplemented with ICSR, or any combination thereof. In some aspects,
suitable
basal media include Click's medium, OpTimizer (CTS ) medium, Stemline T cell
expansion
medium (Sigma-Aldrich), AIM V medium (CTS ), TexMACS medium (Miltenyi
Biotech),
ImmunoCult medium (Stem Cell Technologies), PRIME-XV T-Cell Expansion XSFM
(Irvine
Scientific), Iscoves medium, and/or RPMI-1640 medium. In some aspects, the
basal media
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comprises NaC1 free CTSTm OPTIMIZERTm. In some aspects, suitable basal media
include Click's
medium, OpTimizer (CTS ) medium, Stemline T cell expansion medium (Sigma-
Aldrich),
AIM V medium (CT SS), TexMACS medium (Miltenyi Biotech), ImmunoCult medium
(Stem Cell Technologies), PRIME-XV T-Cell Expansion XSFM (Irvine Scientific),
Iscoves
medium, and/or RPMI-1640 medium. In some aspects, the basal media comprises
NaCl free
CTSTm OpTimizerTm. In some aspects, the basal media comprises one or more
sodium salt in
addition to the NaC1 that is added to control the tonicity, e.g., NaCl added
in combination with
potassium ion
[0177] As used herein, the term "cytokine" refers to small,
secreted proteins released by
cells that have a specific effect on the interactions and communications
between cells. Non-limiting
examples of cytokines include interleukins (e.g., interleukin (IL)-1, IL-2, IL-
4, IL-7, IL-9, IL-13,
IL-15, IL-3, IL-5, IL-6, IL-11, IL-10, IL-20, IL-14, IL-16, IL-17, IL-21, IL-
23, and IL-29),
interferons (IFN; e.g., IFN-u, IFN-13, and IFN-y), tumor necrosis factor (TNF)
family members,
and transforming growth factor (TGF) family members. Some aspects of the
present disclosure are
directed to methods of culturing cells, e.g., T cells and/or NK cells, in a
medium comprising a
cytokine. Some aspects of the present disclosure are directed to methods of
culturing TILs in a
medium comprising a cytokine. Some aspects of the present disclosure are
directed to methods of
expanding TILs in a medium comprising a cytokine. In some aspects, the
cytokine is an interleukin.
In some aspects, the cytokine is selected from IL-2, IL-7, IL-15, IL-21, and a
combination thereof.
IL-2 (UniProtKB ¨ P60568) is produced by T cells in response to antigenic or
mitogenic
stimulation. IL-2 is known to stimulate T cell proliferation and other
activities crucial to regulation
of the immune response. IL-7 (UniProtKB ¨ P13232) is a hematopoietic growth
factor capable of
stimulating the proliferation of lymphoid progenitors. IL-7 is believed to
play a role in proliferation
during certain stages of B-cell maturation. IL-15 (UniProtKB ¨ P40933), like
IL-2, is a cytokine
that stimulates the proliferation of T-lymphocytes. IL-21 (UniProtKB ¨ Q91-
1BE4) is a cytokine
with immunoregulatory activity. IL-21 is thought to promote the transition
between innate and
adaptive immunity and to induce the production of IgG1 and IgG3 in B-cells. IL-
21 may also play
a role in proliferation and maturation of natural killer (NK) cells in synergy
with 1L-15, and IL-21
may regulate proliferation of mature B- and T-cells in response to activating
stimuli. In synergy
with IL-15 and IL-18, IL-15 also stimulates interferon gamma production in T-
cells and NK cells,
and IL-21 may also inhibit dendritic cell activation and maturation during a T-
cell-mediated
immune response.
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[0178] As used herein, the term "higher than" means greater than
but not equal to. For
example, "higher than 5 mM" means any amount that is more than 5 mM, but which
does not
include 5 mM.
[0179] The term "preferentially," as used herein, refers to the
predominant outcome. For
example, if the methods disclosed herein preferentially promote expansion of
CD8-P TILs, it is to
be understood that the predominant product of the expansion is CD8+ TILs. The
term
"preferentially" does not necessarily mean that 100% of, e.g., the resulting
Tits are CD8', rather
the term suggests that CD8+ TILs are expanded to a greater extent than CD8-
Tits.
[0180] As used herein, "administering" refers to the physical
introduction of a therapeutic
agent or a composition comprising a therapeutic agent to a subject, using any
of the various
methods and delivery systems. The different routes of administration for a
therapeutic agent
described herein (e.g., a TIL cultured as described herein) include
intravenous, intraperitoneal,
intramuscular, subcutaneous, spinal or other parenteral routes of
administration, for example by
injection or infusion.
[0181] The phrase "parenteral administration" as used herein
means modes of
administration other than enteral and topical administration, usually by
injection, and includes,
without limitation, intravenous, intraperitoneal, intramuscular,
intraarterial, intrathecal,
intralymphatic, intralesional, intracapsular, intraorbital, intracardiac,
intradermal, transtracheal,
intratracheal, pulmonary, subcutaneous, subcuticular, intraarticular,
subcapsular, subarachnoid,
intraventricular, intravitreal, epidural, and intrasternal injection and
infusion, as well as in vivo
electroporation.
[0182] Alternatively, a therapeutic agent described herein
(e.g., a TIL cultured as described
herein) can be administered via a non-parenteral route, such as a topical,
epidermal, or mucosal
route of administration, for example, intranasally, orally, vaginally,
rectally, sublingually, or
topically. Administering can also be performed, for example, once, a plurality
of times, and/or over
one or more extended periods.
[0183] As used herein, the term "antigen" refers to any natural
or synthetic immunogenic
substance, such as a protein, peptide, or hapten. As used herein, the term
"cognate antigen" refers
to an antigen which an immune cell (e.g., a TIL) recognizes and thereby,
induces the activation of
the immune cell (e.g., triggering intracellular signals that induce effector
functions, such as
cytokine production, and/or for proliferation of the cell).
[0184] A "cancer" refers a broad group of various diseases
characterized by the
uncontrolled growth of abnormal cells in the body. Unregulated cell division
and growth results in
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the formation of malignant tumors that invade neighboring tissues and can also
metastasize to
distant parts of the body through the lymphatic system or bloodstream.
"Cancer" as used herein
refers to primary, metastatic and recurrent cancers.
[0185] The term "hematological malignancy" or "hematological
cancer" refers to
mammalian cancers and tumors of the hematopoietic and lymphoid tissues. Non-
limiting examples
of hematological malignancies include those affecting tissues of the blood,
bone marrow, lymph
nodes, and lymphatic system, including acute lymphoblastic leukemia (ALL),
chronic lymphocytic
lymphoma (CLL), small lymphocytic lymphoma (SLL), acute myelogenous leukemia
(AML),
chronic myelogenous leukemia (CIVIL), acute monocytic leukemia (AMoL),
Hodgkin's
lymphoma, and non-Hodgkin's lymphomas. Hematological malignancies are also
referred to as
"liquid tumors." Liquid tumor cancers include, but are not limited to,
leukemias, myelomas, and
lymphomas, as well as other hematological malignancies. TILs obtained from
liquid tumors may
also be referred to herein as marrow infiltrating lymphocytes (MILs).
[0186] A "solid tumor," as used herein, refers to an abnormal
mass of tissue. Solid tumors
may be benign or malignant. Nonlimiting examples of solid tumors include
sarcomas, carcinomas,
and lymphomas, such as cancers of the lung, breast, prostate, colon, rectum,
and bladder. The tissue
structure of a solid tumor includes interdependent tissue compartments
including the parenchyma
(cancer cells) and the supporting stromal cells in which the cancer cells are
dispersed, and which
may provide a supporting microenvironment.
[0187] As used herein, the term "immune response" refers to a
biological response within
a vertebrate against foreign agents, which response protects the organism
against these agents and
diseases caused by them. An immune response is mediated by the action of a
cell of the immune
system (e.g., a T lymphocyte (e.g., a TIL), B lymphocyte, natural killer (NK)
cell, macrophage,
eosinophil, mast cell, dendritic cell or neutrophil) and soluble
macromolecules produced by any of
these cells or the liver (including antibodies, cytokines, and complement)
that results in selective
targeting, binding to, damage to, destruction of, and/or elimination from the
vertebrate's body of
invading pathogens, cells or tissues infected with pathogens, cancerous or
other abnormal cells, or,
in cases of autoimmunity or pathological inflammation, normal human cells or
tissues. An immune
reaction includes, e.g., activation or inhibition of a T cell, e.g., an
effector T cell or a Th cell, such
as a CD4+ or CD8 '1Th, or the inhibition of a Treg cell. As used herein, the
terms "T cell" and "T
lymphocytes" are interchangeable and refer to any lymphocytes produced or
processed by the
thymus gland. In some aspects, a TIL is a CDS+ TIL. In some aspects, a TIL is
a CD4+ TIL.
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[0188] As used herein, the term "anti-tumor immune response"
refers to an immune
response against a tumor antigen.
[0189] A "subject" includes any human or nonhuman animal. The
term "nonhuman animal"
includes, but is not limited to, vertebrates such as nonhuman primates, sheep,
dogs, and rodents
such as mice, rats and guinea pigs. In some aspects, the subject is a human.
The terms "subject"
and "patient" are used interchangeably herein. As used herein, the phrase
"subject in need thereof'
includes subjects, such as mammalian subjects, that would benefit, e.g., from
administration of
immune cells, e.g., Tits, cultured as described herein to control tumor
growth.
[0190] The term "therapeutically effective amount" or
"therapeutically effective dosage"
refers to an amount of an agent (e.g., a TIL cultured as described herein)
that provides the desired
biological, therapeutic, and/or prophylactic result That result can be
reduction, amelioration,
palliation, lessening, delaying, and/or alleviation of one or more of the
signs, symptoms, or causes
of a disease, or any other desired alteration of a biological system. In
reference to solid tumors, an
effective amount comprises an amount sufficient to cause a tumor to shrink
and/or to decrease the
growth rate of the tumor (such as to suppress tumor growth) or to prevent or
delay other unwanted
cell proliferation. In some aspects, an effective amount is an amount
sufficient to delay tumor
development. In some aspects, an effective amount is an amount sufficient to
prevent or delay
tumor recurrence. An effective amount can be administered in one or more
administrations.
[0191] The effective amount of the composition (e.g., cells
cultured as described herein)
can, for example, (i) reduce the number of cancer cells; (ii) reduce tumor
size; (iii) inhibit, delay,
slow to some extent and can stop cancer cell infiltration into peripheral
organs; (iv) inhibit (i.e.,
slow to some extent and can stop tumor metastasis); (v) inhibit tumor growth;
(vi) prevent or delay
occurrence and/or recurrence of tumor; and/or (vii) relieve to some extent one
or more of the
symptoms associated with the cancer.
[0192] In some aspects, a "therapeutically effective amount" is
the amount of a
composition disclosed herein (e.g., T cells cultured as described herein),
which is clinically proven
to effect a significant decrease in cancer or slowing of progression
(regression) of cancer, such as
an advanced solid tumor. The ability of a therapeutic agent of the present
disclosure (e.g., T cells
cultured as described herein) to promote disease regression can be evaluated
using a variety of
methods known to the skilled practitioner, such as in human subjects during
clinical trials, in
animal model systems predictive of efficacy in humans, or by assaying the
activity of the agent in
Iii vitro assays.
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[0193] The terms "effective" and "effectiveness" with regard to
a treatment include both
pharmacological effectiveness and physiological safety. Pharmacological
effectiveness refers to
the ability of a composition disclosed herein (e.g., cells cultured as
described herein) to promote
cancer regression in the patient. Physiological safety refers to the level of
toxicity, or other adverse
physiological effects at the cellular, organ, and/or organism level (adverse
effects) resulting from
administration of a composition disclosed herein (e.g., cells cultured as
described herein).
[0194] As used herein, the term "tumor reactive" refers to the
ability of an immune cell,
e.g., a TIL, to target and kill a tumor cell. As used herein, the term "tumor
specific" refers to a
tumor reactive immune cell, e.g., TIL, that specifically targets a tumor cell.
[0195] As used herein, the term "putative tumor reactive" refers
to immune cells (e.g.,
TILs) or clones that are potentially tumor reactive based on functional or
phenotype characteristics
(e.g., expression of genes such as CXCL13, 4-1BB, PD-1, and TIGIT).
[0196] As used herein, the term "T cell receptor" or "TCR"
refers to a heterodimer
composed of 2 different transmembrane polypeptide chains: an a chain and a 1
chain, each
consisting of a constant region, which anchors the chain inside the T-cell
surface membrane, and
a variable region, which recognizes and binds to the antigen presented by
MFICs. The TCR
complex is associated with 6 polypeptides forming 2 heterodimers, CD3yE and
CD3oE, and 1
homodimer CD3 which together forms the CD3 complex. T-cell receptor-engineered
T-cell
therapy utilizes the modification of T cells that retain these complexes to
specifically target the
antigens expressed by particular tumor cells. As used herein, the term "TCR"
includes naturally
occurring TCRs and engineered TCRs.
[0197] As used herein, the terms "ug" and "uM" are used
interchangeably with "ag" and
"aM," respectively.
[0198] As used herein, the term "research scale," "MRM research
scale process," or
"research scale TIL" refers to TILs cultured and/expanded in MRM with lower
target TIL yields
and final product volumes compared to a large scale MRM process. In some
aspects, different
ratios of TIL to PBMCs can be used compared to the large scale MRM process.
For instance, in
some aspects, the ratio of TIL to PBMCs for a research scale MRM process is
greater compared to
the large scale MRM process. In some aspects, culture and/or expansion times
can be longer for
research scale TIL than for large scale TIL. In some aspects, a dynamic REP
step is excluded from
the research scale process.
[0199] As used herein, the terms "large scale," "MRM large scale
process," or "large scale
TIL" refer to TILs cultured and/expanded in MRM with higher target TIL yields
and final product
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volumes compared to a research scale M_RIVI process. In some aspects,
different ratios of TIL to
PBMCs can be used compared to the research scale MR1\4 process. For instance,
in some aspects,
the ratio of TIL to PBMCs for a large scale MR1VI process is less that the
corresponding ratio for
the research scale MRM process. In some aspects, culture and/or expansion
times can be shorter
for large scale TIL than for research scale TIL. In some aspects, a dynamic
REP step is included
in the large scale process.
[0200] Various aspects described herein are described in further
detail in the following
subsections.
11. Methods of the Disclosure
[0201] The present disclosure is directed to methods of
culturing immune cells, e.g., TILs,
ex vivo or in vitro. In some aspects, the methods of the present disclosure
comprise culturing or
placing immune cells, e.g., TILs, in a culture condition, wherein the culture
(e.g., certain ion
concentrations, tonicity of the medium, cytokines, and or any combination
thereof) is capable of
enhancing the expansion of CDS+ TILs. In some aspects, the culture (e.g.,
certain ion
concentrations, tonicity of the medium, cytokines, and or any combination
thereof) is capable of
reducing, limiting, or preventing the differentiation of the immune cells,
e.g., the TILs (e.g., CD8+
TILs and/or CD4 TILs), thereby affecting or improving their use in a cell
therapy. In some aspects,
the present disclosure comprises culturing of TILs in a metabolic
reprogramming media that is
high in potassium concentration. Increased potassium was surprisingly found to
correlate with
increased expansion of CD8+ TILs that have increased expression of stem-like
markers and
increased clonal diversity, while maintaining tumor-reactivity (e.g., tumor
specificity), as
compared to conventional methods using lower potassium levels, e.g., less than
about 40 mM
potassium ion, e.g., 5 mM potassium ion. Further, though exceedingly high
concentrations of
potassium (e.g.,> 80 mM, >90 mM, or > 100 mM) reduced TIL expansion, the
methods described
herein yielded therapeutically effective numbers of TILs following culture
conditions, e.g.,
durations, consistent with conventional methods.
[0202] The use of immunotherapy strategies has demonstrated
considerable clinical
efficacy in the treatment of certain types of advanced cancer. Immune
checkpoint blockade (ICB)
can result in objective and sometimes durable responses in patients with
metastatic melanoma.
Certain cohorts of colon cancer, lung cancer patients and small proportions of
patients with
additional malignancies can also benefit from ICB. Chimeric antigen receptor
(CAR) T cell therapy
has mediated dramatic clinical responses in patients with blood cell
malignancies, most notably B
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cell-lineage tumors that can be targeted with CD19 or B cell maturation
antigen (BCMA) CARs.
Treatment with T cells transduced with T cell receptors (TCRs) that recognize
shared, non-mutated
tumor antigens such as NY-ESO-1 can also mediate clinical responses in
patients who express
TCR matched human leukocyte antigens (HLAs). However, in spite of these
notable successes, the
vast majority of patients with advanced cancers still do not benefit from
immunotherapy treatments
and will eventually succumb to their illness.
[0203] TIL therapy has also shown a potential in mediating
clinical responses in patients
with advanced cancer. Emerging evidence has demonstrated that TILs are a
heterogenous
population composed of both tumor-reactive and non-specific bystander cells.
This heterogenous
population of TILs causes difficulty and unwanted effects in the TIL therapy
and/or dilution of the
efficacy of the TIL therapy as the non-specific bystander cells in the
heterogenous population are
not preferred. Bystander cells are nonspecific T cells, which can dilute the
diversity of reactive
TILs. Bystander cells include TILs that recognize epitopes that are not tumor
related. In addition,
the efficacy of TIL therapy has demonstrated diverse responses in patients
with melanoma,
advanced cervical, lung, breast, and/or gastrointestinal cancers.
[0204] In some aspects, the present disclosure provides methods
of reducing the
heterogeneity of TIL population ex vivo or in vitro for an in vivo therapy. In
some aspects, the
methods disclosed herein enrich for a particular type of a TIL population,
e.g., CD8+ TILs and/or
tumor-reactive CD8+ TILs. In some aspects the methods disclosed herein enrich
for stem-like T
cell populations, e.g., stem-like tumor-reactive TILs and/or stem-like tumor-
reactive CD8+ TILs.
[0205] Not being bound by any theory, the present disclosure
sets forth a method of
enriching a TIL population with a particular cell type, i.e., tumor-reactive
TIL, CD8+ TIL, tumor-
reactive CD8+ TIL, stem-like tumor-reactive TIL, stem-like CD8+ TIL, and/or
stem-like tumor-
reactive CD8+ TIL, using a hyperkalemic medium. Therefore, some aspects of the
present
disclosure are directed to methods of culturing TILs ex vivo or in vitro
comprising placing a
heterogeneous population of TILs in a hyperkalemic medium comprising potassium
ion at a
concentration higher than 40 mM. In some aspects, the heterogeneous population
of TILs is
enriched in CDS+ TILs after being placed in the hyperkalemic medium.
[0206] Some aspects of the present disclosure are directed to
methods of increasing a
number or percentage of CDS+ TILs (e.g., tumor reactive, e.g., tumor specific,
CD8+ TILs) ex vivo
or in vitro comprising culturing a heterogeneous population of TILs in a
hyperkalemic medium
comprising potassium ion at a concentration of at least 5 mM. Other aspects of
the present
disclosure are directed to methods of preparing a CD8tenriched (e.g., tumor
reactive CD8+-
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enriched) population of TILs, comprising culturing a heterogeneous population
of TILs ex vivo or
in vitro in a hyperkalemic medium comprising potassium ion at a concentration
of at least 5 mM.
[0207] Some aspects of the present disclosure are directed to
methods of increasing a
number or percentage of tumor reactive TILs ex vivo or in vitro comprising
culturing a
heterogeneous population of TILs in a hyperkalemic medium comprising potassium
ion at a
concentration of at least 5 mM. Other aspects of the present disclosure are
directed to methods of
preparing a tumor reactive-enriched population of TILs, comprising culturing a
heterogeneous
population of Tits ex vivo or in vitro in a hyperkalemic medium comprising
potassium ion at a
concentration of at least 5 mM.
[0208] Some aspects of the present disclosure are directed to
methods of increasing a
number or percentage of stem-like TILs (e.g., stem-like tumor reactive TILs,
stem-like CD8+ TILs,
or stem-like tumor reactive CD8+ TILs) ex vivo or in vitro comprising
culturing a heterogeneous
population of TILs in a hyperkalemic medium comprising potassium ion at a
concentration of at
least 5 mM. Other aspects of the present disclosure are directed to methods of
preparing a
population of TILs enriched for stem-like TILs (e.g., stem-like tumor reactive
TILs, stem-like
CD8+ TILs, or stem-like tumor reactive CD8+ TILs), comprising culturing a
heterogeneous
population of TILs ex vivo or in vitro in a hyperkalemic medium comprising
potassium ion at a
concentration of at least 5 mM.
[0209] In some aspects, the methods and/or compositions
disclosed herein increase the
clonal diversity of TILs in culture, as compared to TILs cultured under
control conditions (e.g., in
a media comprising potassium ion at a concentration of less than about 5 mM).
[0210] Clonal diversity can be assessed using any methods. In
some aspects, clonal
diversity is assessed using a subset of TILs cultured according to the methods
disclosed herein.
Non-limiting examples of methods of assessing clonal diversity of a population
of TILs can be
found, for example, in Venturi et al., J. Inimunolog. Mtd. 321:182-95 (2007),
which is incorporated
by reference herein in its entirety. In some aspects, clonal diversity is
assessed using
IMMUNOSEQ (ADAPTIVE BIOTECHNOLOGIES0). In some aspects, clonal diversity is
assessed using TCR deep sequencing. In certain aspects, the clonal diversity
is assessed by
sequencing TCR13 CDR3 seqeunces in total RNA isolated from the population of
TILs (e.g., cDNA
prepared from the total RNA). In some aspects, clonal diversity is assessed
using Simpsons
cl onality .
[0211] In some aspects, TILs cultured according to the methods
disclosed herein have a
clonal diversity that is the same as the clonal diversity of TILs in a tumor
sample. In some aspects,
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the TILs cultured according to the methods disclosed herein have a clonal
diversity that is at least
about 99% to about 100%, at least about 98% to about 100%, at least about 97%
to about 100%,
at least about 96% to about 100%, at least about 95% to about 100%, at least
about 94% to about
100%, at least about 93% to about 100%, at least about 92% to about 100%, at
least about 91% to
about 100%, at least about 90% to about 100%, at least about 85% to about
100%, at least about
80% to about 100%, at least about 75% to about 100%, at least about 70% to
about 100%, at least
about 65% to about 100%, at least about 60% to about 100%, at least about 55%
to about 100%,
at least about 50% to about 100%, at least about 45% to about 100%, or at
least about 40% to about
100% of the clonal diversity of TILs in a tumor sample. In certain aspects,
the TILs cultured
according to the methods disclosed herein have a clonal diversity that is at
least about 95% to about
100% of the clonal diversity of TILs in a tumor sample. In certain aspects,
the TILs cultured
according to the methods disclosed herein have a clonal diversity that is at
least about 90% to about
100% of the clonal diversity of TILs in a tumor sample. In certain aspects,
the TILs cultured
according to the methods disclosed herein have a clonal diversity that is at
least about 85% to about
100% of the clonal diversity of TILs in a tumor sample. In certain aspects,
the TILs cultured
according to the methods disclosed herein have a clonal diversity that is at
least about 80% to about
100% of the clonal diversity of Tits in a tumor sample. In certain aspects,
the TILs cultured
according to the methods disclosed herein have a clonal diversity that is at
least about 75% to about
100% of the clonal diversity of Tits in a tumor sample. In certain aspects,
the TILs cultured
according to the methods disclosed herein have a clonal diversity that is at
least about 70% to about
100% of the clonal diversity of TILs in a tumor sample. In certain aspects,
the TILs cultured
according to the methods disclosed herein have a clonal diversity that is at
least about 60% to about
100% of the clonal diversity of Tits in a tumor sample. In certain aspects,
the TILs cultured
according to the methods disclosed herein have a clonal diversity that is at
least about 50% to about
100% of the clonal diversity of Tits in a tumor sample. In certain aspects,
the TILs cultured
according to the methods disclosed herein have a clonal diversity that is at
least about 40% to about
100% of the clonal diversity of TILs in a tumor sample.
[0212] In some aspects, clonal diversity is assessed using
Simpsons clonality (pi2 where,
pi is the proportional abundance of clone i in a given sample). Simpsons
clonality is commonly
used to assess for productive rearrangements within a sample thus measuring
the magnitude of the
clone frequency distribution (see, e.g., Venturi et al., J. Imrnunol. Iffeth.
32/ :182-95 (2007), which
is incorporated by reference herein in its entirety). The values of the
Simpsons clonality range from
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0 to 1, where values approaching 1 represent a less clonally diverse and thus
a more monoclonal
TIL population.
[0213] In some aspects, the clonal diversity of TILs cultured
according to the methods
disclosed herein have a clonal diversity score of less than about 0.5, less
than about 0.45, less than
about 0.4, less than about 0.35, less than about 0.3, less than about 0.275,
less than about 0.25, less
than about 0.225, less than about 0.2, less than about 0.175, less than about
0.15, less than about
0.125, less than about 0.1, less than about 0.075, less than about 0.07, less
than about 0.06, or less
than about 0.05 as measured by Simpsons clonality. In some aspects, the clonal
diversity of Tits
cultured according to the methods disclosed herein have a clonal diversity
score of less than about
0.5, as measured by Simpsons clonality. In some aspects, the clonal diversity
of TILs cultured
according to the methods disclosed herein have a clonal diversity score of
less than about 0.4, as
measured by Simpsons clonality. In some aspects, the clonal diversity of TILs
cultured according
to the methods disclosed herein have a clonal diversity score of less than
about 0.3, as measured
by Simpsons clonality. In some aspects, the clonal diversity of TILs cultured
according to the
methods disclosed herein have a clonal diversity score of less than about
0.275, as measured by
Simpsons clonality. In some aspects, the clonal diversity of TILs cultured
according to the methods
disclosed herein have a clonal diversity score of less than about 0.25, as
measured by Simpsons
clonality. In some aspects, the clonal diversity of Tits cultured according to
the methods disclosed
herein have a clonal diversity score of less than about 0.24, as measured by
Simpsons clonality. In
some aspects, the clonal diversity of TILs cultured according to the methods
disclosed herein have
a clonal diversity score of less than about 0.23, as measured by Simpsons
clonality. In some
aspects, the clonal diversity of TILs cultured according to the methods
disclosed herein have a
clonal diversity score of less than about 0.22, as measured by Simpsons
clonality. In some aspects,
the clonal diversity of TILs cultured according to the methods disclosed
herein have a clonal
diversity score of less than about 0.21, as measured by Simpsons clonality. In
some aspects, the
clonal diversity of TILs cultured according to the methods disclosed herein
have a clonal diversity
score of less than about 0.2, as measured by Simpsons clonality. In some
aspects, the clonal
diversity of TILs cultured according to the methods disclosed herein have a
clonal diversity score
of less than about 0.19, as measured by Simpsons clonality. In some aspects,
the clonal diversity
of TILs cultured according to the methods disclosed herein have a clonal
diversity score of less
than about 0.18, as measured by Simpsons clonality. In some aspects, the
clonal diversity of TILs
cultured according to the methods disclosed herein have a clonal diversity
score of less than about
0.17, as measured by Simpsons clonality. In some aspects, the clonal diversity
of TILs cultured
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according to the methods disclosed herein have a clonal diversity score of
less than about 0.16, as
measured by Simpsons clonality. In some aspects, the clonal diversity of TILs
cultured according
to the methods disclosed herein have a clonal diversity score of less than
about 0.15, as measured
by Simpsons clonality. In some aspects, the clonal diversity of TILs cultured
according to the
methods disclosed herein have a clonal diversity score of less than about
0.14, as measured by
Simpsons clonality. In some aspects, the clonal diversity of TILs cultured
according to the methods
disclosed herein have a clonal diversity score of less than about 0.13, as
measured by Simpsons
clonality. In some aspects, the clonal diversity of TILs cultured according to
the methods disclosed
herein have a clonal diversity score of less than about 0.12, as measured by
Simpsons clonality. In
some aspects, the clonal diversity of TILs cultured according to the methods
disclosed herein have
a clonal diversity score of less than about 0.11, as measured by Simpsons
clonality. In some
aspects, the clonal diversity of TILs cultured according to the methods
disclosed herein have a
clonal diversity score of less than about 0.1, as measured by Simpsons
clonality. In some aspects,
the clonal diversity of TILs cultured according to the methods disclosed
herein have a clonal
diversity score of less than about 0.09, as measured by Simpsons clonality. In
some aspects, the
clonal diversity of TILs cultured according to the methods disclosed herein
have a clonal diversity
score of less than about 0.08, as measured by Simpsons clonality. In some
aspects, the clonal
diversity of TILs cultured according to the methods disclosed herein have a
clonal diversity score
of less than about 0.07, as measured by Simpsons clonality. In some aspects,
the clonal diversity
of TILs cultured according to the methods disclosed herein have a clonal
diversity score of less
than about 0.06, as measured by Simpsons clonality. In some aspects, the
clonal diversity of TILs
cultured according to the methods disclosed herein have a clonal diversity
score of less than about
0.05, as measured by Simpsons clonality.
[0214] In some aspects, the present disclosure includes a method
of expanding TILs
obtained from a human subject comprising:
a. culturing the TILs in initial TIL culture media ("Initial TIL Culturing");
b. culturing the TTLs in secondary TIL culture media ("Second TIE Culturing");
and
c. culturing the TILs in third (or final) TIL culture media ("Final TIL
Culturing"),
wherein the initial TIL culture media, the secondary TIL culture media, and/or
the third
TIL culture media are hyperkalemic. In some aspects, the Final TIL Culturing
further
comprises T cell stimulation or activation. In some aspects, the Second TIL
Culturing
further comprises T cell stimulation or activation.
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[0215] In some aspects, the present disclosure includes a method
of expanding Tits
obtained from a human subject comprising:
a. culturing the TILs in initial TIL culture media ("Initial TIL Culturing");
and
b. expanding the TILs in secondary TIL culture media (-Second TIL Expansion");
wherein the initial TIL culture media and/or the secondary TIL culture media
are hyperkalemic.
[0216] In some aspects, the present disclosure includes a method
of expanding Tits
obtained from a human subject comprising:
a. culturing the Tits in initial TIL culture media ("Initial TIL Culturing");
b. expanding the TILs in secondary TM culture media ("Second TM Expansion");
and
c. expanding the TILs in third (or final) TIL culture media ("Final TM
Expansion"),
wherein the initial TM culture media, the secondary TM culture media, and/or
the third TIL culture
media are hyperkalemic.
[0217] In some aspects, only the initial TIL culture media are
hyperkalemic. In some
aspects, only the secondary TIL culture media are hyperkalemic. In some
aspects, both the initial
TIL culture media and the secondary TIL culture media are hyperkalemic. In
some aspects, the
initial TM culture media and the secondary TIL culture media are hyperkalemic
and the third TM
culture media are not hyperkalemic. In some aspects, the initial TM culture
media further
comprises IL-2, IL-21, or both. In some aspects the initial TIL culture, the
secondary TIL culture
and the third or final TM culture comprises IL-2 with or without IL-21.
[0218] In some aspects, the initial TM culture media, the
secondary TIL culture and/or the
third or final TIL culture further comprises a T cell supplement, a serum
replacement, glutamine,
a glutamine substitute (e.g., Glutamax (L-alanine-L-glutamine)), non-essential
amino acids, an
antibiotics (e.g., Penicillin, Streptomycin, or both), an anti-fungal agent
(e.g., FUNGINTm), and/or
sodium pyruvate.
[0219] In some aspects, the TILs are cultured in the initial TIL
culture media up to about
six days, about seven days, about eight days, about nine 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
or about 19 days. In some aspects the TILs are cultured in the initial TM
culture media for about
14 days to about 19 days.
[0220] In some aspects, the TILs in the second TM Culturing are
stimulated with a CD3
agonist, a CD28 agonist, or both in the secondary TIL culture media in (b). In
some aspects, the
T1Ls in the second TM Culturing are further stimulated with a CD27 ligand in
the secondary TIL
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culture media. In some aspects, the TILs in the second TIL Culturing are
further stimulated with a
4-1BB ligand in the secondary TIL culture media.
[0221] In some aspects, the TILs in the second TIL Expansion are
cultured for at least
about 6 days, at least about 7 days, at least about 8 days, at least about 9
days, at least about 10
days, at least about 11 days after the stimulation or activation.
[0222] In some aspects, the TILs in the second TIL Expansion are
cultured for about 6 days
to about 12 days, about 7 days to about 11 days, about 7 days to about 10
days, about 8 days to
about 12 days, after stimulation or activation.
[0223] In some aspects, the TILs in the third or final TlL
Expansion are cultured for at least
about 7 days, at least about 8 days, at least about 9 days, at least about 10
days, at least about 11
days, at least about 12 days, at least about 13 days, at least about 14 days,
at least about 15 days
after the second stimulation or activation.
[0224] In some aspects, the TILs in the third or final TIL
Expansion are cultured for about
7 days to about 14 days, about 7 days to about 12 days, about 7 days to about
11 days, about 8 days
to about 14 days, about 8 days to about 13 days, about 8 days to about 12
days, after the second
stimulation or activation.
[0225] The present disclosure also provides culturing the TILs
in the metabolic
reprogramming media disclosed herein, the cell culture disclosed herein, or
the cell bag or
bioreactor disclosed herein as an initial TIL culture. In some aspects, the
initial TIL culture
culturing is maintained for at least about six days, at least about seven
days, at least about eight
days, at least about 9 days, at least about 10 days, at least about 11 days,
at least about 12 days, at
least about 13 days, at least about 14 days, at least about 15 days, at least
about 16 days, at least
about 17 days, at least about 18 days, at least abour 19 days. In some
aspects, the initial TIL culture
culturing is maintained for 14 days to about 19 days.
[0226] The present methods can further be developed into a
secondary TIL expansion. In
order to start a secondary TIE expansion, the TILs are stimulated or activated
with a CD3 agonist
and/or a CD28 agonist, e.g., TRANSACTTm. In some aspects, the TILs in the
media are further
stimulated with a CD27 ligand. In some aspects, the TILs in the media are
further stimulated with
a 4-1BB ligand. In some aspects, the second TIL expansion is maintained for at
least about 6 days,
at least about 7 days, at least about 8 days, at least about 9 days, at least
about 10 days, at least
about 11 days. In some aspects, the secondary TIL expansion culturing is
maintained for about 7
days (about one week).
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[0227] In some aspects, the Tits are cultured in secondary TIL
culture media until cell
yield in the secondary expansion reaches at least about 1x107 to at least
about 50x107, at least about
2x107 to at least about 40x107, at least about 3x107 to at least about 30x107,
at least about 4x107 to
at least about 25x107, at least about 5x107 to at least about 20x107, at least
about 1x107 to at least
about 20x107, at least about 2x107 to at least about 20x107, at least about
3x107 to at least about
20x107, or at least about 4x107 to at least about 20x107 cells. In some
aspects, the TILs are cultured
in secondary TIL culture media until cell yield in the secondary expansion
reaches at least about
5x107 to at least about 20x107 cells. In some aspects, the TILs are cultured
in secondary T1L culture
media until cell yield in the secondary expansion reaches at least about lx
i07, at least about 2x107,
at least about 3x107, at least about 4x107, at least about 5x107, at least
about 6x107, at least about
7x107, at least about 8x107, at least about 9x107, at least about 10x107, at
least about 11x107, at
least about 12x107, at least about 13x107, at least about 14x107, at least
about 15x107, at least about
16x107, at least about 17x107, at least about 18x107, at least about 19x107,
or at least about 20x107
cells. In some aspects, the TILs are cultured in secondary TIL culture media
until cell yield in the
secondary expansion reaches at least about 5x107 cells. In some aspects, the
TILs are cultured in
secondary TIL culture media until cell yield in the secondary expansion
reaches at least about
6x107 cells. In some aspects, the TILs are cultured in secondary TIL culture
media until cell yield
in the secondary expansion reaches at least about 7x107 cells. In some
aspects, the TILs are cultured
in secondary TIL culture media until cell yield in the secondary expansion
reaches at least about
8x107 cells. In some aspects, the TILs are cultured in secondary TIL culture
media until cell yield
in the secondary expansion reaches at least about 9x107 cells. In some
aspects, the TILs are cultured
in secondary TIL media until cell yield in the secondary expansion reaches at
least about 10x107
cells. In some aspects, the TILs are cultured in secondary TIL culture media
until cell yield in the
secondary expansion reaches at least about 15x107 cells. In some aspects, the
TILs are cultured in
secondary TIL culture media until cell yield in the secondary expansion
reaches at least about
20x107 cells.
[0228] After the secondary TIL expansion, the TfLs can be
expanded further in the final
expansion stage. In order to start the final TIL expansion, the TILs from the
second TIL expansion
culture are transferred to control media (i.e., non-hyperkalemic media). At
the start of the final TIL
expansion culture, the TILs are further stimulated with a CD3 agonist and/or a
CD28 agonist e.g.,
TRANSACTTm. In some aspects, the TILs in the media are further stimulated with
a CD27 ligand.
In some aspects, the TILs in the media are further stimulated with a 4-1BB
ligand.
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[0229] In some aspects, the Tits are cultured in secondary TIL
culture media until cell
yield in the secondary expansion reaches at least about 1x107 to at least
about 50x107, at least about
2x107 to at least about 40x107, at least about 3x107 to at least about 30x107,
at least about 4x107 to
at least about 25x107, at least about 5x107 to at least about 20x107, at least
about 1x107 to at least
about 20x107, at least about 2x107 to at least about 20x107, at least about
3x107 to at least about
20x107, or at least about 4x107 to at least about 20x107 cells. In some
aspects, the TILs are cultured
in secondary TIL culture media until cell yield in the secondary expansion
reaches at least about
5x107 to at least about 20x107 cells. In some aspects, the TThs are cultured
in secondary T1L culture
media until cell yield in the secondary expansion reaches at least about lx
i07, at least about 2x107,
at least about 3x107, at least about 4x107, at least about 5x107, at least
about 6x107, at least about
7x107, at least about 8x107, at least about 9x107, at least about 10x107, at
least about 11x107, at
least about 12x107, at least about 13x107, at least about 14x107, at least
about 15x107, at least about
16x107, at least about 17x107, at least about 18x107, at least about 19x107,
or at least about 20x107
cells. In some aspects, the TILs are cultured in secondary TIL culture media
until cell yield in the
secondary expansion reaches at least about 5x107 cells. In some aspects, the
TILs are cultured in
secondary TIL culture media until cell yield in the secondary expansion
reaches at least about
6x107 cells. In some aspects, the TILs are cultured in secondary TIL culture
media until cell yield
in the secondary expansion reaches at least about 7x107 cells. In some
aspects, the TILs are cultured
in secondary TIL culture media until cell yield in the secondary expansion
reaches at least about
8x107 cells. In some aspects, the TILs are cultured in secondary TIL culture
media until cell yield
in the secondary expansion reaches at least about 9x107 cells. In some
aspects, the TILs are cultured
in secondary TIL culture media until cell yield in the secondary expansion
reaches at least about
10x107 cells. In some aspects, the TILs are cultured in secondary TIL culture
media until cell yield
in the secondary expansion reaches at least about 15x107 cells. In some
aspects, the TILs are
cultured in secondary TIL culture media until cell yield in the secondary
expansion reaches at least
about 20x107 cells.
[0230] In some aspects, TILs are subjected to a final expansion.
In some aspects, the final
expansion comprises a stimulation. In some aspects the stimulation is the same
as the stimulation
used during the secondary expansion. In some aspects, the TILs are stimulated
during the final
expansion by culturing the cells in a medium comprising TRANSACTTm with or
without 4-1BBL
and/or CD27L. In some aspects, the TILs are stimulated during the final
expansion by culturing
the cells in a medium comprising TRANSACTTm and 4-1BBL and/or CD27L In some
aspects,
the TILs are stimulated during the final expansion by culturing the cells in a
medium comprising
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at least about 1:100 TRANSACTTm, at least about 1 mg/m1 4-1BBL, and at least
about 5 ig/m1
CD27L.
[0231] In some aspects, the final expansion step is carried out
in static GREX. In some
aspects, the final expansion is carried out in a stirred tank. In some
aspects, the final expansion is
continued until the cell yield in the final TIL culture media reaches at least
about 40x109 to at least
about 100x109, at least about 40x109 to at least about 90x109, at least about
40x109 to at least about
80x109, at least about 40x109 to at least about 70x109, atleast about 40x109
to at least about 60x109,
at least about 40x109 to at least about 50x109, at least about 10x109 to at
least about 100x109, at
least about 20x109 to at least about 100x109, at least about 30x109 to at
least about 100x109, at least
about 30x109 to at least about 50x109, or at least about 35x109 to at least
about 45x109 cells. In
some aspects, the final expansion is continued until the cell yield in the
final TIL culture media
reaches at least about 40x109 to at least about 100x109 cells. In some
aspects, the final expansion
is continued until the cell yield in the final TIL culture media reaches at
least about 40x109, at least
about 45x109, at least about 50x109, at least about 55x109, at least about
60x109, at least about
65x109, at least about 70x109, at least about 75x109, at least about 80x109,
at least about 85x109,
at least about 90x109, at least about 95x109, or at least about 100x109 cells.
In some aspects, the
final expansion is continued until the cell yield in the final TIL culture
media reaches at least about
40x109 cells. In some aspects, the final expansion is continued until the cell
yield in the final TIL
culture media reaches at least about 50x109 cells. In some aspects, the final
expansion is continued
until the cell yield in the final TIL culture media reaches at least about
60x109 cells. In some
aspects, the final expansion is continued until the cell yield in the final
TIL culture media reaches
at least about 70x109 cells. In some aspects, the final expansion is continued
until the cell yield in
the final TIL culture media reaches at least about 80x109 cells. In some
aspects, the final expansion
is continued until the cell yield in the final T1L culture media reaches at
least about 90x109 cells.
In some aspects, the final expansion is continued until the cell yield in the
final TIL culture media
reaches at least about 100x109 cells.
[0232] In some aspects, the final expansion is continued until
the cell yield in the final TIL
culture media for at least about 7 to at least about 21 days. In some aspects,
the final expansion is
continued until the cell yield in the final TIL culture media for at least
about 7 days. In some
aspects, the final expansion is continued until the cell yield in the final
TIL culture media for at
least about 8 days. In some aspects, the final expansion is continued until
the cell yield in the final
TIT, culture media for at least about 9 days. In some aspects, the final
expansion is continued until
the cell yield in the final TIL culture media for at least about 10 days. In
some aspects, the final
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expansion is continued until the cell yield in the final TIL culture media for
at least about 11 days.
In some aspects, the final expansion is continued until the cell yield in the
final TIL culture media
for at least about 12 days. In some aspects, the final expansion is continued
until the cell yield in
the final TIL culture media for at least about 13 days. In some aspects, the
final expansion is
continued until the cell yield in the final TIL culture media for at least
about 14 days. In some
aspects, the final expansion is continued until the cell yield in the final
TIL culture media for at
least about 15 days. In some aspects, the final expansion is continued until
the cell yield in the final
TIL culture media for at least about 16 days. In some aspects, the final
expansion is continued until
the cell yield in the final Tit culture media for at least about 17 days. In
some aspects, the final
expansion is continued until the cell yield in the final TIL culture media for
at least about 18 days.
In some aspects, the final expansion is continued until the cell yield in the
final TIL culture media
for at least about 19 days. In some aspects, the final expansion is continued
until the cell yield in
the final TIL culture media for at least about 20 days. In some aspects, the
final expansion is
continued until the cell yield in the final TIL culture media for at least
about 21 days.
[0233] In some aspects, the hyperkalemic medium is not
hypotonic. In some aspects, the
hyperkalemic medium is not isotonic. In some aspects, the hyperkalemic medium
is not hypertonic.
[0234] In some aspects, the heterogeneous population of TILs
comprises CD4+ TILs and
CD8+ TILs. In some aspects, the heterogeneous population of TILs is obtained
from one or more
tumor sample obtained from a subject. Any tumor sample obtained from a subject
can be used in
the methods disclosed herein. In some aspects, the tumor sample comprises a
tumor biopsy. In
some aspects, the tumor biopsy comprises a punch biopsy. In some aspects, the
tumor sample
comprises tumor tissue obtained during a tumor resection surgery. In some
aspects, the tumor
sample comprises a core needle biopsy. In some aspects, the tumor sample is
collected taken from
an inflamed tumor, e.g., a tumor comprising a high number of TILs.
[0235] In some aspects, the tumor sample is plated and subjected
to an initial TIL culture.
In some aspects, the initial TIL culture comprises culturing the tumor sample
in the metabolic
reprogramming medium, e.g., hyperkalemic medium. Any methods for TIL expansion
from a
tumor sample can be used in the methods disclosed herein. In some aspects, the
tumor sample is
fractionated prior to plating and initial TIL culture. In some aspects, the
initial TIL culture lasts for
at least about 7 days, at least about 8 days, at least about 9 days, at least
about 10 days, at least
about 11 days, at least about 12 days, at least about 13 days, at least about
14 days, at least about
15 days, at least about 16 days, at least about 17 days, at least about 18
days, at least about 19 days,
at least about 20 days, at least about 21 days, at least about 22 days, at
least about 23 days, at least
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about 24 days, at least about 25 days, at least about 26 days, at least about
27 days, or at least about
28 days. In some aspects, the initial TIL culture lasts at least about 14 days
to about 19 days. In
some aspects the initial TIL culture lasts at least about 14 days.
[0236] In some aspects, the proportion of CDS+ TILs (e.g., tumor
reactive CD8+ TILs
and/or stem-like CD8+ TILs) to non-CD8+ Tits is increased following the
initial TIL culture, as
compared to the proportion of CD8+ Tits to non-CD8+ TILs prior to the initial
TIL culture. In
some aspects, the proportion of CD8 TILs (e.g., tumor reactive CD8+ TILs
and/or stem-like CD8+
TILs) to non-CD8+ TILs is increased by at least about 1.5-fold, at least about
2-fold, at least about
3-fold, at least about 3.5-fold, at least about 4-fold, at least about 4.5-
fold, at least about 5-fold, at
least about 6-fold, at least about 7-fold, at least about 8-fold, at least
about 9-fold, at least about
10-fold, at least about 15-fold, at least about 20-fold, at least about 25-
fold, at least about 30-fold,
at least about 35-fold, at least about 40-fold, at least about 45-fold, at
least about 50-fold, at least
about 60-fold, at least about 70-fold, at least about 80-fold, at least about
90-fold, or at least about
100-fold. In some aspects, the proportion of CD8+ TILs (e.g., tumor reactive
CD8+ TILs and/or
stem-like CD8+ TILs) to non-CD8+ TILs is increased by at least about 50-fold.
[0237] In some aspects, following culture of the heterogeneous
population of TILs, at least
about 10%, at least about 20%, at least about 25%, at least about 30%, at
least about 35%, at least
about 40%, at least about 45%, at least about 50%, at least about 55%, at
least about 60%, at least
about 65%, at least about 70%, at least about 75%, or at least about 80% of
the TILs in the
population are CD8+ TILs (e.g., tumor reactive CD8+ TILs and/or stem-like CD8+
TILs). In some
aspects, following culture of the heterogeneous population of TILs, at least
about 50% of the TILs
in the population are CD8+ TILs (e.g., tumor reactive CD8+ TILs and/or stem-
like CD8+ TILs).
In some aspects, following culture of the heterogeneous population of TILs, at
least about 25% of
the TILs in the population are CD8+ TILs (e.g., tumor reactive CD8+ TILs
and/or stem-like CD8+
TILs). In some aspects, following culture of the heterogeneous population of
TILs, at least about
75% of the TILs in the population are CD8 TILs (e.g., tumor reactive CD8+
TILs and/or stem-
like CD8+ TILs).
[0238] In some aspects, the TILs are stimulated or activated
following the initial TIL
culture. Any methods for expansion and/or stimulation of TILs can be used
during the stimulation
of the TILs. In some aspects, the TILs are stimulated following the initial
TIL culture. In some
aspects, the TILs are stimulated by subjecting the TILs to TRANSACTTm TIL
expansion, TIL
rapid expansion protocol, or a combination thereof. In some aspects, the TILs
are stimulated in a
hyperkalemic medium disclosed herein.
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[0239] In some aspects, a population of immune cells, e.g., Tits
(e.g., CD8+ TILs (e.g.,
tumor reactive CD8+ TILs)), cultured using the methods disclosed herein
exhibits an increased
number of stem-like Tits relative to a population of cells cultured using
conventional methods,
e.g., in a medium having less than about 40 mM potassium ion. In some aspects,
the immune cells,
e.g., TILs (e.g., CDS+ TILs (e.g., tumor reactive CD8+ TILs)), exhibit
increased expression of
markers characteristic of stem-like cells relative to the starting population
of cells. In some aspects,
the starting population of cells comprises cells obtained from a human
subject. In some aspects,
the starting population of cells comprises TILs obtained from a human subject.
[0240] Increased cell multipotency can be measured using any
methods. In some aspects,
cell sternness is measured by antibody staining followed by gated flow
cytometry. In some aspects,
the cell sternness is measured by autophagy flux_ In some aspects, the cell
sternness is measured
by glucose uptake. In some aspects, the cell sternness is measured by fatty
acid uptake. In some
aspects, the cell sternness is measured by mitochondrial biomass. In some
aspects, the cell sternness
is measured by RNA quantification/expression analysis (e.g., microarray, qPCR
(TaqMan), RNA-
Seq., single-cell RNA-Seq., or any combinations thereof). In some aspects, the
cell sternness is
measured by (e.g., transcripts that are linked to) a metabolism assay (e.g., a
Seahorse metabolism
assay, analysis of extracellular acidification rate (ECAR); analysis of oxygen
consumption rate
(OCR); analysis of spare respiratory capacity; and/or analysis of
mitochondrial membrane
potential). In some aspects, sternness is measured using one or more in vivo
functional assays (e.g.,
assaying cell persistence, antitumor capacity, antitumor clearance, viral
clearance, multipotency,
cytokine release, cell killing, or any combination thereof).
[0241] In some aspects, the differentiation status of the immune
cells, e.g., TILs (e.g., CD8+
TILs (e.g., tumor reactive CD8+ TILs)), is characterized by increased numbers
of cells expressing
markers typical of less differentiated cells. In some aspects, an increase in
the number of stem-like
immune cells, e.g., TILs (e.g., CD8+ TILs (e.g., tumor reactive CD8+ TILs)),
is characterized by
increased numbers of immune cells, e.g., TILs (e.g., CD8+ TILs (e.g., tumor
reactive CD8+ TILs)),
expressing markers typical of TN and/or Tscm cells. In some aspects, an
increase in the number of
stem-like immune cells, e.g., TILs (e.g., CDS+ TILs (e.g., tumor reactive CD8+
TILs)), is
characterized by increased numbers of immune cells, e.g., TILs (e.g., CD8
TILs (e.g., tumor
reactive CD8+ TILs)), expressing markers typical of Tscm cells. In some
aspects, the population
of immune cells, e.g., TILs (e.g., CD8+ TILs (e.g., tumor reactive CD8+
TILs)), exhibits an
increased number of immune cells, e.g., TILs (e.g., CD8 TILs (e.g., tumor
reactive CD8+ TILs)),
that express CD45RA. In some aspects, the population of immune cells, e.g.,
TILs (e.g., CD8+
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TILs (e.g., tumor reactive CD8+ TILs)), exhibits an increased number of immune
cells, e.g., TILs
(e.g., CD8+ TILs (e.g., tumor reactive CD8+ TILs)), that express CCR7. In some
aspects, the
population of TILs exhibits an increased number of immune cells, e.g., TILs
(e.g., CD8+ TILs (e.g.,
tumor reactive CD8+ TILs)), that express CD62L. In some aspects, the
population of TILs exhibits
an increased number of immune cells, e.g., TILs (e.g., CD8+ TILs (e.g., tumor
reactive CD8+
TILs)), that express CD28. In some aspects, the population of immune cells,
e.g., Tits (e.g., CD8+
TILs (e.g., tumor reactive CD8+ TILs)), exhibits an increased number of immune
cells, e.g., TILs
(e.g., CDS+ TILs (e.g., tumor reactive CD8+ Tits)), that express CD95. In some
aspects, the
immune cells, e.g., TILs (e.g., CD8+ TILs (e.g., tumor reactive CD8+ TILs)),
are CD45R010. In
some aspects, the immune cells, e.g., TILs (e.g., CD8+ TILs (e.g, tumor
reactive CD8+ TILs)), do
not express CD45RO. In some aspects, the population of immune cells, e.g.,
TILs (e.g., CD8+ TILs
(e.g., tumor reactive CD8+ TILs)), exhibits an increased number of immune
cells, e.g., TILs (e.g.,
CDS+ TILs (e.g., tumor reactive CD8+ TILs)), that are CD8+, CD45RA+, CCR7+,
and CD62L+. In
some aspects, the population of immune cells, e.g., TILs (e.g., CD8 TILs
(e.g., tumor reactive
CD8+ TILs)), exhibits an increased number of immune cells, e.g-., TILs
CD8+ TILs (e.g.,
tumor reactive CD8+ TILs)), that are CD8', CD95', CD45RAt CCR7+, and CD62L+.
In some
aspects, the population of immune cells, e.g., TILs (e.g., CD8 TILs (e.g.,
tumor reactive CD8+
TILs)), exhibits an increased number of cells that express TCF7. In some
aspects, the population
of immune cells, e.g., TILs (e.g., CD8+ TILs (e.g., tumor reactive CD8+
TILs)), exhibits an
increased number of immune cells, e.g., TILs (e.g., CD8 TILs (e.g., tumor
reactive CD8+ TILs)),
that are CD8-, CD45RA+, CCR7+, CD62L+, and TCF7+. In some aspects, the
population of
immune cells, e.g., TILs (e.g., CD8+ TILs (e.g., tumor reactive CD8+ TILs)),
exhibits an increased
number of immune cells, e.g., TILs (e.g., CD8 TILs (e.g., tumor reactive CD8+
TILs)), that are
CD8+, CD95+, CD45RA , CCR7+, CD62L+, and TCF7+. In some aspects, the immune
cells, e.g.,
TILs (e.g., CD8+ TILs (e.g., tumor reactive CD8+ TILs)), express CD3. In some
aspects, the
population of immune cells, e.g., TILs (e.g., CD8+ TILs (e.g., tumor reactive
CD8+ TILs)), exhibits
an increased number of immune cells, e.g., TILs (e.g., CD8 TILs (e.g., tumor
reactive CD8+
TILs)), that are CD8+, CD3+, CD45RA+, CCR7+, CD62L+, TCF7 . In some aspects,
the population
of immune cells, e.g., TILs (e.g., CD8 TILs (e.g., tumor reactive CD8+
TILs)), exhibits an
increased number of immune cells, e.g., TILs (e.g., CD8' TILs (e.g., tumor
reactive CD8+ TILs)),
that are CDS+, CD3+, CD95+, CD45RA+, CCR7+, CD62L+, TCF7 . In some aspects,
the immune
cells, e.g., Tits (e.g., CD8+ TILs (e.g., tumor reactive CD8+ TILs)), express
CD27. In some
aspects, the population of immune cells, e.g., TILs (e.g., CD8' TILs (e.g.,
tumor reactive CD8+
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TILs)), exhibits an increased number of immune cells, e.g., TILs (e.g., CD8+
TILs (e.g., tumor
reactive CD8+ TILs)), that are CD8+, CD27+, CD3+, CD95-, CD45RA , CCR7+,
CD62L+, TCF7 .
In some aspects, the population of immune cells, e.g., TILs (e.g., CDS+ TILs
(e.g., tumor reactive
CD8+ TILs)), exhibits an increased number of immune cells, e.g., TILs (e.g.,
CDS+ TILs (e.g.,
tumor reactive CD8+ TILs)), that are CDS+, CD27 , CD3+, CD95 , CD45RA+, CCR7+,
CD62L+,
TC177+. In some aspects, the population of immune cells, e.g., TILs (e.g.,
CDS+ TILs (e.g., tumor
reactive CD8+ TILs)), exhibits an increased number of Tscm cells. In some
aspects, the population
of immune cells, e.g., TILs (e.g., CD8+ TILs (e.g., tumor reactive CD8+
Tits)), exhibits an
increased number of TN cells. In some aspects, the population of immune cells,
e.g., TILs (e.g.,
CD8+ TILs (e.g., tumor reactive CD8+ TILs)), exhibits an increased number of
Tscm and TN cells.
In some aspects, the population of cell exhibits an increased number of stem-
like TILs
[0242]
In some aspects, the number of stem-like immune cells, e.g., TILs
(e.g., CD8+ TILs
(e.g., tumor reactive CD8+ TILs)), in the culture is increased by at least
about 5%, at least about
10%, at least about 15%, at least about 20%, at least about 25%, at least
about 30%, at least about
35%, at least about 40%, at least about 45%, at least about 50%, at least
about 60%, at least about
70%, at least about 80%, at least about 90%, or at least about 100%, relative
to the number of stem-
like immune cells, e.g., TILs (e.g., CD8+ TILs (e.g, tumor reactive CD8+
TILs)), prior to culture.
In some aspects, the number of stem-like immune cells, e.g., TILs (e.g., CDS+
TILs (e.g., tumor
reactive CD8+ TILs)), in the culture is increased by at least about 1.5-fold,
at least about 2-fold, at
least about 2.5-fold, at least about 3-fold, at least about 3.5-fold, at least
about 4-fold, at least about
4.5-fold, at least about 5-fold, at least about 6-fold, at least about 7-fold,
at least about 8-fold, at
least about 9-fold, at least about 10-fold, at least about 15-fold, or at
least about 20-fold, relative
to the number of stem-like immune cells, e.g., TILs (e.g., CD8 TILs (e.g,
tumor reactive CD8+
TILs)), prior to culture.
[0243]
In some aspects, following culture of immune cells, e.g., TILs (e.g.,
CD8+ TILs
(e.g., tumor reactive CD8+
s)), according to the methods disclosed herein, stem-like CDS+ Tits
(e.g., stem-like tumor reactive CD8+ TILs) constitute at least about 1%, at
least about 2%, at least
about 3%, at least about 4%, at least about 5%, at least about 10%, at least
about 15%, of the total
number of CD8+ TILs in the culture.
[0244]
In some aspects, following culture of TILs according to the methods
disclosed
herein, stem-like TILs constitute at least about 10% to at least about 70% of
the total number of
TILs in the culture. In some aspects, following culture of TILs according to
the methods disclosed
herein, stem-like TILs constitute at least about 10%, at least about 20%, at
least about 30%, at least
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about 40%, at least about 50%, at least about 60%, or at least about 70% of
the total number of
CD8+ TILs in the culture. In some aspects, following culture of TILs according
to the methods
disclosed herein, stem-like TILs constitute at least about 10%, at least about
20%, at least about
30%, at least about 40%, at least about 50%, at least about 60%, or at least
about 70% of the total
number of CD4+ TILs in the culture.
[0245] In some aspects, following culture of TILs according to
the methods disclosed
herein, at least about 10% to at least about 40% of the total number of TILs
in the culture are CD39-
/CD69- Tits. In some aspects, following culture of TThs according to the
methods disclosed herein,
at least about 10%, at least about 15%, at least about 20%, at least about
25%, at least about 30%,
at least about 35%, or at least about 40% of the total number of TILs in the
culture are CD39-
/CD69- TILs.
[0246] In some aspects, following culture of TILs according to
the methods disclosed
herein, at least about 10% to at least about 70% of the total number of TILs
in the culture are CD39-
/TCF7+ TILs. In some aspects, following culture of Tits according to the
methods disclosed herein,
at least about 10%, at least about 15%, at least about 20%, at least about
25%, at least about 30%,
at least about 35%, or at least about 40% of the total number of TILs in the
culture are CD39-
/TCF7+ TILs. In some aspects the TILs are CD4+ T cells. In some aspects the
TILs are CD8+ TILs.
[0247] In some aspects, upon adoptive transfer of the immune
cells, e.g., TILs (e.g., CD8+
TILs (e.g., tumor reactive CD8+ TILs)), cultured according to the methods
disclosed herein, the
transferred cells exhibit decreased cell exhaustion, as compared to cells
cultured using
conventional culture conditions. In some aspects, upon adoptive transfer of
the cultured TILs, the
transferred CD8tenriched TILs persist for a longer period of time in vivo, as
compared to TILs
cultured using conventional culture conditions. Such increased persistence
refers to the ability of
the TIL to infilitrate and function in the tumor microenvironment, ability to
resist exhaustion, and
the persistence of sternness to ensure continued expansion and durability of
response. In some
aspects, immune cells, e.g. T cells, cultured according to the methods
disclosed herein, are stem-
like cells. Such cells are capable of self-renewal, proliferation and
differentiation. In some aspects,
immune cells, e.g. T cells, cultured according to the methods disclosed
herein, are stem-like cells
which also express effector-like markers. In some aspects, immune cells, e.g.
T cells, cultured
according to the methods disclosed herein, are stem-like cells which also
maintain the ability to
target and kill tumor cells.
[0248] In some aspects, the transferred CD8 -enriched TILs, have
a greater in vivo
efficacy, e.g., tumor-killing activity, as compared to TILs cultured using
conventional culture
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conditions. In some aspects, a lower dose of the CD8tenriched TILs cultured
according to the
methods disclosed herein is needed to elicit a response, e.g., decreased tumor
volume, in a subject
as compared to cells cultured using conventional culture conditions.
[0249] In some aspects, the TILs are cultured in the metabolic
reprogramming media, e.g.,
hyperkalemic medium disclosed herein for the entirety of ex vivo culture,
e.g., from the time the
tumor sample is first plated through the entire expansion process, and until
administration. In some
aspects, the TILs are cultured in the medium disclosed herein for the duration
of expansion.
[0250] In some aspects, the metabolic reprogramming media, e.g.,
hyperkalemic culture
medium comprises a mitochondrial fuel. In some aspects, the metabolic
reprogramming media,
e.g., hyperkalemic culture medium, comprises 0-Acetyl-L-carnitine
hydrochloride. In some
aspects, the metabolic reprogramming media, e.g., hyperkalemic culture medium,
comprises at
least about 0.1 mM, at least about 0.5 mM, at least about 1.0 mM, at least
about 5 mM, or at least
about 10 mM O-Acetyl-L-carnitine hydrochloride. In some aspects, the metabolic
reprogramming
media, e.g., hyperkalemic culture medium, comprises at least about 1.0 mM O-
Acetyl-L-carnitine
hydrochloride.
[0251] In some aspects, the metabolic reprogramming media, e.g.,
hyperkalemic culture
medium, comprises inhibitor of glycolysis-mediated metabolism, e.g., a kinase
inhibitor, e.g., a
phosphoinositide 3-kinase inhibitor. In some aspects, the metabolic
reprogramming media, e.g.,
hyperkalemic culture medium, comprises a phosphatidylinosito1-3- kinase (PI3K)
inhibitor, e.g.,
idelalisib (e.g., CAL-101; Selleckchem). In some aspects, the metabolic
reprogramming media,
e.g., hyperkalemic culture medium, comprises at least about 0.1 mM, at least
about 0.5 mM, at
least about 1.0 mM, at least about 5 mM, or at least about 10 mM idelalisib.
In some aspects, the
metabolic reprogramming media, e.g., hyperkalemic culture medium, comprises at
least about 1.0
mM idelalisib.
[0252] In some aspects, the metabolic reprogramming media, e.g.,
hyperkalemic culture
medium, further comprises one or more of (i) one or more cell expansion
agents, (ii) sodium ion,
(iii) one or more saccharides, (iv) calcium ion, and (v) one or more
cytokines.
II.A. Potassium
[0253] Some aspects of the present disclosure are directed to
methods of culturing TILs ex
vivo or in vitro comprising placing a heterogeneous population of TILs in a
metabolic
reprogramming media, e.g., hyperkalemic medium. Some aspects of the present
disclosure are
directed to methods of increasing a number or percentage of CD8+ TILs ex vivo
or in vitro
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comprising culturing a heterogeneous population of TILs in a metabolic
reprogramming media,
e.g., hyperkalemic medium. Other aspects of the present disclosure are
directed to methods of
preparing a CD8+-enriched population of tumor infiltrating lymphocytes (TILs),
comprising
culturing a heterogeneous population of TILs ex vivo or in vitro in a
metabolic reprogramming
media, e.g., hyperkalemic medium. In some aspects, the concentration of
potassium ion is at least
about 30 mM to at least about 100 mM. In some aspects, the concentration of
potassium ion is at
least about 30 mM, at least about 35 mM, at least about 40 mM, at least about
45 mM, at least
about 50 mM, at least about 55 mM, at least about 60 mM, at least about 65 mM,
at least about 70
mM, at least about 75 mM, at least about 80 mM, at least about 85 mM, at least
about 90 mM, at
least about 95 mM, or at least about 100 Mm. In some aspects, the
concentration of potassium ion
is at least about 50 mM. In some aspects, the concentration of potassium ion
is about 40 mM. In
some aspects, the concentration of potassium ion is about 45 mM. In some
aspects, the
concentration of potassium ion is about 50 mM.
[0254] In some aspects, the concentration of potassium ion is at
least about 55 mM, at least
about 60 mM, at least about 65 mM, at least about 70 mM, at least about 75 mM,
at least about 80
mM, at least about 85 mM, at least about 90 mM, at least about 95 mM, or at
least about 100 mM,
at least about 105 mM, at least about 110 mM, at least about 115 mM, at least
about 120 mM. In
some aspects, the concentration of potassium ion is about 55 mM, about 60 mM,
about 65 mM,
about 70 mM, about 75 mM, about 80 mM, about 85 mM, about 90 mM, about 95 mM,
about 100
mM, about 105 mM, about 110 mM, about 115 mM, about 120 mM. In some aspects,
the
concentration of potassium ion is about 55 mM. In some aspects, the
concentration of potassium
ion is about 60 mM. In some aspects, the concentration of potassium ion is
about 65 mM. In some
aspects, the concentration of potassium ion is about 70 mM. In some aspects
the concentration of
potassium ion is about 40 mM to about 90 mM.
[0255] In some aspects, the concentration of potassium ion is
about 40 mM to about 90
mM. In some aspects, the concentration of potassium ion is about 40 mM to
about 85 mM, about
40 mM to about 80 mM, about 40 mM to about 75 mM, about 40 mM to about 70 mM,
about 40
mM to about 65 mM, about 40 mM to about 60 mM, about 40 mM to about 55 mM, or
about 40
mM to about 50 mM. In some aspects, the concentration of potassium ion is
about 50 mM to about
90 mM, about 50 mM to about 85 mM, about 50 mM to about 80 mM, about 50 mM to
about 75
mM, about 50 mM to about 70 mM, about 50 mM to about 65 mM, about 50 mM to
about 60 mM,
or about 50 mM to about 55 mM.
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[0256] In some aspects, the concentration of potassium ion is
about 50 mM to about 100
mM. In some aspects, the concentration of potassium ion is about 50 mM to
about 100 mM, about
50 mM to about 95 mM, about 50 mM to about 90 mM, about 50 mM to about 85 mM,
about 50
mM to about 80 mM, about 50 mM to about 75 mM, about 50 mM to about 70 mM,
about 50 mM
to about 65 mM, about 50 mM to about 60 mM, or about 50 mM to about 55 mM.
[0257] In some aspects, the concentration of potassium ion is
about 55 mM to about 100
mM. In some aspects, the concentration of potassium ion is about 55 mM to
about 100 mM, about
55 mM to about 95 mM, about 55 mM to about 90 mM, about 55 mM to about 85 mM,
about 55
mM to about 80 mM, about 55 mM to about 75 mM, about 55 mM to about 70 mM,
about 55 mM
to about 65 mM, or about 55 mM to about 60 mM.
[0258] In some aspects, the concentration of potassium ion is
about 60 mM to about 100
mM. In some aspects, the concentration of potassium ion is about 60 mM to
about 100 mM, about
60 mM to about 95 mM, about 60 mM to about 90 mM, about 60 mM to about 85 mM,
about 60
mM to about 80 mM, about 60 mM to about 75 mM, about 60 mM to about 70 mM, or
about 60
mM to about 65 mM.
[0259] In some aspects, the concentration of potassium ion is
about 65 mM to about 100
mM. In some aspects, the concentration of potassium ion is about 65 mM to
about 100 mM, about
65 mM to about 95 mM, about 65 mM to about 90 mM, about 65 mM to about 85 mM,
about 65
mM to about 80 mM, about 65 mM to about 75 mM, or about 65 mM to about 70 mM.
[0260] In some aspects, the concentration of potassium ion is
about 70 mM to about 100
mM. In some aspects, the concentration of potassium ion is about 70 mM to
about 100 mM, about
70 mM to about 95 mM, about 70 mM to about 90 mM, about 70 mM to about 85 mM,
about 70
mM to about 80 mM, or about 70 mM to about 75 mM.
[0261] In some aspects, the concentration of potassium ion is
about 75 mM to about 100
mM. In some aspects, the concentration of potassium ion is about 75 mM to
about 100 mM, about
75 mM to about 95 mM, about 75 mM to about 90 mM, about 75 mM to about 85 mM,
or about
75 mM to about 80 mM.
[0262] In some aspects, the concentration of potassium ion is
about 80 mM to about 100
mM. In some aspects, the concentration of potassium ion is about 80 mM to
about 100 mM, about
80 mM to about 95 mM, about 80 mM to about 90 mM, or about 80 mM to about 85
mM.
[0263] In some aspects, the concentration of potassium ion is
about 85 mM to about 100
mM. In some aspects, the concentration of potassium ion is about 85 mM to
about 100 mM, about
85 mM to about 95 mM, or about 85 mM to about 90 mM.
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[0264] In some aspects, the concentration of potassium ion is
about 90 mM to about 100
mM. In some aspects, the concentration of potassium ion is about 90 mM to
about 95 mM.
[0265] In some aspects, the concentration of potassium ion is
about 95 mM to about 100
mM.
[0266] In some aspects, the concentration of potassium ion is
about 50 mM to about 90
mM. In some aspects, the concentration of potassium ion is about 50 mM to
about 80 mM. In some
aspects, the concentration of potassium ion is about 60 mM to about 90 mM. In
some aspects, the
concentration of potassium ion is about 60 mM to about 80 mM. In some aspects,
the concentration
of potassium ion is about 70 mM to about 90 mM. In some aspects, the
concentration of potassium
ion is about 70 mM to about 80 mM. In some aspects, the concentration of
potassium ion is about
80 mM to about 90 mM. In some aspects, the medium is hypertonic. In some
aspects, the medium
is isotonic. In some aspects, the medium comprises at least about 50 mM
potassium ion and less
than about 90 mM NaCl. In some aspects, the total concentration of potassium
ion and NaCl is
between 110 mM and 140 mM.
[0267] In some aspects, the concentration of potassium ion is
about 50 mM to about 55
mM. In some aspects, the concentration of potassium ion is about 55 mM to
about 60 mM. In some
aspects, the concentration of potassium ion is about 60 mM to about 65 mM. In
some aspects, the
concentration of potassium ion is about 65 mM to about 70 mM. In some aspects,
the concentration
of potassium ion is about 70 mM to about 75 mM. In some aspects, the
concentration of potassium
ion is about 75 mM to about 80 mM. In some aspects, the concentration of
potassium ion is about
80 mM to about 85 mM. In some aspects, the concentration of potassium ion is
about 85 mM to
about 90 mM. In some aspects, the concentration of potassium ion is about 90
mM to about 95
mM. In some aspects, the concentration of potassium ion is about 95 mM to
about 100 mM. In
some aspects, the concentration of potassium ion is about 100 mM to about 105
mM. In some
aspects, the concentration of potassium ion is about 105 mM to about 110 mM.
In some aspects,
the concentration of potassium ion is about 110 mM to about 115 mM. In some
aspects, the
concentration of potassium ion is about 115 mM to about 120 mM.
[0268] In some aspects, the concentration of potassium ion is
about 40 mM to about 90
mM. In some aspects, the concentration of potassium ion is about 40 mM to
about 80 mM. In some
aspects, the concentration of potassium ion is about 40 mM to about 70 mM. In
some aspects, the
concentration of potassium ion is about 50 mM to about 90 mM. In some aspects,
the concentration
of potassium ion is about 50 mM to about 80 mM. In some aspects, the
concentration of potassium
ion is about 50 mM to about 70 mM. In some aspects, the concentration of
potassium ion is about
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55 mM to about 90 mM. In some aspects, the concentration of potassium ion is
about 55 mM to
about 80 mM. In some aspects, the concentration of potassium ion is about 55
mM to about 70
mM. In some aspects, the concentration of potassium ion is about 60 mM to
about 90 mM. In some
aspects, the concentration of potassium ion is about 60 mM to about 80 mM. In
some aspects, the
concentration of potassium ion is about 60 mM to about 70 mM. In some aspects,
the concentration
of potassium ion is about 65 mM to about 90 mM. In some aspects, the
concentration of potassium
ion is about 65 mM to about 80 mM. In some aspects, the concentration of
potassium ion is about
65 mM to about 70 mM.
[0269] In some aspects, the concentration of potassium ion is
higher than about 40 mM. In
some aspects, the concentration of potassium ion is about 40 mM. In some
aspects, the
concentration of potassium ion is higher than about 41 mM. In some aspects,
the concentration of
potassium ion is about 41 mM. In some aspects, the concentration of potassium
ion is higher than
about 42 mM. In some aspects, the concentration of potassium ion is about 42
mM. In some
aspects, the concentration of potassium ion is higher than about 43 mM. In
some aspects, the
concentration of potassium ion is about 43 mM. In some aspects, the
concentration of potassium
ion is higher than about 44 mM. In some aspects, the concentration of
potassium ion is about 44
mM. In some aspects, the concentration of potassium ion is higher than about
45 mM. In some
aspects, the concentration of potassium ion is about 45 mM. In some aspects,
the concentration of
potassium ion is higher than about 46 mM. In some aspects, the concentration
of potassium ion is
about 46 mM. In some aspects, the concentration of potassium ion is higher
than about 47 mM. In
some aspects, the concentration of potassium ion is about 47 mM. In some
aspects, the
concentration of potassium ion is higher than about 48 mM. In some aspects,
the concentration of
potassium ion is about 48 mM. In some aspects, the concentration of potassium
ion is higher than
about 49 mM. In some aspects, the concentration of potassium ion is about 49
mM.
[0270] In some aspects, the concentration of potassium ion is
higher than about 50 mM. In
some aspects, the concentration of potassium ion is about 50 mM. In some
aspects, the
concentration of potassium ion is higher than about 51 mM. In some aspects,
the concentration of
potassium ion is about 51 mM. In some aspects, the concentration of potassium
ion is higher than
about 52 mM. In some aspects, the concentration of potassium ion is about 52
mM. In some
aspects, the concentration of potassium ion is higher than about 53 mM. In
some aspects, the
concentration of potassium ion is about 53 mM. In some aspects, the
concentration of potassium
ion is higher than about 54 mM. In some aspects, the concentration of
potassium ion is about 54
mM. In some aspects, the concentration of potassium ion is higher than about
55 mM. In some
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aspects, the concentration of potassium ion is about 55 mM. In some aspects,
the concentration of
potassium ion is higher than about 56 mM. In some aspects, the concentration
of potassium ion is
about 56 mM. In some aspects, the concentration of potassium ion is higher
than about 57 mM. In
some aspects, the concentration of potassium ion is about 57 mM. In some
aspects, the
concentration of potassium ion is higher than about 58 mM. In some aspects,
the concentration of
potassium ion is about 58 mM. In some aspects, the concentration of potassium
ion is higher than
about 59 mM. In some aspects, the concentration of potassium ion is about 59
mM.
[0271] In some aspects, the concentration of potassium ion is
higher than about 60 mM. In
some aspects, the concentration of potassium ion is about 60 mM. In some
aspects, the
concentration of potassium ion is higher than about 61 mM. In some aspects,
the concentration of
potassium ion is about 61 mM. In some aspects, the concentration of potassium
ion is higher than
about 62 mM. In some aspects, the concentration of potassium ion is about 62
mM. In some
aspects, the concentration of potassium ion is higher than about 63 mM. In
some aspects, the
concentration of potassium ion is about 63 mM. In some aspects, the
concentration of potassium
ion is higher than about 64 mM. In some aspects, the concentration of
potassium ion is about 64
mM. In some aspects, the concentration of potassium ion is higher than about
65 mM. In some
aspects, the concentration of potassium ion is about 65 mM. In some aspects,
the concentration of
potassium ion is higher than about 66 mM. In some aspects, the concentration
of potassium ion is
about 66 mM. In some aspects, the concentration of potassium ion is higher
than about 67 mM. In
some aspects, the concentration of potassium ion is about 67 mM. In some
aspects, the
concentration of potassium ion is higher than about 68 mM. In some aspects,
the concentration of
potassium ion is about 68 mM. In some aspects, the concentration of potassium
ion is higher than
about 69 mM. In some aspects, the concentration of potassium ion is about 69
mM.
[0272] In some aspects, the concentration of potassium ion is
higher than about 70 mM. In
some aspects, the concentration of potassium ion is about 70 mM. In some
aspects, the
concentration of potassium ion is higher than about 71 mM. In some aspects,
the concentration of
potassium ion is about 71 mM. In some aspects, the concentration of potassium
ion is higher than
about 72 mM. In some aspects, the concentration of potassium ion is about 72
mM. In some
aspects, the concentration of potassium ion is higher than about 73 mM. In
some aspects, the
concentration of potassium ion is about 73 mM. In some aspects, the
concentration of potassium
ion is higher than about 74 mM. In some aspects, the concentration of
potassium ion is about 74
mM. In some aspects, the concentration of potassium ion is higher than about
75 mM. In some
aspects, the concentration of potassium ion is about 75 mM. In some aspects,
the concentration of
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potassium ion is higher than about 76 mM. In some aspects, the concentration
of potassium ion is
about 76 mM. In some aspects, the concentration of potassium ion is higher
than about 77 mM. In
some aspects, the concentration of potassium ion is about 77 mM. In some
aspects, the
concentration of potassium ion is higher than about 78 mM. In some aspects,
the concentration of
potassium ion is about 78 mM. In some aspects, the concentration of potassium
ion is higher than
about 79 mM. In some aspects, the concentration of potassium ion is about 79
mM.
[0273] In some aspects, the concentration of potassium ion is
higher than about 80 mM. In
some aspects, the concentration of potassium ion is about 80 mM. In some
aspects, the
concentration of potassium ion is higher than about 81 mM. In some aspects,
the concentration of
potassium ion is about 81 mM. In some aspects, the concentration of potassium
ion is higher than
about 82 mM. In some aspects, the concentration of potassium ion is about 82
mM. In some
aspects, the concentration of potassium ion is higher than about 83 mM. In
some aspects, the
concentration of potassium ion is about 83 mM. In some aspects, the
concentration of potassium
ion is higher than about 84 mM. In some aspects, the concentration of
potassium ion is about 84
mM. In some aspects, the concentration of potassium ion is higher than about
85 mM. In some
aspects, the concentration of potassium ion is about 85 mM. In some aspects,
the concentration of
potassium ion is higher than about 86 mM. In some aspects, the concentration
of potassium ion is
about 86 mM. In some aspects, the concentration of potassium ion is higher
than about 87 mM. In
some aspects, the concentration of potassium ion is about 87 mM. In some
aspects, the
concentration of potassium ion is higher than about 88 mM. In some aspects,
the concentration of
potassium ion is about 88 mM. In some aspects, the concentration of potassium
ion is higher than
about 89 mM. In some aspects, the concentration of potassium ion is about 89
mM.
[0274] In some aspects, the concentration of potassium ion is
higher than about 90 mM. In
some aspects, the concentration of potassium ion is about 90 mM. In some
aspects, the
concentration of potassium ion is higher than about 91 mM. In some aspects,
the concentration of
potassium ion is about 91 mM. In some aspects, the concentration of potassium
ion is higher than
about 92 mM. In some aspects, the concentration of potassium ion is about 92
mM. In some
aspects, the concentration of potassium ion is higher than about 93 mM. In
some aspects, the
concentration of potassium ion is about 93 mM. In some aspects, the
concentration of potassium
ion is higher than about 94 mM. In some aspects, the concentration of
potassium ion is about 94
mM. In some aspects, the concentration of potassium ion is higher than about
95 mM. In some
aspects, the concentration of potassium ion is about 95 mM. In some aspects,
the concentration of
potassium ion is higher than about 96 mM. In some aspects, the concentration
of potassium ion is
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about 96 mM. In some aspects, the concentration of potassium ion is higher
than about 97 mM. In
some aspects, the concentration of potassium ion is about 97 mM. In some
aspects, the
concentration of potassium ion is higher than about 98 mM. In some aspects,
the concentration of
potassium ion is about 98 mM. In some aspects, the concentration of potassium
ion is higher than
about 99 mM. In some aspects, the concentration of potassium ion is about 99
mM.
[0275] In some aspects, the concentration of potassium ion is
higher than about 100 mM.
In some aspects, the concentration of potassium ion is about 100 mM.
[0276] In some aspects, the concentration of potassium ion is
about 50 mM to about 90
mM, and the concentration of NaC1 is less than about 90 mM to about 50 mM. In
some aspects,
the concentration of potassium ion is about 50 mM to about 80 mM, and the
concentration of NaCl
is less than about 90 mM to about 60 mM. In some aspects, the concentration of
potassium ion is
about 60 mM to about 90 mM, and the concentration of NaCl is less than about
90 mM to about
60 mM. In some aspects, the concentration of potassium ion is about 60 mM to
about 80 mM, and
the concentration of NaCl is less than about 80 mM to about 60 mM. In some
aspects, the
concentration of potassium ion is about 70 mM to about 90 mM, and the
concentration of NaC1 is
less than about 70 mM to about 50 mM. In some aspects, the concentration of
potassium ion is
about 70 mM to about 80 mM, and the concentration of NaCl is less than about
70 mM to about
60 mM. In some aspects, the concentration of potassium ion is about 80 mM to
about 90 mM, and
the concentration of NaCl is less than about 60 mM to about 50 mM. In some
aspects, the total
concentration of potassium ion and NaCl is between 110 mM and 140 mM.
[0277] In some aspects, the concentration of potassium ion is
about 50 mM to about 55
mM. In some aspects, the concentration of potassium ion is about 50 mM to
about 55 mM, and the
concentration of NaCl is less than about 90 mM to about 85 mM. In some
aspects, the concentration
of potassium ion is about 55 mM to about 60 mM. In some aspects, the
concentration of potassium
ion is about 55 mM to about 60 mM, and the concentration of NaCl is less than
about 85 mM to
about 80 mM. In some aspects, the concentration of potassium ion is about 60
mM to about 65
mM. In some aspects, the concentration of potassium ion is about 60 mM to
about 65 mM, and the
concentration of NaCl is less than about 80 mM to about 75 mM. In some
aspects, the concentration
of potassium ion is about 65 mM to about 70 mM. In some aspects, the
concentration of potassium
ion is about 65 mM to about 70 mM, and the concentration of NaCl is less than
about 75 mM to
about 70 mM. In some aspects, the concentration of potassium ion is about 70
mM to about 75
mM. In some aspects, the concentration of potassium ion is about 70 mM to
about 75 mM, and the
concentration of NaCl is less than about 70 mM to about 65 mM. In some
aspects, the concentration
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of potassium ion is about 75 mM to about 80 mM. In some aspects, the
concentration of potassium
ion is about 75 mM to about 80 mM, and the concentration of NaCl is less than
about 65 mM to
about 60 mM. In some aspects, the concentration of potassium ion is about 80
mM to about 85
mM. In some aspects, the concentration of potassium ion is about 80 mM to
about 85 mM, and the
concentration of NaCl is less than about 60 mM to about 55 mM. In some
aspects, the concentration
of potassium ion is about 85 mM to about 90 mM. In some aspects, the
concentration of potassium
ion is about 85 mM to about 90 mM, and the concentration of NaCl is less than
about 55 mM to
about 50 mM. In some aspects, the concentration of potassium ion is about 90
mM to about 95
mM. In some aspects, the concentration of potassium ion is about 90 mM to
about 95 mM, and the
concentration of NaCl is less than about 50 mM to about 45 mM. In some
aspects, the concentration
of potassium ion is about 95 mM to about 100 mM. In some aspects, the
concentration of potassium
ion is about 95 mM to about 100 mM, and the concentration of NaCl is less than
about 45 mM to
about 40 mM. In some aspects, the concentration of potassium ion is about 100
mM to about 105
mM. In some aspects, the concentration of potassium ion is about 100 mM to
about 105 mM, and
the concentration of NaCl is less than about 40 mM to about 35 mM. In some
aspects, the
concentration of potassium ion is about 105 mM to about 110 mM. In some
aspects, the
concentration of potassium ion is about 105 mM to about 110 mM, and the
concentration of NaCl
is less than about 35 to about 30. In some aspects, the concentration of
potassium ion is about 110
mM to about 115 mM. In some aspects, the concentration of potassium ion is
about 110 mM to
about 115 mM, and the concentration of NaCl is less than about 30 mM to about
25 mM. In some
aspects, the concentration of potassium ion is about 115 mM to about 120 mM.
In some aspects,
the concentration of potassium ion is about 115 mM to about 120 mM, and the
concentration of
NaCl is less than about 25 mM to about 20 mM. In some aspects, the total
concentration of
potassium ion and NaCl is between 110 mM and 140 mM.
[0278] In some aspects, the concentration of potassium ion is
higher than about 40 mM,
wherein the total concentration of potassium ion and NaC1 in the medium is
between 110 mM and
140 mM. In some aspects, the concentration of potassium ion is about 40 mM,
wherein the total
concentration of potassium ion and NaCl in the medium is between 110 mM and
140 mM. In some
aspects, the concentration of potassium ion is higher than about 41 mM,
wherein the total
concentration of potassium ion and NaC1 in the medium is between 110 mM and
140 mM. In some
aspects, the concentration of potassium ion is about 41 mM, wherein the total
concentration of
potassium ion and NaCl in the medium is between 110 mM and 140 mM. In some
aspects, the
concentration of potassium ion is higher than about 42 mM, wherein the total
concentration of
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potassium ion and NaCl in the medium is between 110 mM and 140 mM. In some
aspects, the
concentration of potassium ion is about 42 mM, wherein the total concentration
of potassium ion
and NaCl in the medium is between 110 mM and 140 mM. In some aspects, the
concentration of
potassium ion is higher than about 43 mM, wherein the total concentration of
potassium ion and
NaCl in the medium is between 110 mM and 140 mM. In some aspects, the
concentration of
potassium ion is about 43 mM, wherein the total concentration of potassium ion
and NaCl in the
medium is between 110 mM and 140 mM. In some aspects, the concentration of
potassium ion is
higher than about 44 mM, wherein the total concentration of potassium ion and
NaCl in the medium
is between 110 mM and 140 mM. In some aspects, the concentration of potassium
ion is about 44
mM, wherein the total concentration of potassium ion and NaCl in the medium is
between 110 mM
and 140 mM. In some aspects, the concentration of potassium ion is higher than
about 45 mM,
wherein the total concentration of potassium ion and NaC1 in the medium is
between 110 mM and
140 mM. In some aspects, the concentration of potassium ion is about 45 mM,
wherein the total
concentration of potassium ion and NaCl in the medium is between 110 mM and
140 mM. In some
aspects, the concentration of potassium ion is higher than about 46 mM,
wherein the total
concentration of potassium ion and NaCl in the medium is between 110 mM and
140 mM. In some
aspects, the concentration of potassium ion is about 46 mM, wherein the total
concentration of
potassium ion and NaCl in the medium is between 110 mM and 140 mM. In some
aspects, the
concentration of potassium ion is higher than about 47 mM, wherein the total
concentration of
potassium ion and NaCl in the medium is between 110 mM and 140 mM. In some
aspects, the
concentration of potassium ion is about 47 mM, wherein the total concentration
of potassium ion
and NaCl in the medium is between 110 mM and 140 mM. In some aspects, the
concentration of
potassium ion is higher than about 48 mM, wherein the total concentration of
potassium ion and
NaCl in the medium is between 110 mM and 140 mM. In some aspects, the
concentration of
potassium ion is about 48 mM, wherein the total concentration of potassium ion
and NaCl in the
medium is between 110 mM and 140 mM. In some aspects, the concentration of
potassium ion is
higher than about 49 mM, wherein the total concentration of potassium ion and
NaCl in the medium
is between 110 mM and 140 mM. In some aspects, the concentration of potassium
ion is about 49
mM, wherein the total concentration of potassium ion and NaCl in the medium is
between 110 mM
and 140 mM.
[0279] In some aspects, the concentration of potassium ion is
higher than about 50 mM,
wherein the total concentration of potassium ion and NaC1 in the medium is
between 110 mM and
140 mM. In some aspects, the concentration of potassium ion is about 50 mM,
wherein the total
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concentration of potassium ion and NaCl in the medium is between 110 mM and
140 mM. In some
aspects, the concentration of potassium ion is higher than about 51 mM,
wherein the total
concentration of potassium ion and NaCl in the medium is between 110 mM and
140 mM. In some
aspects, the concentration of potassium ion is about 51 mM, wherein the total
concentration of
potassium ion and NaCl in the medium is between 110 mM and 140 mM. In some
aspects, the
concentration of potassium ion is higher than about 52 mM, wherein the total
concentration of
potassium ion and NaCl in the medium is between 110 mM and 140 mM. In some
aspects, the
concentration of potassium ion is about 52 mM, wherein the total concentration
of potassium ion
and NaCl in the medium is between 110 mM and 140 mM. In some aspects, the
concentration of
potassium ion is higher than about 53 mM, wherein the total concentration of
potassium ion and
NaCl in the medium is between 110 mM and 140 mM. In some aspects, the
concentration of
potassium ion is about 53 mM, wherein the total concentration of potassium ion
and NaCl in the
medium is between 110 mM and 140 mM. In some aspects, the concentration of
potassium ion is
higher than about 54 mM, wherein the total concentration of potassium ion and
NaCl in the medium
is between 110 mM and 140 mM. In some aspects, the concentration of potassium
ion is about 54
mM, wherein the total concentration of potassium ion and NaCl in the medium is
between 110 mM
and 140 mM. In some aspects, the concentration of potassium ion is higher than
about 55 mM,
wherein the total concentration of potassium ion and NaC1 in the medium is
between 110 mM and
140 mM. In some aspects, the concentration of potassium ion is about 55 mM,
wherein the total
concentration of potassium ion and NaCl in the medium is between 110 mM and
140 mM. In some
aspects, the concentration of potassium ion is higher than about 56 mM,
wherein the total
concentration of potassium ion and NaCl in the medium is between 110 mM and
140 mM. In some
aspects, the concentration of potassium ion is about 56 mM, wherein the total
concentration of
potassium ion and NaCl in the medium is between 110 mM and 140 mM. In some
aspects, the
concentration of potassium ion is higher than about 57 mM, wherein the total
concentration of
potassium ion and NaCl in the medium is between 110 mM and 140 mM. In some
aspects, the
concentration of potassium ion is about 57 mM, wherein the total concentration
of potassium ion
and NaCl in the medium is between 110 mM and 140 mM. In some aspects, the
concentration of
potassium ion is higher than about 58 mM, wherein the total concentration of
potassium ion and
NaC1 in the medium is between 110 mM and 140 mM. In some aspects, the
concentration of
potassium ion is about 58 mM, wherein the total concentration of potassium ion
and NaC1 in the
medium is between 110 mM and 140 mM. In some aspects, the concentration of
potassium ion is
higher than about 59 mM, wherein the total concentration of potassium ion and
NaCl in the medium
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is between 110 mM and 140 mM. In some aspects, the concentration of potassium
ion is about 59
mM, wherein the total concentration of potassium ion and NaCl in the medium is
between 110 mM
and 140 mM.
[0280] In some aspects, the concentration of potassium ion is
higher than about 60 mM,
wherein the total concentration of potassium ion and NaC1 in the medium is
between 110 mM and
140 mM. In some aspects, the concentration of potassium ion is about 60 mM,
wherein the total
concentration of potassium ion and NaCl in the medium is between 110 mM and
140 mM. In some
aspects, the concentration of potassium ion is higher than about 61 mM,
wherein the total
concentration of potassium ion and NaCl in the medium is between 110 mM and
140 mM. In some
aspects, the concentration of potassium ion is about 61 mM, wherein the total
concentration of
potassium ion and NaCl in the medium is between 110 mM and 140 mM. In some
aspects, the
concentration of potassium ion is higher than about 62 mM, wherein the total
concentration of
potassium ion and NaCl in the medium is between 110 mM and 140 mM. In some
aspects, the
concentration of potassium ion is about 62 mM, wherein the total concentration
of potassium ion
and NaCl in the medium is between 110 mM and 140 mM. In some aspects, the
concentration of
potassium ion is higher than about 63 mM, wherein the total concentration of
potassium ion and
NaCl in the medium is between 110 mM and 140 mM. In some aspects, the
concentration of
potassium ion is about 63 mM, wherein the total concentration of potassium ion
and NaCl in the
medium is between 110 mM and 140 mM. In some aspects, the concentration of
potassium ion is
higher than about 64 mM, wherein the total concentration of potassium ion and
NaCl in the medium
is between 110 mM and 140 mM. In some aspects, the concentration of potassium
ion is about 64
mM, wherein the total concentration of potassium ion and NaCl in the medium is
between 110 mM
and 140 mM. In some aspects, the concentration of potassium ion is higher than
about 65 mM,
wherein the total concentration of potassium ion and NaC1 in the medium is
between 110 mM and
140 mM. In some aspects, the concentration of potassium ion is about 65 mM,
wherein the total
concentration of potassium ion and NaCI in the medium is between 110 mM and
140 mM. In some
aspects, the concentration of potassium ion is higher than about 66 mM,
wherein the total
concentration of potassium ion and NaCl in the medium is between 110 mM and
140 mM. In some
aspects, the concentration of potassium ion is about 66 mM, wherein the total
concentration of
potassium ion and NaC1 in the medium is between 110 mM and 140 mM. In some
aspects, the
concentration of potassium ion is higher than about 67 mM, wherein the total
concentration of
potassium ion and NaCl in the medium is between 110 mM and 140 mM. In some
aspects, the
concentration of potassium ion is about 67 mM, wherein the total concentration
of potassium ion
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and NaC1 in the medium is between 110 mM and 140 mM. In some aspects, the
concentration of
potassium ion is higher than about 68 mM, wherein the total concentration of
potassium ion and
NaCl in the medium is between 110 mM and 140 mM. In some aspects, the
concentration of
potassium ion is about 68 mM, wherein the total concentration of potassium ion
and NaCl in the
medium is between 110 mM and 140 mM. In some aspects, the concentration of
potassium ion is
higher than about 69 mM, wherein the total concentration of potassium ion and
NaCl in the medium
is between 110 mM and 140 mM. In some aspects, the concentration of potassium
ion is about 69
mM, wherein the total concentration of potassium ion and NaCl in the medium is
between 110 mM
and 140 mM.
[0281] In some aspects, the concentration of potassium ion is
higher than about 70 mM,
wherein the total concentration of potassium ion and NaC1 in the medium is
between 110 mM and
140 mM. In some aspects, the concentration of potassium ion is about 70 mM,
wherein the total
concentration of potassium ion and NaCl in the medium is between 110 mM and
140 mM. In some
aspects, the concentration of potassium ion is higher than about 71 mM,
wherein the total
concentration of potassium ion and NaCl in the medium is between 110 mM and
140 mM. In some
aspects, the concentration of potassium ion is about 71 mM, wherein the total
concentration of
potassium ion and NaCl in the medium is between 110 mM and 140 mM. In some
aspects, the
concentration of potassium ion is higher than about 72 mM, wherein the total
concentration of
potassium ion and NaCl in the medium is between 110 mM and 140 mM. In some
aspects, the
concentration of potassium ion is about 72 mM, wherein the total concentration
of potassium ion
and NaCl in the medium is between 110 mM and 140 mM. In some aspects, the
concentration of
potassium ion is higher than about 73 mM, wherein the total concentration of
potassium ion and
NaCl in the medium is between 110 mM and 140 mM. In some aspects, the
concentration of
potassium ion is about 73 mM, wherein the total concentration of potassium ion
and NaCl in the
medium is between 110 mM and 140 mM. In some aspects, the concentration of
potassium ion is
higher than about 74 mM, wherein the total concentration of potassium ion and
NaCl in the medium
is between 110 mM and 140 mM. In some aspects, the concentration of potassium
ion is about 74
mM, wherein the total concentration of potassium ion and NaCl in the medium is
between 110 mM
and 140 mM. In some aspects, the concentration of potassium ion is higher than
about 75 mM,
wherein the total concentration of potassium ion and NaC1 in the medium is
between 110 mM and
140 mM. In some aspects, the concentration of potassium ion is about 75 mM,
wherein the total
concentration of potassium ion and NaCl in the medium is between 110 mM and
140 mM. In some
aspects, the concentration of potassium ion is higher than about 76 mM,
wherein the total
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concentration of potassium ion and NaCl in the medium is between 110 mM and
140 mM. In some
aspects, the concentration of potassium ion is about 76 mM, wherein the total
concentration of
potassium ion and NaCl in the medium is between 110 mM and 140 mM. In some
aspects, the
concentration of potassium ion is higher than about 77 mM, wherein the total
concentration of
potassium ion and NaCl in the medium is between 110 mM and 140 mM. In some
aspects, the
concentration of potassium ion is about 77 mM, wherein the total concentration
of potassium ion
and NaCl in the medium is between 110 mM and 140 mM. In some aspects, the
concentration of
potassium ion is higher than about 78 mM, wherein the total concentration of
potassium ion and
NaCl in the medium is between 110 mM and 140 mM. In some aspects, the
concentration of
potassium ion is about 78 mM, wherein the total concentration of potassium ion
and NaCl in the
medium is between 110 mM and 140 mM. In some aspects, the concentration of
potassium ion is
higher than about 79 mM, wherein the total concentration of potassium ion and
NaCl in the medium
is between 110 mM and 140 mM. In some aspects, the concentration of potassium
ion is about 79
mM, wherein the total concentration of potassium ion and NaCl in the medium is
between 110 mM
and 140 mM.
[0282] In some aspects, the concentration of potassium ion is
higher than about 80 mM,
wherein the total concentration of potassium ion and NaC1 in the medium is
between 110 mM and
140 mM. In some aspects, the concentration of potassium ion is about 80 mM,
wherein the total
concentration of potassium ion and NaCl in the medium is between 110 mM and
140 mM. In some
aspects, the concentration of potassium ion is higher than about 81 mM,
wherein the total
concentration of potassium ion and NaCl in the medium is between 110 mM and
140 mM. In some
aspects, the concentration of potassium ion is about 81 mM, wherein the total
concentration of
potassium ion and NaCl in the medium is between 110 mM and 140 mM. In some
aspects, the
concentration of potassium ion is higher than about 82 mM, wherein the total
concentration of
potassium ion and NaCl in the medium is between 110 mM and 140 mM. In some
aspects, the
concentration of potassium ion is about 82 mM, wherein the total concentration
of potassium ion
and NaC1 in the medium is between 110 mM and 140 mM. In some aspects, the
concentration of
potassium ion is higher than about 83 mM, wherein the total concentration of
potassium ion and
NaCl in the medium is between 110 mM and 140 mM. In some aspects, the
concentration of
potassium ion is about 83 mM, wherein the total concentration of potassium ion
and NaC1 in the
medium is between 110 mM and 140 mM. In some aspects, the concentration of
potassium ion is
higher than about 84 mM, wherein the total concentration of potassium ion and
NaCl in the medium
is between 110 mM and 140 mM. In some aspects, the concentration of potassium
ion is about 84
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mM, wherein the total concentration of potassium ion and NaCl in the medium is
between 110 mM
and 140 mM. In some aspects, the concentration of potassium ion is higher than
about 85 mM,
wherein the total concentration of potassium ion and NaC1 in the medium is
between 110 mM and
140 mM. In some aspects, the concentration of potassium ion is about 85 mM,
wherein the total
concentration of potassium ion and NaCl in the medium is between 110 mM and
140 mM. In some
aspects, the concentration of potassium ion is higher than about 86 mM,
wherein the total
concentration of potassium ion and NaCl in the medium is between 110 mM and
140 mM. In some
aspects, the concentration of potassium ion is about 86 mM, wherein the total
concentration of
potassium ion and NaCl in the medium is between 110 mM and 140 mM. In some
aspects, the
concentration of potassium ion is higher than about 87 mM, wherein the total
concentration of
potassium ion and NaCl in the medium is between 110 mM and 140 mM. In some
aspects, the
concentration of potassium ion is about 87 mM, wherein the total concentration
of potassium ion
and NaCl in the medium is between 110 mM and 140 mM. In some aspects, the
concentration of
potassium ion is higher than about 88 mM, wherein the total concentration of
potassium ion and
NaCl in the medium is between 110 mM and 140 mM. In some aspects, the
concentration of
potassium ion is about 88 mM, wherein the total concentration of potassium ion
and NaCl in the
medium is between 110 mM and 140 mM. In some aspects, the concentration of
potassium ion is
higher than about 89 mM, wherein the total concentration of potassium ion and
NaCl in the medium
is between 110 mM and 140 mM. In some aspects, the concentration of potassium
ion is about 89
mM, wherein the total concentration of potassium ion and NaCl in the medium is
between 110 mM
and 140 mM.
[0283] In some aspects, the concentration of potassium ion is
higher than about 90 mM,
wherein the total concentration of potassium ion and NaC1 in the medium is
between 110 mM and
140 mM. In some aspects, the concentration of potassium ion is about 90 mM,
wherein the total
concentration of potassium ion and NaCl in the medium is between 110 mM and
140 mM. In some
aspects, the concentration of potassium ion is higher than about 91 mM,
wherein the total
concentration of potassium ion and NaCI in the medium is between 110 mM and
140 mM. In some
aspects, the concentration of potassium ion is about 91 mM, wherein the total
concentration of
potassium ion and NaCl in the medium is between 110 mM and 140 mM. In some
aspects, the
concentration of potassium ion is higher than about 92 mM, wherein the total
concentration of
potassium ion and NaC1 in the medium is between 110 mM and 140 mM. In some
aspects, the
concentration of potassium ion is about 92 mM, wherein the total concentration
of potassium ion
and NaCl in the medium is between 110 mM and 140 mM. In some aspects, the
concentration of
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potassium ion is higher than about 93 mM, wherein the total concentration of
potassium ion and
NaCl in the medium is between 110 mM and 140 mM. In some aspects, the
concentration of
potassium ion is about 93 mM, wherein the total concentration of potassium ion
and NaCl in the
medium is between 110 mM and 140 mM. In some aspects, the concentration of
potassium ion is
higher than about 94 mM, wherein the total concentration of potassium ion and
NaCl in the medium
is between 110 mM and 140 mM. In some aspects, the concentration of potassium
ion is about 94
mM, wherein the total concentration of potassium ion and NaCl in the medium is
between 110 mM
and 140 mM. In some aspects, the concentration of potassium ion is higher than
about 95 mM,
wherein the total concentration of potassium ion and NaC1 in the medium is
between 110 mM and
140 mM. In some aspects, the concentration of potassium ion is about 95 mM,
wherein the total
concentration of potassium ion and NaCl in the medium is between 110 mM and
140 mM. In some
aspects, the concentration of potassium ion is higher than about 96 mM,
wherein the total
concentration of potassium ion and NaCl in the medium is between 110 mM and
140 mM. In some
aspects, the concentration of potassium ion is about 96 mM, wherein the total
concentration of
potassium ion and NaCl in the medium is between 110 mM and 140 mM. In some
aspects, the
concentration of potassium ion is higher than about 97 mM, wherein the total
concentration of
potassium ion and NaCl in the medium is between 110 mM and 140 mM. In some
aspects, the
concentration of potassium ion is about 97 mM, wherein the total concentration
of potassium ion
and NaCl in the medium is between 110 mM and 140 mM. In some aspects, the
concentration of
potassium ion is higher than about 98 mM, wherein the total concentration of
potassium ion and
NaCl in the medium is between 110 mM and 140 mM. In some aspects, the
concentration of
potassium ion is about 98 mM, wherein the total concentration of potassium ion
and NaCl in the
medium is between 110 mM and 140 mM. In some aspects, the concentration of
potassium ion is
higher than about 99 mM, wherein the total concentration of potassium ion and
NaCl in the medium
is between 110 mM and 140 mM. In some aspects, the concentration of potassium
ion is about 99
mM, wherein the total concentration of potassium ion and NaCl in the medium is
between 110 mM
and 140 mM.
[0284] In some aspects, the concentration of potassium ion is
higher than about 100 mM,
wherein the total concentration of potassium ion and NaC1 in the medium is
between 110 mM and
140 mM. In some aspects, the concentration of potassium ion is about 100 mM,
wherein the total
concentration of potassium ion and NaC1 in the medium is between 110 mM and
140 mM. In some
aspects, the concentration of potassium ion is higher than about 101 mM,
wherein the total
concentration of potassium ion and NaCl in the medium is between 110 mM and
140 mM. In some
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aspects, the concentration of potassium ion is about 101 mM, wherein the total
concentration of
potassium ion and NaCl in the medium is between 110 mM and 140 mM. In some
aspects, the
concentration of potassium ion is higher than about 102 mM, wherein the total
concentration of
potassium ion and NaCl in the medium is between 110 mM and 140 mM. In some
aspects, the
concentration of potassium ion is about 102 mM, wherein the total
concentration of potassium ion
and NaCl in the medium is between 110 mM and 140 mM. In some aspects, the
concentration of
potassium ion is higher than about 103 mM, wherein the total concentration of
potassium ion and
NaCl in the medium is between 110 mM and 140 mM. In some aspects, the
concentration of
potassium ion is about 103 mM, wherein the total concentration of potassium
ion and NaCl in the
medium is between 110 mM and 140 mM. In some aspects, the concentration of
potassium ion is
higher than about 104 mM, wherein the total concentration of potassium ion and
NaCl in the
medium is between 110 mM and 140 mM. In some aspects, the concentration of
potassium ion is
about 104 mM, wherein the total concentration of potassium ion and NaC1 in the
medium is
between 110 mM and 140 mM. In some aspects, the concentration of potassium ion
is higher than
about 105 mM, wherein the total concentration of potassium ion and NaC1 in the
medium is
between 110 mM and 140 mM. In some aspects, the concentration of potassium ion
is about 105
mM, wherein the total concentration of potassium ion and NaCl in the medium is
between 110 mM
and 140 mM. In some aspects, the concentration of potassium ion is higher than
about 106 mM,
wherein the total concentration of potassium ion and NaC1 in the medium is
between 110 mM and
140 mM. In some aspects, the concentration of potassium ion is about 106 mM,
wherein the total
concentration of potassium ion and NaC1 in the medium is between 110 mM and
140 mM. In some
aspects, the concentration of potassium ion is higher than about 107 mM,
wherein the total
concentration of potassium ion and NaC1 in the medium is between 110 mM and
140 mM. In some
aspects, the concentration of potassium ion is about 107 mM, wherein the total
concentration of
potassium ion and NaCl in the medium is between 110 mM and 140 mM. In some
aspects, the
concentration of potassium ion is higher than about 108 mM, wherein the total
concentration of
potassium ion and NaC1 in the medium is between 110 mM and 140 mM. In some
aspects, the
concentration of potassium ion is about 108 mM, wherein the total
concentration of potassium ion
and NaCl in the medium is between 110 mM and 140 mM. In some aspects, the
concentration of
potassium ion is higher than about 109 mM, wherein the total concentration of
potassium ion and
NaC1 in the medium is between 110 mM and 140 mM. In some aspects, the
concentration of
potassium ion is about 109 mM, wherein the total concentration of potassium
ion and NaCl in the
medium is between 110 mM and 140 mM.
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[0285] In some aspects, the concentration of potassium ion is
higher than about 110 mM,
wherein the total concentration of potassium ion and NaCl in the medium is
between 110 mM and
140 mM. In some aspects, the concentration of potassium ion is about 110 mM,
wherein the total
concentration of potassium ion and NaC1 in the medium is between 110 mM and
140 mM. In some
aspects, the concentration of potassium ion is higher than about 111 mM,
wherein the total
concentration of potassium ion and NaC1 in the medium is between 110 mM and
140 mM. In some
aspects, the concentration of potassium ion is about 111 mM, wherein the total
concentration of
potassium ion and NaCl in the medium is between 110 mM and 140 mM. In some
aspects, the
concentration of potassium ion is higher than about 112 mM, wherein the total
concentration of
potassium ion and NaCl in the medium is between 110 mM and 140 mM. In some
aspects, the
concentration of potassium ion is about 112 mM, wherein the total
concentration of potassium ion
and NaCl in the medium is between 110 mM and 140 mM. In some aspects, the
concentration of
potassium ion is higher than about 113 mM, wherein the total concentration of
potassium ion and
NaCl in the medium is between 110 mM and 140 mM. In some aspects, the
concentration of
potassium ion is about 113 mM, wherein the total concentration of potassium
ion and NaCl in the
medium is between 110 mM and 140 mM. In some aspects, the concentration of
potassium ion is
higher than about 114 mM, wherein the total concentration of potassium ion and
NaCl in the
medium is between 110 mM and 140 mM. In some aspects, the concentration of
potassium ion is
about 114 mM, wherein the total concentration of potassium ion and NaC1 in the
medium is
between 110 mM and 140 mM. In some aspects, the concentration of potassium ion
is higher than
about 115 mM, wherein the total concentration of potassium ion and NaC1 in the
medium is
between 110 mM and 140 mM. In some aspects, the concentration of potassium ion
is about 115
mM, wherein the total concentration of potassium ion and NaCl in the medium is
between 110 mM
and 140 mM. In some aspects, the concentration of potassium ion is higher than
about 116 mM,
wherein the total concentration of potassium ion and NaCl in the medium is
between 110 mM and
140 mM. In some aspects, the concentration of potassium ion is about 116 mM,
wherein the total
concentration of potassium ion and NaCI in the medium is between 110 mM and
140 mM. In some
aspects, the concentration of potassium ion is higher than about 117 mM,
wherein the total
concentration of potassium ion and NaC1 in the medium is between 110 mM and
140 mM. In some
aspects, the concentration of potassium ion is about 117 mM, wherein the total
concentration of
potassium ion and NaC1 in the medium is between 110 mM and 140 mM. In some
aspects, the
concentration of potassium ion is higher than about 118 mM, wherein the total
concentration of
potassium ion and NaCl in the medium is between 110 mM and 140 mM. In some
aspects, the
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concentration of potassium ion is about 118 mM, wherein the total
concentration of potassium ion
and NaCl in the medium is between 110 mM and 140 mM. In some aspects, the
concentration of
potassium ion is higher than about 119 mM, wherein the total concentration of
potassium ion and
NaCl in the medium is between 110 mM and 140 mM. In some aspects, the
concentration of
potassium ion is about 119 mM, wherein the total concentration of potassium
ion and NaCl in the
medium is between 110 mM and 140 mM.
[0286] In some aspects, the concentration of potassium ion is
higher than about 120 mM,
wherein the total concentration of potassium ion and NaC1 in the medium is
between 110 mM and
140 mM. In some aspects, the concentration of potassium ion is about 120 mM,
wherein the total
concentration of potassium ion and NaC1 in the medium is between 110 mM and
140 mM. In some
aspects, the concentration of potassium ion is higher than about 121 mM,
wherein the total
concentration of potassium ion and NaC1 in the medium is between 110 mM and
140 mM. In some
aspects, the concentration of potassium ion is about 121 mM, wherein the total
concentration of
potassium ion and NaCl in the medium is between 110 mM and 140 mM. In some
aspects, the
concentration of potassium ion is higher than about 122 mM, wherein the total
concentration of
potassium ion and NaCl in the medium is between 110 mM and 140 mM. In some
aspects, the
concentration of potassium ion is about 122 mM, wherein the total
concentration of potassium ion
and NaCl in the medium is between 110 mM and 140 mM. In some aspects, the
concentration of
potassium ion is higher than about 123 mM, wherein the total concentration of
potassium ion and
NaCl in the medium is between 110 mM and 140 mM. In some aspects, the
concentration of
potassium ion is about 123 mM, wherein the total concentration of potassium
ion and NaCl in the
medium is between 110 mM and 140 mM. In some aspects, the concentration of
potassium ion is
higher than about 124 mM, wherein the total concentration of potassium ion and
NaCl in the
medium is between 110 mM and 140 mM. In some aspects, the concentration of
potassium ion is
about 124 mM, wherein the total concentration of potassium ion and NaC1 in the
medium is
between 110 mM and 140 mM. In some aspects, the concentration of potassium ion
is higher than
about 125 mM, wherein the total concentration of potassium ion and NaC1 in the
medium is
between 110 mM and 140 mM. In some aspects, the concentration of potassium ion
is about 125
mM, wherein the total concentration of potassium ion and NaCl in the medium is
between 110 mM
and 140 mM. In some aspects, the concentration of potassium ion is higher than
about 126 mM,
wherein the total concentration of potassium ion and NaC1 in the medium is
between 110 mM and
140 mM. In some aspects, the concentration of potassium ion is about 126 mM,
wherein the total
concentration of potassium ion and NaC1 in the medium is between 110 mM and
140 mM. In some
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aspects, the concentration of potassium ion is higher than about 127 mM,
wherein the total
concentration of potassium ion and NaCl in the medium is between 110 mM and
140 mM. In some
aspects, the concentration of potassium ion is about 127 mM, wherein the total
concentration of
potassium ion and NaCl in the medium is between 110 mM and 140 mM. In some
aspects, the
concentration of potassium ion is higher than about 128 mM, wherein the total
concentration of
potassium ion and NaCl in the medium is between 110 mM and 140 mM. In some
aspects, the
concentration of potassium ion is about 128 mM, wherein the total
concentration of potassium ion
and NaCl in the medium is between 110 mM and 140 mM. In some aspects, the
concentration of
potassium ion is higher than about 129 mM, wherein the total concentration of
potassium ion and
NaCl in the medium is between 110 mM and 140 mM. In some aspects, the
concentration of
potassium ion is about 129 mM, wherein the total concentration of potassium
ion and NaCl in the
medium is between 110 mM and 140 mM. In some aspects, the concentration of
potassium ion is
higher than about 130 mM, wherein the total concentration of potassium ion and
NaCl in the
medium is between 110 mM and 140 mM. In some aspects, the concentration of
potassium ion is
about 130 mM, wherein the total concentration of potassium ion and NaC1 in the
medium is
between 110 mM and 140 mM.
[0287] In some aspects, the hyperkalemic medium comprising a
high concentration of
potassium ion can be prepared by adding a sufficient amount of a potassium
salt in a medium. In
some aspects, non-limiting examples of potassium salt include potassium
aminetrichloroplatinate,
potassium aquapentachlororuthenate, potassium bis(oxalato)platinate(II)
dihydrate, potassium
bisulfate, potassium borohydride, potassium bromide, potassium carbonate,
potassium chloride,
potassium chromate, potassium dichromate, potassium dicyanoargentate,
potassium
dicyanoaurate, potassium fluoride, potassium fluorosulfate, potassium
hexachloroiridate,
potassium hexachloroosmate, potassium hexachloropalladate, potassium
hexachloroplatinate,
potassium hexachlororhenate, potassium hexacyanochromate, potassium
hexacyanoferrate,
potassium hexacyanoruthenate(II) hydrate, potassium hexafluoroantimonate,
potassium
hexafluoronickel ate, potassium hexafluorophosphate, potassium
hexafluorotitanate, potassium
hexafluorozirconate, potassium hexahydroxoantimonate, potassium
hexaiodoplatinate, potassium
hexaiodorhenate, potassium hydroxide, potassium iodate, potassium iodide,
potassium manganate,
potassium metavanadate, potassium molybdate, potassium nitrate, potassium
nitrosodisulfonate,
potassium osmate(VI) di hydrate, potassium pentachloronitrosylruth en ate,
potassi urn perch] orate,
potassium perrhenate, potassium perruthenate, potassium persulfate, potassium
phosphate dibasic,
potassium phosphate monobasic, potassium pyrophosphate, potassium
selenocyanate, potassium
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selenocyanate, potassium stannate trihydrate, potassium sulfate, potassium
tellurate hydrate,
potassium tellurite, potassium tetrab orate tetrahydrate, potassium
tetrabromoaurate, potassium
tetrabromopalladate, potassium tetrachloropalladate, potassium
tetrachloroplatinate, potassium
tetracyanopalladate, potassium tetracyanoplatinate, potassium tetrafluorob
orate, potassium
tetranitroplatinate, potassium tetrathionate, potassium p-
toluenethiosulfonate, and potassium
hydroxycitrate tribasic monohydrate. In some aspects, the potassium salt
comprises potassium
chloride (KC1). In some aspects, the potassium salt comprises potassium
gluconate. In certain
aspects, the potassium salt comprises potassium citrate. In certain aspects,
the potassium salt
comprises potassium hydroxycitrate. In some aspects, the potassium salt
comprises a combination
of any of the potassium salts disclosed herein.
II.B. Cell Expansion Agents
[0288] In some aspects, the metabolic reprogramming media, e.g.,
hyperkalemic medium,
further comprises a cell expansion agent. As used herein, a "cell expansion
agent" refers to an
agent, e.g., small molecule, polypeptide, or any combination thereof, that
promotes the in vitro
and/or ex vivo growth and proliferation of cultured immune cells, e.g., TILs.
In some aspects, the
cell expansion agent comprises a PI3K inhibitor. In some aspects, the medium
further comprises
an AKT inhibitor. In some aspects, the medium further comprises a PI3K
inhibitor and an AKT
inhibitor. In some aspects, the PI3K inhibitor comprises LY294002. In some
aspects, the PI3K
inhibitor comprises IC87114. In some aspects, the PI3K inhibitor comprises
idelalisib (see, e.g.,
Peterson et al., Blood Adv. 2(3):210-23 (2018)). In some aspects, the medium
further comprises a
GSK3B inhibitor. In some aspects, the GSK3B inhibitor comprises TWS119. In
some aspects, the
medium further comprises an ACLY inhibitor. In some aspects, the ACLY
inhibitor comprises
potassium hydroxycitrate tribasic monohydrate. In some aspects, the PI3K
inhibitor comprises
hydroxyl citrate. In some aspects, the PI3K inhibitor comprises pi ctilisib.
In some aspects, the
PI3K inhibitor comprises CAL-101. In some aspects, the AKT inhibitor comprises
MK2206,
A443654, or AKTi-VIII (CAS 612847-09-3)
II.C. Sodium
[0289] In some aspects, the metabolic reprogramming media, e.g.,
hyperkalemic medium,
further comprises sodium ion (e.g., NaC1). In some aspects, the metabolic
reprogramming media
comprises sodium ion (e.g, NaCl) at a concentration of less than about 115 mM.
In some aspects
the metabolic reprogramming media, comprises sodium ion (e.g., NaCl) at a
concentration of 40
mM to about 80 mM.
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[0290] In some aspects, the target concentration of sodium is
reached by starting with a
basal medium comprising a higher concentration of sodium ion (e.g., NaCl) and
diluting the
solution to reach the target concentration of sodium ion (e.g., NaC1). In some
aspects, the target
concentration of sodium is reached by raising the concentration of sodium ion
(e.g., NaCl) by
adding one or more sodium salts (e.g., more NaC1). Non-limiting examples of
sodium salts include
sodium (meta)periodate, sodium arsenyl tartrate hydrate, sodium azide, sodium
benzyloxide,
sodium bromide, sodium carbonate, sodium chloride, sodium chromate, sodium
cyclohexanebutyrate, sodium ethanethiolate, sodium fluoride, sodium
fluorophosphate, sodium
formate, sodium hexachloroiridate(III) hydrate, sodium hexachloroiridate(IV)
hexahydrate,
sodium hexachloroplatinate(IV) hexahydrate, sodium hexachlororhodate(III),
sodium
hexafluoroaluminate, sodium hexafluoroantimonate(V), sodium
hexafluoroarsenate(V), sodium
hexafluoroferrate(III), sodium hexafluorophosphate, sodium hexafluorosilicate,
sodium
hex ahy droxyplatinate(IV), sodium he xametap ho sphate, sodium hydrogen di fl
uori de, sodium
hydrogen sulfate, sodium hydrogencyanamide, sodium hydroxide, sodium iodide,
sodium
metaborate tetrahydrate, sodium metasilicate nonahydrate, sodium metavanadate,
sodium
molybdate, sodium nitrate, sodium nitrite, sodium oxalate, sodium perborate
monohydrate, sodium
percarbonate, sodium perchlorate, sodium periodate, sodium permanganate,
sodium perrhenate,
sodium phosphate, sodium pyrophosphate, sodium selenate, sodium selenite,
sodium stannate,
sodium sulfate, sodium tellurite, sodium tetraborate, sodium
tetrachloroaluminate, sodium
tetrachloroaurate(III), sodium tetrachl oropalladate(II), sodium
tetrachloroplatinate(II), sodium
thiophosphate tribasic, sodium thiosulfate, sodium thiosulfate pentahydrate,
sodium yttrium
oxyfluoride, Trisodium trimetaphosphate, and any combination thereof. In some
aspects, the
sodium salt comprises sodium chloride (NaCl). In some aspects, the sodium salt
comprises sodium
gluconate. In certain aspects, the sodium salt comprises sodium bicarbonate.
In certain aspects, the
sodium salt comprises sodium hydroxycitrate. In certain aspects, the sodium
salt comprises sodium
phosphate.
[0291] In some aspects, the concentration of the sodium ion
(e.g., NaC1) is less than that of
the basal medium. In some aspects, the concentration of the sodium ion (e.g.,
NaCl) is reduced as
the concentration of potassium ion is increased. In some aspects, the
concentration of the sodium
ion (e.g., NaC1) is from about 25 mM to about 115 mM. In some aspects, the
concentration of the
sodium ion (e.g., NaC1) is from about 25 mM to about 100 mM, about 30 mM to
about 40 mM,
about 30 mM to about 50 mM, about 30 mM to about 60 mM, about 30 mM to about
70 mM, about
30 mM to about 80 mM, about 40 mM to about 50 mM, about 40 mM to about 60 mM,
about 40
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mM to about 70 mM, about 40 mM to about 80 mM, about 50 mM to about 55 mM,
about 50 mM
to about 60 mM, about 50 mM to about 65 mM, about 50 mM to about 70 mM, about
50 mM to
about 75 mM, about 50 mM to about 80 mM, about 55 mM to about 60 mM, about 55
mM to about
65 mM, about 55 mM to about 70 mM, about 55 mM to about 75 mM, about 55 mM to
about 80
mM, about 60 mM to about 65 mM, about 60 mM to about 70 mM, about 60 mM to
about 75 mM,
about 60 mM to about 80 mM, about 70 mM to about 75 mM, about 70 mM to about
80 mM, or
about 75 mM to about 80 mM. In certain aspects, the concentration of the
sodium ion (e.g., NaC1)
is from about 40 mM to about 80 mM. In some aspects, the concentration of the
sodium ion (e.g.,
NaC1) is from about 50 mM to about 85 mM. In some aspects, the concentration
of the sodium ion
(e.g., NaC1) is from about 55 mM to about 80 mM. In some aspects, the
concentration of the sodium
ion (e.g., NaC1) is from about 30 mM to about 35 mM. In some aspects, the
concentration of the
sodium ion (e.g., NaC1) is from about 35 mM to about 40 mM. In some aspects,
the concentration
of the sodium ion (e.g., NaC1) is from about 40 mM to about 45 mM. In some
aspects, the
concentration of the sodium ion (e.g., NaC1) is from about 45 mM to about 50
mM. In some aspects,
the concentration of the sodium ion (e.g., NaC1) is from about 50 mM to about
55 mM. In some
aspects, the concentration of the sodium ion (e.g., NaC1) is from about 55 mM
to about 60 mM. In
some aspects, the concentration of the sodium ion (e.g., NaC1) is from about
60 mM to about 65
mM. In some aspects, the concentration of the sodium ion (e.g., NaC1) is from
about 65 mM to
about 70 mM. In some aspects, the concentration of the sodium ion (e.g., NaC1)
is from about 70
mM to about 75 mM. In some aspects, the concentration of the sodium ion (e.g.,
NaC1) is from
about 75 mM to about 80 mM. In some aspects, the concentration of the sodium
ion (e.g., NaC1)
is from about 80 mM to about 85 mM.
[0292] In some aspects, the concentration of the sodium ion
(e.g., NaC1) is about 30 mM,
about 35 mM, about 40 mM, about 45 mM, about 50 mM, about 55 mM, about 60 mM,
about 65
mM, about 70 mM, about 75 mM, about 80 mM, about 85 mM, or about 90 mM. In
some aspects,
the concentration of sodium ion (e.g., NaC1) is about 40 mM. In some aspects,
the concentration
of sodium ion (e.g., NaC1) is about 45 mM. In some aspects, the concentration
of sodium ion (e.g.,
NaC1) is about 50 mM. In some aspects, the concentration of sodium ion (e.g.,
NaC1) is about 55
mM. In some aspects, the concentration of sodium ion (e.g., NaC1) is about 60
mM. In some
aspects, the concentration of sodium ion (e.g., NaC1) is about 65 mM. In some
aspects, the
concentration of sodium ion (e.g., NaC1) is about 70 mM. In some aspects, the
concentration of
sodium ion (e.g., NaC1) is about 75 mM. In some aspects, the concentration of
sodium ion (e.g.,
NaC1) is about 80 mM.
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[0293] In some aspects, the medium comprises about 40 mM to
about 90 mM potassium
ion and about 40 mM to about 80 mM sodium ion (e.g., NaC1).
[0294] In some aspects, the medium comprises about 50 mM to
about 75 mM potassium
ion and about 80 mM to about 90 mM sodium ion (e.g., NaC1). In some aspects,
the medium
comprises about 55 mM to about 75 mM potassium ion and about 80 mM to about 90
mM sodium
ion (e.g., NaC1). In some aspects, the medium comprises about 60 mM to about
75 mM potassium
ion and about 80 mM to about 90 mM sodium ion (e.g., NaC1). In some aspects,
the medium
comprises about 65 mM to about 75 mM potassium ion and about 80 mM to about 85
mM sodium
ion (e.g., NaC1). In some aspects, the medium comprises about 65 mM potassium
ion and about 80
mM to about 85 mM sodium ion (e.g., NaC1). In some aspects, the medium
comprises about 66
mM potassium ion and about 80 mM to about 85 mM sodium ion (e.g., NaC1)_ In
some aspects,
the medium comprises about 67 mM potassium ion and about 80 mM to about 85 mM
sodium ion
(e.g., NaC1). In some aspects, the medium comprises about 68 mM potassium ion
and about 80
mM to about 85 mM sodium ion (e.g., NaC1). In some aspects, the medium
comprises about 69
mM potassium ion and about 80 mM to about 85 mM sodium ion (e.g., NaC1). In
some aspects,
the medium comprises about 70 mM potassium ion and about 80 mM to about 85 mM
sodium ion
(e.g., NaC1). In some aspects, the medium comprises about 71 mM potassium ion
and about 80
mM to about 85 mM sodium ion (e.g., NaC1). In some aspects, the medium
comprises about 72
mM potassium ion and about 80 mM to about 85 mM sodium ion (e.g., NaC1). In
some aspects,
the medium comprises about 73 mM potassium ion and about 80 mM to about 85 mM
sodium ion
(e.g., NaC1). In some aspects, the medium comprises about 74 mM potassium ion
and about 80
mM to about 85 mM sodium ion (e.g., NaC1). In some aspects, the medium
comprises about 75
mM potassium ion and about 80 mM to about 85 mM sodium ion (e.g., NaC1). In
some aspects,
the medium comprises about 65 mM potassium ion and about 80 mM sodium ion
(e.g., NaC1). In
some aspects, the medium comprises about 65 mM potassium ion and about 85 mM
sodium ion
(e.g., NaC1). In some aspects, the medium comprises about 65 mM potassium ion
and about 90
mM sodium ion (e.g., NaC1). In some aspects, the medium comprises about 70 mM
potassium ion
and about 80 mM sodium ion (e.g., NaC1). In some aspects, the medium comprises
about 70 mM
potassium ion and about 85 mM sodium ion (e.g., NaC1). In some aspects, the
medium comprises
about 70 mM potassium ion and about 90 mM sodium ion (e.g., NaC1). In some
aspects, the
medium comprises about 75 mM potassium ion and about 80 mM sodium ion (e.g.,
NaC1). In some
aspects, the medium comprises about 75 mM potassium ion and about 85 mM sodium
ion (e.g.,
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NaCl). In some aspects, the medium comprises about 75 mM potassium ion and
about 90 mM
sodium ion (e.g., NaCl).
[0295] In some aspects, the medium comprises about 40 mM to
about 90 mM potassium
ion and about 30 mM to about 109 mM NaC1, wherein the concentration of NaC1
(mM) is equal to
or lower than (135 ¨ potassium ion concentration). In some aspects, the medium
comprises about
40 mM potassium ion and less than or equal to about 95 mM NaCl (e.g., about 95
mM, about 94
mM, about 93 mM, about 92 mM, about 91 mM, about 90 mM, about 85 mM, about 80
mM, about
75 mM, about 70 mM, about 65 mM, about 60 mM, about 55 mM, or about 50 mM
NaCl),In some
aspects, the medium comprises about 45 mM potassium ion and less than or equal
to about 90 mM
NaCl (e.g., about 90 mM, about 89 mM, about 88 mM, about 87 mM, about 86 mM,
about 85 mM,
about 80 mM, about 75 mM, about 70 mM, about 65 mM, about 60 mM, about 55 mM,
or about
50 mM NaCl). In some aspects, the medium comprises about 50 mM potassium ion
and less than
or equal to about 85 mM NaCl (e.g., about 85 mM, about 84 mM, about 83 mM,
about 82 mM,
about 81 mM, about 80 mM, about 75 mM, about 70 mM, about 65 mM, about 60 mM,
about 55
mM, or about 50 mM NaCl). In some aspects, the medium comprises about 55 mM
potassium ion
and less than or equal to about 80 mM NaCl (e.g., about 80 mM, about 79 mM,
about 78 mM,
about 77 mM, about 76 mM, about 75 mM, about 70 mM, about 65 mM, about 60 mM,
about 55
mM, or about 50 mM NaCl). In some aspects, the medium comprises about 60 mM
potassium ion
and less than or equal to about 75 mM NaCl (e.g., about 75 mM, about 74 mM,
about 73 mM,
about 72 mM, about 71 mM, about 70 mM, about 65 mM, about 60 mM, about 55 mM,
or about
50 mM NaCl). In some aspects, the medium comprises about 65 mM potassium ion
and less than
or equal to about 70 mM NaCl (e.g., about 70 mM, about 69 mM, about 68 mM,
about 67 mM,
about 66 mM, about 65 mM, about 60 mM, about 55 mM, or about 50 mM NaCl). In
some aspects,
the medium comprises about 70 mM potassium ion and less than or equal to about
70 mM NaCl
(e.g., about 65 mM, about 64 mM, about 63 mM, about 62 mM, about 61 mM, about
60 mM, about
55 mM, or about 50 mM NaCl). In some aspects, the medium comprises about 75 mM
potassium
ion and less than or equal to about 60 mM NaC1 (e.g., about 60 mM, about 59
mM, about 58 mM,
about 57 mM, about 56 mM, about 55 mM, about 50 mM, about 45 mM, or about 40
mM NaCl).
In some aspects, the medium comprises about 80 mM potassium ion and less than
or equal to about
55 mM NaC1 (e.g., about 55 mM, about 54 mM, about 53 mM, about 52 mM, about 51
mM, about
50 mM, about 45 mM, about 40 mM, or about 35 mM NaC1) In some aspects, the
medium
comprises about 85 mM potassium ion and less than or equal to about 50 mM NaCl
(e.g., about 50
mM, about 49 mM, about 48 mM, about 47 mM, about 46 mM, about 45 mM, about 40
mM, about
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35 mM, or about 30 mM NaCl). In some aspects, the medium comprises about 90 mM
potassium
ion and less than or equal to about 45 mM NaCl (e.g., about 45 mM, about 44
mM, about 43 mM,
about 42 mM, about 41 mM, about 40 mM, about 35 mM, about 30 mM, or about 25
mM NaCl).
In some aspects, the medium comprises about 70 mM potassium ion and about 60
mM NaCl. In
some aspects, the medium comprises about 70 mM potassium ion and about 61 mM
NaCl. In some
aspects, the medium comprises about 70 mM potassium ion and about 62 mM NaCl.
[0296] In some aspects, the medium comprises about 50 mM
potassium ion and about 75
mM NaCl. In some aspects, the medium is hypotonic. In some aspects, the medium
is isotonic.
[0297] Some aspects of the present disclosure are directed to
methods of culturing cells,
e.g., pluripotent, multipotent, and/or immune cells (e.g., T cells, NK cells,
and/or TILs),
comprising placing the cells in a medium comprising (i) potassium ion at a
concentration higher
than 40 mM and (ii) NaCl at a concentration of less than about 100 mM. Certain
aspects of the
present disclosure are directed to methods of culturing T cells, comprising
placing the T cells in a
medium comprising (i) potassium ion at a concentration of at least about 50 mM
and (ii) NaCl at
a concentration of less than about 90 mM.
II.D. Saccharides
[0298] In some aspects, the metabolic reprograming media (MRM),
e.g., hyperkalemic
media, comprises a saccharide. In some aspects, the MRM is hypotonic. In some
aspects, the MRM
is isotonic. In some aspects, the target concentration of the saccharide is
reached by starting with
a basal medium comprising a higher concentration of the saccharide and
diluting the solution to
reach the target concentration of the saccharide. In some aspects, the target
concentration of the
saccharide is reached by raising the concentration of the saccharide by adding
the saccharide until
the desired concentration is reached.
[0299] In some aspects, the saccharide is a monosaccharide, a
disaccharide, or a
polysaccharide. In some aspects, the saccharide is selected from glucose,
fructose, galactose,
mannose, maltose, sucrose, lactose, trehalose, or any combination thereof. In
some aspects, the
saccharide is glucose. In some aspects, the MRM comprises (i) potassium ion at
a concentration of
at least about 30 mM to at least about 100 mM and (ii) glucose. In some
aspects, the MRM
comprises (i) potassium ion at a concentration higher than 40 mM and (ii)
glucose. In some aspects,
the MRM comprises (i) potassium ion at a concentration of at least about 30 mM
to at least about
100 mM and (ii) mannose. In some aspects, the 1V1R1VI comprises (i) potassium
ion at a
concentration of higher than 40 mM and (ii) mannose. In some aspects, the MRM
comprises (i)
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potassium ion at a concentration of at least about 50 mM and (ii) mannose. In
some aspects, the
MRIVI is hypotonic. In some aspects, the MRM is isotonic. In some aspects, the
MRM comprises
(i) potassium ion at a concentration higher than 40 mM and (ii) glucose;
wherein the total
concentration of potassium ion and NaC1 is between 110 mM and 140 mM. In some
aspects, the
MR1\4 comprises (i) potassium ion at a concentration higher than 50 mM and
(ii) glucose; wherein
the total concentration of potassium ion and NaCl is between 110 mM and 140
mM. In some
aspects, the MRM comprises (i) potassium ion at a concentration of at least
about 40 mM and (ii)
mannose; wherein the total concentration of potassium ion and NaC1 is between
110 mM and 140
mM. In some aspects, the MRM comprises (i) potassium ion at a concentration of
at least about 50
mM and (ii) mannose; wherein the total concentration of potassium ion and NaC1
is between 110
mM and 140 mM. In some aspects, the MRM comprises (i) potassium ion at a
concentration higher
than 40 mM and (ii) glucose; wherein the total concentration of potassium ion
and NaCl is between
110 mM and 140 mM. In some aspects, the MRM comprises (i) potassium ion at a
concentration
higher than 50 mM and (ii) glucose; wherein the total concentration of
potassium ion and NaC1 is
between 110 m1VI and 140 mM. In some aspects, the MRM comprises (i) potassium
ion at a
concentration of at least about 40 mM and (ii) mannose; wherein the total
concentration of
potassium ion and NaCl is between 110 mM and 140 mM.In some aspects, the MRM
comprises
(i) potassium ion at a concentration of at least about 50 mM and (ii) mannose;
wherein the total
concentration of potassium ion and NaCl is between 110 mM and 140 mM.
[0300] In some aspects, the concentration of the saccharide,
e.g., glucose, is about 10mM
to about 24 mM. In some aspects, the concentration of the saccharide, e.g.,
glucose, is less than
about 24 mM. In some aspects, the concentration of the saccharide, e.g.,
glucose, is more than
about 10 mM. In some aspects, the concentration of the saccharide, e.g.,
glucose, is about 5 mM.
In some aspects, the concentration of the saccharide, e.g., glucose, is from
about 5 mM to about 20
mM. In some aspects, the concentration of the saccharide, e.g., glucose, is
from about 10 mM to
about 20 mM. In some aspects, the concentration of the saccharide, e.g.,
glucose, is from about 10
mM to about 25 mM, about 10 mM to about 20 mM, about 10 mM to about 5 mM,
about 15 mM
to about 25 mM, about 15 mM to about 20 mM, about 15 mM to about 19 mM, about
15 mM to
about 18 mM, about 15 mM to about 17 mM, about 15 mM to about 16 mM, about 16
mM to about
20 mM, about 16 mM to about 19 mM, about 16 mM to about 18 mM, about 16 mM to
about 17
mM, about 17 mM to about 20 mM, about 17 mM to about 19 mM, or about 17 mM to
about 18
mM. In some aspects, the concentration of the saccharide, e.g., glucose, is
from about 5 mM to
about 20 mM. In some aspects, the concentration of the saccharide, e.g.,
glucose, is from about 10
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mM to about 20 mM. In some aspects, the concentration of the saccharide, e.g.,
glucose, is from
about 10 mM to about 15 mM. In some aspects, the concentration of the
saccharide, e.g., glucose,
is from about 14 mM to about 14.5 mM. In some aspects, the concentration of
the saccharide, e.g.,
glucose, is from about 14.5 mM to about 15 mM. In some aspects, the
concentration of the
saccharide, e.g., glucose, is from about 15 mM to about 15.5 mM. In some
aspects, the
concentration of the saccharide, e.g., glucose, is from about 15.5 mM to about
16 mM. In some
aspects, the concentration of the saccharide, e.g , glucose, is from about 16
mM to about 16.5 mM.
In some aspects, the concentration of the saccharide, e.g., glucose, is from
about 16.5 mM to about
17 mM. In some aspects, the concentration of the saccharide, e.g., glucose, is
from about 17 mM
to about 17.5 mM. In some aspects, the concentration of the saccharide, e.g.,
glucose, is from about
17.5 mM to about 18 mM.
[0301] In some aspects, the concentration of the saccharide,
e.g., glucose, is about 5 mM,
about 6 mM, about 7 mM, about 8 mM, about 9 mM, about 10 mM, is about 10.5 mM,
about 11
mM, about 11.5 mM, about 12 mM, about 12.5 mM, about 13 mM, about 13.5 mM,
about 14 mM,
about 14.5 mM, about 15 mM, about 15.5 mM, about 16 mM, about 16.5 mM, about
17 mM, about
17.5 mM, about 18 mM, about 18.5 mM, about 19 mM, about 19.5 mM, about 20 mM,
about 20.5
mM, about 21 mM, about 22 mM, about 23 mM, about 24 mM, or about 25 mM.
[0302] In some aspects, the concentration of the saccharide,
e.g., glucose, is about 5 mM.
In some aspects, the concentration of the saccharide, e.g., glucose, is about
6 mM. In some aspects,
the concentration of the saccharide, e.g., glucose, is about 7 mM. In some
aspects, the concentration
of the saccharide, e.g., glucose, is about 8 mM. In some aspects, the
concentration of the saccharide,
e.g., glucose, is about 9 mM. In some aspects, the concentration of the
saccharide, e.g., glucose, is
about 10 mM. In some aspects, the concentration of the saccharide, e.g.,
glucose, is about 10.5
mM. In some aspects, the concentration of the saccharide, e.g., glucose, is
about 11 mM. In some
aspects, the concentration of the saccharide, e.g., glucose, is about 11.5 mM.
In some aspects, the
concentration of the saccharide, e.g., glucose, is about 12 mM. In some
aspects, the concentration
of the saccharide, e.g., glucose, is about 12.5 mM. In some aspects, the
concentration of the
saccharide, e.g., glucose, is about 13 mM. In some aspects, the concentration
of the saccharide,
e.g., glucose, is about 13.5 mM. In some aspects, the concentration of the
saccharide, e.g., glucose,
is about 14 mM. In some aspects, the concentration of the saccharide, e.g.,
glucose, is about 14.5
mM. In some aspects, the concentration of the saccharide, e.g., glucose, is
about 15 mM. In some
aspects, the concentration of the saccharide, e.g., glucose, is about 15.4 mM.
In some aspects, the
concentration of the saccharide, e.g., glucose, is about 15.9 mM. In some
aspects, the concentration
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of the saccharide, e.g., glucose, is about 16.3 mM. In some aspects, the
concentration of the
saccharide, e.g., glucose, is about 16.8 mM. In some aspects, the
concentration of the saccharide,
e.g., glucose, is about 17.2 mM. In some aspects, the concentration of the
saccharide, e.g., glucose,
is about 17.7 mM.
11.E. Calcium
[0303] In some aspects, the MRM, e.g, hyperkalemic media,
comprises calcium ion. In
some aspects, the target concentration of calcium is reached by starting with
a basal medium
comprising a higher concentration of calcium ion and diluting the solution to
reach the target
concentration of calcium ion. In some aspects, the target concentration of
calcium is reached by
raising the concentration of calcium ion by adding one or more calcium salts.
Non-limiting
examples of calcium salts include calcium bromide, calcium carbonate, calcium
chloride, calcium
cyanamide, calcium fluoride, calcium hydride, calcium hydroxide, calcium
iodate, calcium iodide,
calcium nitrate, calcium nitrite, calcium oxalate, calcium perchlorate
tetrahydrate, calcium
phosphate monobasic, calcium phosphate tribasic, calcium sulfate, calcium
thiocyanate
tetrahydrate, hydroxyapatite, and any combination thereof In some aspects, the
calcium salt
comprises calcium chloride (CaCl2). In some aspects, the calcium salt
comprises calcium
gluconate.
[0304] In some aspects, the concentration of the calcium ion is
less than that of the basal
medium. In some aspects, the concentration of the calcium ion is greater than
that of the basal
medium. In some aspects, the concentration of calcium ion is more than about
0.4 mM. In some
aspects, the concentration of calcium ion is less than about 2.8 mM. In some
aspects, the
concentration of calcium ion is less than about 2.5 mM. In some aspects, the
concentration of
calcium ion is less than about 2.0 mM. In some aspects, the concentration of
calcium ion is less
than about 1.9 mM In some aspects, the concentration of calcium ion is less
than about 1.8 mM.
In some aspects, the concentration of calcium ion is less than about 1.7 mM.
In some aspects, the
concentration of calcium ion is less than about 1.6 mM. In some aspects, the
concentration of
calcium ion is less than about 1.5 mM. In some aspects, the concentration of
calcium ion is less
than about 1.4 mM. In some aspects, the concentration of calcium ion is less
than about 1.3 mM.
In some aspects, the concentration of calcium ion is less than about 1.2 mM.
In some aspects, the
concentration of calcium ion is less than about 1.1 mM. In some aspects, the
concentration of
calcium ion is less than about 1.0 mM.
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[0305] In some aspects, the concentration of calcium ion is from
about 0.4 mM to about
2.8 mM, about 0.4 mM to about 2.7 mM, about 0.4 mM to about 2.5 mM, about 0.5
mM to about
2.0 mM, about 1.0 mM to about 2.0 mM, about 1.1 mM to about 2.0 mM, about 1.2
mM to about
2.0 mM, about 1.3 mM to about 2.0 mM, about 1.4 mM to about 2.0 mM, about 1.5
mM to about
2.0 mM, about 1.6 mM to about 2.0 mM, about 1.7 mM to about 2.0 mM, about 1.8
mM to about
2.0 mM, about 0.8 to about 0.9 mM, about 0.8 to about 1.0 mM, about 0.8 to
about 1.1 mM, about
0.8 to about 1.2 mM, about 0.8 to about 1.3 mM, about 0.8 to about 1.4 mM,
about 0.8 to about
1.5 mM, about 0.8 to about 1.6 mM, about 0.8 to about 1.7 mM, about 0.8 to
about 1.8 mM, about
0.9 to about 1.0 mM, about 0.9 to about 1.1 mM, about 0.9 to about 1.2 mM,
about 0.9 to about
1.3 mM, about 0.9 to about 1.4 mM, about 0.9 to about 1.5 mM, about 0.9 to
about 1.6 mM, about
0.9 to about 1.7 mM, about 0.9 to about 1.8 mM, about 1.0 to about 1.1 mM,
about 1.0 to about
1.2 mM, about 1.0 to about 1.3 mM, about 1.0 to about 1.4 mM, about 1.0 to
about 1.5 mM, about
1.0 to about 1.6 mM, about 1.0 to about 1.7 mM, about 1.0 to about 1.8 mM,
about 1.1 to about
1.2 mM, about 1.1 to about 1.3 mM, about 1.1 to about 1.4 mM, about 1.1 to
about 1.5 mM, about
1.1 to about 1.6 mM, about 1.1 to about 1.7 mM, about 1.1 to about 1.8 mM,
about 1.2 to about
1.3 mM, about 1.2 to about 1.4 mM, about 1.2 to about 1.5 mM, about 1.2 to
about 1.6 mM, about
1.2 to about 1.7 mM, about 1.2 to about 1.8 mM, about 1.3 to about 1.4 mM,
about 1.3 to about
1.5 mM, about 1.3 to about 1.6 mM, about 1.3 to about 1.7 mM, about 1.3 to
about 1.8 mM, about
1.4 to about 1.5 mM, about 1.4 to about 1.6 mM, about 1.4 to about 1.7 mM,
about 1.4 to about
1.8 mM, about 1.5 to about 1.6 mM, about 1.5 to about 1.7 mM, about 1.5 to
about 1.8 mM, about
1.6 to about 1.7 mM, about 1.6 to about 1.8 mM, or about 1.7 to about 1.8 mM.
[0306] In some aspects, the concentration of calcium ion is from
about 0.8 mM to about
1.8 mM. In some aspects, the concentration of calcium ion is from about 0.9 mM
to about 1.8 mM.
In some aspects, the concentration of calcium ion is from about 1.0 mM to
about 1.8 mM. In some
aspects, the concentration of calcium ion is from about 1.1 mM to about 1.8
mM. In some aspects,
the concentration of calcium ion is from about 1.2 mM to about 1.8 mM. In some
aspects, the
concentration of calcium ion is from about 0.8 mM to about 1.8 mM. In some
aspects, the
concentration of calcium ion is from about 0.8 mM to about 0.9 mM. In some
aspects, the
concentration of calcium ion is from about 0.9 mM to about 1.0 mM. In some
aspects, the
concentration of calcium ion is from about 1.0 mM to about 1.1 mM. In some
aspects, the
concentration of calcium ion is from about 1.1 mM to about 1.2 mM. In some
aspects, the
concentration of calcium ion is from about 1.2 mM to about 1.3 mM. In some
aspects, the
concentration of calcium ion is from about 1.3 mM to about 1.4 mM. In some
aspects, the
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concentration of calcium ion is from about 1.4 mM to about 1.5 mM. In some
aspects, the
concentration of calcium ion is from about 1.5 mM to about 1.6 mM. In some
aspects, the
concentration of calcium ion is from about 1.7 mM to about 1.8 mM.
[0307] In some aspects, the concentration of calcium ion is
about 0.6 mM, about 0.7 mM,
about 0.8 mM, about 0.9 mM, about 1.0 mM, about 1.1 mM, about 1.2 mM, about
1.3 mM, about
1.4 mM, about 1.5 mM, about 1.6 mM, about 1.7 mM, about 1.8 mM, about 1.9 mM,
or about 2.0
mM. In some aspects, the concentration of calcium ion is about 0.6 mM. In some
aspects, the
concentration of calcium ion is about 0.7 mM. In some aspects, the
concentration of calcium ion
is about 0.8 mM. In some aspects, the concentration of calcium ion is about
0.9 mM. In some
aspects, the concentration of calcium ion is about 1.0 mM. In some aspects,
the concentration of
calcium ion is about 1.1 mM. In some aspects, the concentration of calcium ion
is about 1.2 mM.
In some aspects, the concentration of calcium ion is about 1.3 mM. In some
aspects, the
concentration of calcium ion is about 1.4 mM. In some aspects, the
concentration of calcium ion
is about 1.5 mM. In some aspects, the concentration of calcium ion is about
1.6 mM. In some
aspects, the concentration of calcium ion is about 1.7 mM. In some aspects,
the concentration of
calcium ion is about 1.8 mM.
[0308] In some aspects, the MRM comprises about 40 mM to about
90 mM potassium ion
and about 0.5 mM to about 2.8 mM calcium ion. In some aspects, the MRM
comprises about 40
mM to about 90 mM potassium ion, NaCl, and about 0.5 mM to about 2.8 mM
calcium ion; wherein
the total concentration of potassium ion and NaC1 is between 110 mM and 140
mM.
II.F. Cytokines
[0309] In some aspects, the MRM comprises a cytokine. In some
aspects, the MRM is
hypotonic. In some aspects, the MRM is isotonic. In some aspects, the cytokine
is selected from
IL-2, IL-7, IL-15, IL-21, and any combination thereof In some aspects, the MRM
does not
comprise IL-2. In some aspects, the MRM comprises IL2 and IL21. In some
aspects, the MRM
comprises IL2, IL21, and IL15.
[0310] The cytokine can be added to the MRM at any point. In
some aspects, the cytokine
is added to the MRM before the TILs (e.g., the tumor sample), are added to the
medium. In some
aspects, the TILs (e.g., the tumor sample), are cultured in the MR1VI
comprising (i) potassium at a
concentration disclosed herein, and (ii) a cytokine throughout T1L culture
including expansion. In
some aspects, the TILs (e.g., the tumor sample), are cultured in the MRM
comprising (i) potassium
at a concentration disclosed herein, and (ii) a cytokine throughout TIL
expansion.
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[0311] In some aspects, the MR1\4 comprises (i) at least about
30 mM to at least about 100
mM potassium ion and (ii) IL-2. In some aspects, the MRM comprises (i) more
than 40 mM
potassium ion and (ii) IL-2. In some aspects, the MRM comprises (i) at least
about 50 mM
potassium ion and (ii) IL-2. In some aspects, the MRM comprises (i) at least
about 30 mM to at
least about 100 mM potassium ion and (ii) IL-7. In some aspects, the MRM
comprises (i) more
than 40 mM potassium ion and (ii) IL-7. In some aspects, the MRM comprises (i)
at least about 50
mM potassium ion and (ii) IL-7. In some aspects, the MRM comprises (i) at
least about 30 mM to
at least about 100 mM potassium ion and (ii) IL-15. In some aspects, the MRM
comprises (i) more
than 40 mM potassium ion and (ii) IL-15. In some aspects, the MRM comprises
(i) at least about
50 mM potassium ion and (ii) IL-15. In some aspects, the MRM comprises (i) at
least about 30
mM to at least about 100 mM potassium ion and (ii) IL-21 In some aspects, the
MRM comprises
(i) more than 40 mM potassium ion and (ii) IL-21. In some aspects, the MRM
comprises (i) at least
about 50 mM potassium ion and (ii) IL-21. In some aspects, the MRM does not
comprise IL-7
and/or IL-15.
[0312] In some aspects, the MRM comprises (i) at least about 30
mM to at least about 100
mM potassium ion, (ii) NaCl, and (iii) IL-2; wherein the total concentration
of potassium ion and
NaCl is from 110 mM to 140 mM. In some aspects, the MRM comprises (i) more
than 40 mM
potassium ion, (ii) NaCl, and (iii) IL-2; wherein the total concentration of
potassium ion and NaCl
is from 110 mM to 140 mM. In some aspects, the MRM comprises (i) at least
about 50 mM
potassium ion, (ii) NaCl, and (iii) IL-2; wherein the total concentration of
potassium ion and NaCl
is from 110 mM to 140 mM. In some aspects, the MRM comprises (i) at least
about 30 mM to at
least about 100 mM potassium ion, (ii) NaCl, and (iii) IL-7; wherein the total
concentration of
potassium ion and NaCl is from 110 mM to 140 mM. In some aspects, the MRM
comprises (i)
more than 40 mM potassium ion, (ii) NaCl, and (iii) IL-7; wherein the total
concentration of
potassium ion and NaC1 is from 110 mM to 140 mM. In some aspects, the MRM
comprises (i) at
least about 50 mM potassium ion, (ii) NaC1, and (iii) IL-7; wherein the total
concentration of
potassium ion and NaC1 is from 110 mM to 140 mM. In some aspects, the MRM
comprises (i) at
least about 30 mM to at least about 100 mM potassium ion, (ii) NaCl, and (iii)
IL-15; wherein the
total concentration of potassium ion and NaCl is from 110 mM to 140 mM. In
some aspects, the
MRM comprises (i) more than 40 mM potassium ion, (ii) NaC1, and (iii) IL-15;
wherein the total
concentration of potassium ion and NaC1 is from 110 mM to 140 mM. In some
aspects, the MRM
comprises (i) at least about 50 mM potassium ion, (ii) NaCl, and (iii) IL-15;
wherein the total
concentration of potassium ion and NaCl is from 110 mM to 140 mM. In some
aspects, the MRM
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comprises (i) at least about 30 mM to at least about 100 mM potassium ion,
(ii) NaCl, and (iii) IL-
21; wherein the total concentration of potassium ion and NaCl is from 110 mM
to 140 mM. In
some aspects, the MIRM comprises (i) more than 40 mM potassium ion, (ii) NaCl,
and (iii) IL-21;
wherein the total concentration of potassium ion and NaCl is from 110 mM to
140 mM. In some
aspects, the MRM comprises (i) at least about 50 mM potassium ion, (ii) NaCl,
and (iii) IL-21;
wherein the total concentration of potassium ion and NaCl is from 110 mM to
140 mM. In some
aspects, the MRM does not comprise IL-7 and/or IL-15; wherein the total
concentration of
potassium ion and NaCl is from 110 mM to 140 mM.
[0313] In some aspects, the MRM comprises at least about 0.1
ng/mL IL-2. In some
aspects, the MRM comprises from about 50 ng/mL to about 600 ng/mL, about 50
ng/mL to about
500 ng/mL, about 50 ng/mL to about 450 ng/mL, about 50 ng/mL to about 400
ng/mL, about 50
ng/mL to about 350 ng/mL, about 50 ng/mL to about 300 ng/mL, about 100 ng/mL
to about 600
ng/mL, about 100 ng/mL to about 500 ng/mL, about 100 ng/mL to about 450 ng/mL,
about 100
ng/mL to about 400 ng/mL, about 100 ng/mL to about 350 ng/mL, about 100 ng/mL
to about 300
ng/mL, about 200 ng/mL to about 500 ng/mL, about 200 ng/mL to about 450 ng/mL,
about 200
ng/mL to about 400 ng/mL, about 200 ng/mL to about 350 ng/mL, about 200 ng/mL
to about 300
ng/mL, about 250 ng/mL to about 350 ng/mL, about 300 ng/mL to about 600 ng/mL,
about 300
ng/mL to about 500 ng/mL, about 300 ng/mL to about 450 ng/mL, about 300 ng/mL
to about 400
ng/mL, about 300 ng/mL to about 350 ng/mL, about 250 ng/mL to about 300 ng/mL,
or about 275
ng/mL to about 325 ng/mL IL-2.
[0314] In some aspects, the MRM comprises at least about 50
ng/mL, at least about 60
ng/mL, at least about 70 ng/mL, at least about 80 ng/mL, at least about 90
ng/mL, at least about
100 ng/mL, at least about 110 ng/mL, at least about 120 ng/mL, at least about
130 ng/mL, at least
about 140 ng/mL, at least about 150 ng/mL, at least about 160 ng/mL, at least
about 170 ng/mL, at
least about 180 ng/mL, at least about 190 ng/mL, at least about 200 ng/mL, at
least about 210
ng/mL, at least about 220 ng/mL, at least about 230 ng/mL, at least about 240
ng/mL, at least about
250 ng/mL, at least about 260 ng/mL, at least about 270 ng/mL, at least about
280 ng/mL, at least
about 290 ng/mL, at least about 300 ng/mL, at least about 310 ng/mL, at least
about 320 ng/mL, at
least about 330 ng/mL, at least about 340 ng/mL, at least about 350 ng/mL, at
least about 360
ng/mL, at least about 370 ng/mL, at least about 380 ng/mL, at least about 390
ng/mL, at least about
400 ng/mL, at least about 410 ng/mL, at least about 420 ng/mL, at least about
430 ng/mL, at least
about 440 ng/mL, at least about 450 ng/mL, at least about 460 ng/mL, at least
about 470 ng/mL, at
least about 480 ng/mL, at least about 490 ng/mL, at least about 500 ng/mL, at
least about 510
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ng/mL, at least about 520 ng/mL, at least about 530 ng/mL, at least about 540
ng/mL, at least about
550 ng/mL, at least about 560 ng/mL, at least about 570 ng/mL, at least about
580 ng/mL, at least
about 590 ng/mL, or at least about 600 ng/mL IL-2. In some aspects, the MRM
comprises at least
about 50 ng/mL IL-2. In some aspects, the MRIVI comprises at least about 60
ng/mL IL-2. In some
aspects, the MRM comprises at least about 70 ng/mL IL-2. In some aspects, the
MRM_ comprises
at least about 73.6 ng/mL IL-2. In some aspects, the MRM comprises at least
about 75 ng/mL IL-
2. In some aspects, the MRM comprises at least about 80 ng/mL IL-2. In some
aspects, the MRM
comprises at least about 90 ng/mL IL-2. In some aspects, the MRM comprises at
least about 100
ng/mL IL-2. In some aspects, the MRM comprises at least about 200 ng/mL IL-2.
In some aspects,
the MRM comprises at least about 300 ng/mL IL-2. In some aspects, the MRM
comprises at least
about 400 ng/mL IL-2. In some aspects, the MRM comprises at least about 500
ng/mL IL-2. In
some aspects, the MR1VI comprises at least about 600 ng/mL IL-2.
[0315] In some aspects, the MRIVI comprises at least about 1500
IU/mL IL-2. In some
aspects, the MRM comprises from about 1500 IU/mL to about 12,000 IU/mL IL-2.
In some
aspects, the MR1V1 comprises at least about 1500 IU/mL, at least about 1600
IU/mL, at least about
1700 IU/mL, at least about 1800 IU/mL, at least about 1900 IU/mL, at least
about 2000 IU/mL, at
least about 2100 IU/mL, at least about 2200 IU/mL, at least about 2300 IU/mL,
at least about 2400
IU/mL, at least about 2500 IU/mL, at least about 2600 IU/mL, at least about
2700 IU/mL, at least
about 2800 IU/mL, at least about 2900 IU/mL, at least about 3000 IU/mL, at
least about 3100
IU/mL, at least about 3200 IU/mL, at least about 3300 IU/mL, at least about
3400 IU/mL, at least
about 3500 IU/mL, at least about 3600 IU/mL, at least about 3700 IU/mL, at
least about 3800
IU/mL, at least about 3900 IU/mL, at least about 4000 IU/mL, at least about
4100 IU/mL, at least
about 4200 IU/mL, at least about 4300 IU/mL, at least about 4400 IU/mL, at
least about 4500
IU/mL, at least about 4600 IU/mL, at least about 4700 IU/mL, at least about
4800 IU/mL, at least
about 4900 IU/mL, at least about 5000 IU/mL, at least about 5100 IU/mL, at
least about 5200
IU/mL, at least about 5300 IU/mL, at least about 5400 IU/mL, at least about
5500 IU/mL, at least
about 5600 IU/mL, at least about 5700 IU/mL, at least about 5800 IU/mL, at
least about 5900
IU/mL, at least about 6000 IU/mL, at least about 6100 IU/mL, at least about
6200 IU/mL, at least
about 6300 IU/mL, at least about 6400 IU/mL, at least about 6500 IU/mL, at
least about 6600
IU/mL, at least about 6700 IU/mL, at least about 6800 IU/mL, at least about
6900 IU/mL, at least
about 7000 IU/mL IL-2, at least about 7100 IU/mL, at least about 7200 IU/mL,
at least about 7300
IU/mL, at least about 7400 IU/mL, at least about 7500 IU/mL, at least about
7600 IU/mL, at least
about 7700 IU/mL, at least about 7800 IU/mL, at least about 7900 IU/mL, or at
least about 8000
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IU/mL IL-2. In some aspects, the MRM comprises at least about 3000 IU/mL IL-2.
In some
aspects, TILs are cultured in MRM during a second culture (e.g., REP culture),
as described herein,
wherein the MRM comprises about 3000 IU/mL. In some aspects, the MRM comprises
at least
about 6000 IU/mL IL-2. In some aspects, TILs are cultured in MRM during an
initial culture, as
described herein, wherein the MRM comprises about 6000 IU/mL.
[0316] In some aspects, the MRM comprises at least about 0.1
ng/mL IL-21. In some
aspects, the MRM comprises from about 0.1 ng/mL to about 30 ng/mL, about 1
ng/mL to about 30
ng/mL, about 1 ng/mL to about 25 ng/mL, about 1 ng/mL to about 20 ng/mL, about
1 ng/mL to
about 15 ng/mL, about 1 ng/mL to about 10 ng/mL, about 5 ng/mL to about 30
ng/mL, about 5
ng/mL to about 20 ng/mL, about 10 ng/mL to about 30 ng/mL, about 10 ng/mL to
about 20 ng/mL,
or about 15 ng/mL to about 30 ng/mL IL-21.
[0317] In some aspects, the MRM comprises at least about 0.1
ng/mL, at least about 0.5
ng/mL, at least about 1 ng/mL, at least about 2 ng/mL, at least about 3 ng/mL,
at least about 4
ng/mL, at least about 5 ng/mL, at least about 6 ng/mL, at least about 7 ng/mL,
at least about 8
ng/mL, at least about 9 ng/mL, at least about 10 ng/mL, at least about 11
ng/mL, at least about 12
ng/mL, at least about 13 ng/mL, at least about 14 ng/mL, at least about 15
ng/mL, at least about 16
ng/mL, at least about 17 ng/mL, at least about 18 ng/mL, at least about 19
ng/mL, at least about 20
ng/mL, at least about 25 ng/mL, at least about 30 ng/mL, at least about 35
ng/mL, or at least about
40 ng/mL IL-21. In some aspects, the 1VIRIVI comprises at least about 1.0
ng/mL IL-21. In some
aspects, the MRM comprises at least about 2.0 ng/mL IL-21. In some aspects,
the MRM comprises
at least about 3.0 ng/mL IL-21. In some aspects, the MRM comprises at least
about 4.0 ng/mL IL-
21. In some aspects, the MRM comprises at least about 5.0 ng/mL IL-21. In some
aspects, the
MRM comprises at least about 6.0 ng/mL IL-21. In some aspects, the MRM
comprises at least
about 7.0 ng/mL IL-21. In some aspects, the MRNI comprises at least about 8.0
ng/mL IL-21. In
some aspects, the MRM comprises at least about 9.0 ng/mL IL-21. In some
aspects, the MRM
comprises at least about 10 ng/mL IL-21. In some aspects, the MRM comprises at
least about 15
ng/mL IL-21. In some aspects, the MRM comprises at least about 20 ng/mL IL-21.
In some aspects,
the MRM comprises at least about 25 ng/mL IL-21. In some aspects, the MRM
comprises at least
about 30 ng/mL IL-21. In some aspects, the MRM comprises at least about 35
ng/mL IL-21.
[0318] In some aspects, the MRM comprises between about 50 IU/mL
to about 500 IU/mL
of IL-21. In some aspects, the MRM comprises about 50 IU/mL, about 60 IU/mL,
about 70 IU/mL,
about 80 IU/mL, about 90 IU/mL, about 100 IU/mL, about 125 IU/mL, about 150
IU/mL, about
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175 IU/mL, about 200 IU/mL, about 225 IU/mL, about 250 IU/mL, about 275 IU/mL,
about 300
IU/mL, about 350 IU/mL, about 400 IU/mL, about 450 IU/mL, or about 500 IU/mL
of IL-21.
[0319] In some aspects, the MRIVI comprises at least about 0.1
ng/mL IL-7. In some
aspects, the MRM comprises from about 0.1 ng/mL to about 20 ng/mL, about 1
ng/mL to about 20
ng/mL, about 1 ng/mL to about 15 ng/mL, about 1 ng/mL to about 14 ng/mL, about
1 ng/mL to
about 13 ng/mL, about 1 ng/mL to about 12 ng/mL, about 1 ng/mL to about 11
ng/mL, about 1
ng/mL to about 10 ng/mL, about 1 ng/mL to about 9 ng/mL, about 1 ng/mL to
about 8 ng/mL,
about 1 ng/mL to about 7 ng/mL, about 1 ng/mL to about 6 ng/mL, about 1 ng/mL
to about 5
ng/mL, about 1 ng/mL to about 4 ng/mL, about 1 ng/mL to about 3 ng/mL, about 1
ng/mL to about
2 ng/mL, about 5 ng/mL to about 15 ng/mL, about 5 ng/mL to about 10 ng/mL,
about 10 ng/mL to
about 20 ng/mL, about 10 ng/mL to about 15 ng/mL, or about 15 ng/mL to about
20 ng/mL IL-7.
[0320] In some aspects, the MRM comprises at least about 0.1
ng/mL, at least about 0.5
ng/mL, at least about 1 ng/mL, at least about 1.3 ng/mL, at least about 1.5
ng/mL, at least about
1.7 ng/mL, at least about 2 ng/mL, at least about 2.3 ng/mL, at least about
2.5 ng/mL, at least about
2.7 ng/mL, at least about 3 ng/mL, at least about 3.3 ng/mL, at least about
3.5 ng/mL, at least about
3.7 ng/mL, at least about 4 ng/mL, at least about 4.3 ng/mL, at least about
4.5 ng/mL, at least about
4.7 ng/mL, at least about 5 ng/mL, at least about 5.3 ng/mL, at least about
5.5 ng/mL, at least about
5.7 ng/mL, at least about 6 ng/mL, at least about 7 ng/mL, at least about 8
ng/mL, at least about 9
ng/mL, at least about 10 ng/mL, at least about 11 ng/mL, at least about 12
ng/mL, at least about 13
ng/mL, at least about 14 ng/mL, at least about 15 ng/mL, at least about 16
ng/mL, at least about 17
ng/mL, at least about 18 ng/mL, at least about 19 ng/mL, or at least about 20
ng/mL IL-7. In some
aspects, the medium comprises at least about 1.0 ng/mL IL-7. In some aspects,
the MRM comprises
at least about 2.0 ng/mL IL-7. In some aspects, the MRM comprises at least
about 2.3 ng/mL IL-
7. In some aspects, the 1\'IR1VI comprises at least about 2.5 ng/mL IL-7. In
some aspects, the MRM
comprises at least about 2.7 ng/mL IL-7. In some aspects, the MRM comprises at
least about 3.0
ng/mL IL-7. In some aspects, the MRM comprises at least about 3.3 ng/mL IL-7.
In some aspects,
the MRM comprises at least about 3.5 ng/mL IL-7. In some aspects, the MRM
comprises at least
about 3.7 ng/mL IL-7.
[0321] In some aspects, the MRM comprises between about 500
IU/mL to about 1,500
IU/mL of IL-7. In some aspects, the MRM comprises about 500 IU/mL, about 550
IU/mL, about
600 IU/mL, about 650 IU/mL, about 700 IU/mL, about 750 IU/mL, about 800 IU/mL,
about 850
IU/mL, about 900 IU/mL, about 950 IU/mL, about 1,000 IU/mL, about 1,050 IU/mL,
about 1,100
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IU/mL, about 1,150 IU/mL, about 1,200 IU/mL, about 1,250 IU/mL, about 1,300
IU/mL, about
1,350 IU/mL, about 1,400 IU/mL, about 1,450 IU/mL, or about 1,500 IU/mL of IL-
7.
[0322]
In some aspects, the MRM comprises at least about 0.1 ng/mL IL-15. In
some
aspects, the MRM comprises from about 0.1 ng/mL to about 20 ng/mL, about I
ng/mL to about 20
ng/mL, about 1 ng/mL to about 15 ng/mL, about 1 ng/mL to about 14 ng/mL, about
1 ng/mL to
about 13 ng/mL, about 1 ng/mL to about 12 ng/mL, about 1 ng/mL to about 11
ng/mL, about 1
ng/mL to about 10 ng/mL, about 1 ng/mL to about 9 ng/mL, about 1 ng/mL to
about 8 ng/mL,
about 1 ng/mL to about 7 ng/mL, about 1 ng/mL to about 6 ng/mL, about 1 ng/mL
to about 5
ng/mL, about 1 ng/mL to about 4 ng/mL, about 1 ng/mL to about 3 ng/mL, about 1
ng/mL to about
2 ng/mL, about 5 ng/mL to about 15 ng/mL, about 5 ng/mL to about 10 ng/mL,
about 10 ng/mL to
about 20 ng/mL, about 10 ng/mL to about 15 ng/mL, or about 15 ng/mL to about
20 ng/mL IL-15.
[0323]
In some aspects, the MRM comprises at least about 0.1 ng/mL, at least
about 0.2
ng/mL, at least about 0.3 ng/mL, at least about 0.4 ng/mL, at least about 0.5
ng/mL, at least about
0.6 ng/mL, at least about 0.7 ng/mL, at least about 0.8 ng/mL, at least about
0.9 ng/mL, at least
about 1 ng/mL, at least about 2 ng/mL, at least about 3 ng/mL, at least about
4 ng/mL, at least
about 5 ng/mL, at least about 6 ng/mL, at least about 7 ng/mL, at least about
8 ng/mL, at least
about 9 ng/mL, at least about 10 ng/mL, at least about 11 ng/mL, at least
about 12 ng/mL, at least
about 13 ng/mL, at least about 14 ng/mL, at least about 15 ng/mL, at least
about 16 ng/mL, at least
about 17 ng/mL, at least about 18 ng/mL, at least about 19 ng/mL, or at least
about 20 ng/mL IL-
15. In some aspects, the MRM comprises at least about 0.1 ng/mL IL-15. In some
aspects, the
MRM comprises at least about 0.2 ng/mL
In some aspects, the MRM comprises at least
about 0.3 ng/mL In some aspects, the MRM comprises at least about
0.4 ng/mL In
some aspects, the MR1V1 comprises at least about 0.5 ng/mL
In some aspects, the MRM
comprises at least about 0.6 ng/mL IL-15. In some aspects, the MRM comprises
at least about 0.7
ng/mL IL-15. In some aspects, the MRM comprises at least about 0.8 ng/mL IL-
15. In some
aspects, the MRM comprises at least about 0.9 ng/mL IL-15. In some aspects,
the MRM comprises
at least about 1.0 ng/mL IL-15. In some aspects, the MRM comprises at least
about 2.0 ng/mL IL-
15. In some aspects, the MRM comprises at least about 3.0 ng/mL 1L-15. In some
aspects, the
MRM comprises at least about 4.0 ng/mL IL-15. In some aspects, the MRM
comprises at least
about 5.0 ng/mL IL-15. In some aspects, the MRM comprises at least about 6.0
ng/mL IL-15. In
some aspects, the MRM comprises at least about 7.0 ng/mL
In some aspects, the MRM
comprises at least about 8.0 ng/mL IL-15. In some aspects, the MR_M comprises
at least about 9.0
ng/mL IL-15. In some aspects, the MRM comprises at least about 10 ng/mL IL-15.
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[0324]
In some aspects, the MRM comprises between about 50 IU/mL to about
500 IU/mL
of IL-15. In some aspects, the MRM comprises about 50 IU/mL, about 60 IU/mL,
about 70 IU/mL,
about 80 IU/mL, about 90 IU/mL, about 100 IU/mL, about 125 IU/mL, about 150
IU/mL, about
175 IU/mL, about 200 IU/mL, about 225 IU/mL, about 250 IU/mL, about 275 IU/mL,
about 300
IU/mL, about 350 IU/mL, about 400 IU/mL, about 450 IU/mL, or about 500 IU/mL
of IL-15.
[0325]
In some aspects, the MRM comprises at least about 30 mM to at least
about 100
mM potassium ion and about 300 ng/mL IL-2. In some aspects, the MRM comprises
more than 40
mM potassium ion and about 300 ng/mL IL-2. In some aspects, the MRM comprises
at least about
45 mM potassium ion and about 300 ng/mL IL-2 In some aspects, the MRM
comprises at least
about 50 mM potassium ion and about 300 ng/mL IL-2. In some aspects, the MRM
comprises at
least about 55 mM potassium ion and about 300 ng/mL IL-2_ In some aspects, the
MR_M comprises
at least about 60 mM potassium ion and about 300 ng/mL IL-2. In some aspects,
the MRM
comprises at least about 65 mM potassium ion and about 300 ng/mL IL-2. In some
aspects, the
MRM comprises at least about 70 mM potassium ion and about 300 ng/mL IL-2. In
some aspects,
the MRM comprises at least about 75 mM potassium ion and about 300 ng/mL IL-2.
In some
aspects, the MRM comprises at least about 80 mM potassium ion and about 300
ng/mL IL-2. In
some aspects, the MRM comprises at least about 85 mM potassium ion and about
300 ng/mL IL-
2. In some aspects, the MRM comprises at least about 90 mM potassium ion and
about 300 ng/mL
IL-2. In some aspects, the MRM comprises (i) at least about 70 mM potassium
ion, (ii) about 60
mM sodium, (iii) about 1.4 mM calcium, (iv) about 16 mM glucose, and (v) about
10 ng/mL IL-2.
[0326]
In some aspects, the MRM comprises at least about 30 mM to at least
about 100
mM potassium ion, about 300 ng/mL IL-2, and about 290 ng/mL IL-7. In some
aspects, the MRM
comprises more than 40 mM potassium ion and about 300 ng/mL IL-2 and about 290
ng/mL IL-7.
In some aspects, the MRM comprises at least about 45 mM potassium ion, about
300 ng/mL IL-2,
and about 290 ng/mL IL-7. In some aspects, the MRIVI comprisess at least about
40 mM potassium
ion, about 300 ng/mL IL-2, and about 290 ng/mL IL-7. In some aspects, the MRM
comprises at
least about 55 mM potassium ion, about 300 ng/mL IL-2, and about 290 ng/mL
In some
aspects, the MRM comprises at least about 60 mM potassium ion, about 300 ng/mL
IL-2, and about
290 ng/mL IL-7. In some aspects, the MRM comprises at least about 65 mM
potassium ion, about
300 ng/mL IL-2, and about 290 ng/mL IL-7. In some aspects, the MRM comprises
at least about
70 mM potassium ion, about 300 ng/mL IL-2, and about 290 ng/mL IL-7. In some
aspects, the
MR_M comprises at least about 75 mM potassium ion, about 300 ng/mL IL-2, and
about 290 ng/mL
IL-7. In some aspects, the MRM comprises at least about 80 mM potassium ion,
about 300 ng/mL
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IL-2, and about 290 ng/mL IL-7. In some aspects, the MRNI comprises at least
about 85 mM
potassium ion, about 300 ng/mL IL-2, and about 290 ng/mL IL-7. In some
aspects, the MRM
comprises at least about 90 mM potassium ion, about 300 ng/mL IL-2, and about
290 ng/mL IL-7.
In some aspects, the MRM comprises (i) at least about 70 mM potassium ion,
(ii) about 60 mM
sodium, (iii) about 1.4 mM calcium, (iv) about 16 mM glucose, (v) about 300
ng/mL 1L-2, and (vi)
about 290 ng/mL IL-7.
[0327]
In some aspects, the MRM comprises at least about 30 mM to at least
about 100
mM potassium ion, about 300 ng/mL IL-2, and about 0.4 ng/mL IL-15. In some
aspects, the MR_M
comprises more than 40 mM potassium ion, about 300 ng/mL IL-2, and about 0.4
ng/mL IL-15. In
some aspects, the MR_I\4 comprises at least about 45 mM potassium ion, about
300 ng/mL IL-2,
and about 0.4 ng/mL IL-15. In some aspects, the MRM comprises at least about
50 mM potassium
ion, about 300 ng/mL IL-2, and about 0.4 ng/mL IL-15. In some aspects, the MRM
comprises at
least about 55 mM potassium ion, about 300 ng/mL IL-2, and about 0.4 ng/mL IL-
15. In some
aspects, the MRM comprises at least about 60 mM potassium ion, about 300 ng/mL
IL-2, and about
0.4 ng/mL IL-15. In some aspects, the MRM comprises at least about 65 mM
potassium ion, about
300 ng/mL IL-2, and about 0.4 ng/mL IL-15. In some aspects, the MRM comprises
at least about
70 mM potassium ion, about 300 ng/mL IL-2, and about 0.4 ng/mL IL-15. In some
aspects, the
MRM comprises at least about 75 mM potassium ion, about 300 ng/mL IL-2, and
about 0.4 ng/mL
IL-15. In some aspects, the MR_I\4 comprises at least about 80 mM potassium
ion, about 300 ng/mL
IL-2, and about 0.4 ng/mL IL-15. In some aspects, the MRM comprises at least
about 85 mM
potassium ion, about 300 ng/mL IL-2, and about 0.4 ng/mL IL-15. In some
aspects, the MRM
comprises at least about 90 mM potassium ion, about 300 ng/mL IL-2, and about
0.4 ng/mL IL-
15. In some aspects, the MRNI comprises (i) at least about 70 mM potassium
ion, (ii) about 60 mM
sodium, (iii) about 1.4 mM calcium, (iv) about 16 mM glucose, (v) about 300
ng/mL IL-2, and (vi)
about 0.4 ng/mL IL-15.
[0328]
In some aspects, the MRM comprises at least about 30 mM to at least
about 100
mM potassium ion, about 300 ng/mL IL-2, about 290 ng/mL IL-7, and about 0.4
ng/mL In
some aspects, the MRM comprises more than 40 mM potassium ion, about 300 ng/mL
IL-2, about
290 ng/mL IL-7, and about 0.4 ng/mL IL-15. In some aspects, the MRM comprises
at least about
45 mM potassium ion, about 300 ng/mL IL-2, about 290 ng/mL IL-7, and about 0.4
ng/mL IL-15.
In some aspects, the MRM comprises at least about 50 mM potassium ion, about
300 ng/mL
about 290 ng/mL IL-7, and about 0.4 ng/mL IL-15. In some aspects, the MRM
comprises at least
about 55 mM potassium ion, about 300 ng/mL IL-2, about 290 ng/mL IL-7, and
about 0.4 ng/mL
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IL-15. In some aspects, the MR_M comprises at least about 60 mM potassium ion,
about 300 ng/mL
IL-2, about 290 ng/mL IL-7, and about 0.4 ng/mL IL-15. In some aspects, the
MRM comprises at
least about 65 mM potassium ion, about 300 ng/mL IL-2, about 290 ng/mL IL-7,
and about 0.4
ng/mL IL-I5. In some aspects, the MRM comprises at least about 70 mM potassium
ion, about
300 ng/mL IL-2, about 290 ng/mL IL-7, and about 0.4 ng/mL IL-15. In some
aspects, the MRM
comprises at least about 75 mM potassium ion and about 10 ng/mL IL-2, about 1
ng/mL IL-7, and
about 1 ng/mL IL-15. In some aspects, the MRM comprises at least about 80 mM
potassium ion,
about 300 ng/mL IL-2, about 290 ng/mL IL-7, and about 0.4 ng/mL IL-15. In some
aspects, the
MRM comprises at least about 85 mM potassium ion, about 300 ng/mL IL-2, about
290 ng/mL IL-
7, and about 0.4 ng/mL IL-15. In some aspects, the MRM comprises at least
about 90 mM
potassium ion, about 300 ng/mL IL-2, about 290 ng/mL IL-7, and about 0.4 ng/mL
IL-15. In some
aspects, the MRM comprises (i) at least about 70 mM potassium ion, (ii) about
60 mM sodium,
(iii) about 1.4 mM calcium, (iv) about 16 mM glucose, (v) about 300 ng/mL IL-
2, (vi) about 290
ng/mL IL-7, and (vii) about 0.4 ng/mL IL-15.
[0329] In some aspects, the MR1VI comprises at least about 30 mM
to at least about 100 5
mM potassium ion, about 300 ng/mL IL-2, and about 30 ng/mL IL-21. In some
aspects, the MRM
comprises more than 40 mM potassium ion, about 300 ng/mL IL-2, and about 30
ng/mL IL-21. In
some aspects, the MRM comprises at least about 45 mM potassium ion, about 300
ng/mL IL-2,
and about 30 ng/mL IL-21. In some aspects, the MRM comprises at least about 50
mM potassium
ion, about 300 ng/mL IL-2, and about 30 ng/mL IL-21. In some aspects, the MIRM
comprises at
least about 55 mM potassium ion, about 300 ng/mL IL-2, and about 30 ng/mL IL-
21. In some
aspects, the MRM comprises at least about 60 mM potassium ion, about 300 ng/mL
IL-2, and about
30 ng/mL IL-21. In some aspects, the MRM comprises at least about 65 mM
potassium ion, about
300 ng/mL IL-2, and about 30 ng/mL IL-21. In some aspects, the MR_IVI
comprises at least about
70 mM potassium ion, about 300 ng/mL IL-2, and about 30 ng/mL IL-21. In some
aspects, the
MRNI comprises at least about 75 mM potassium ion, about 300 ng/mL IL-2, and
about 30 ng/mL
IL-21. In some aspects, the MRM comprises at least about 80 mM potassium ion,
about 300 ng/mL
IL-2, and about 30 ng/mL IL-21. In some aspects, the MRM comprises at least
about 85 mM
potassium ion, about 300 ng/mL IL-2, and about 30 ng/mL IL-21. In some
aspects, the MRM
comprises at least about 90 mM potassium ion, about 300 ng/mL IL-2, and about
30 ng/mL IL-21.
In some aspects, the MRM comprises (i) at least about 70 mM potassium ion,
(ii) about 60 mM
sodium, (iii) about 1.4 mM calcium, (iv) about 16 mM glucose, (v) about 300
ng/mL IL-2, and (vi)
about 30 ng/mL IL-21.
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[0330] In some aspects, the MRM comprises at least about 30 mM
to at least about 100
mM potassium ion, about 290 ng/mL IL-7, and about 20 ng/mL IL-2L In some
aspects, the MRM
comprises more than 40 mM potassium ion, about 290 ng/mL IL-7, and about 20
ng/mL IL-21. In
some aspects, the MRM comprises at least about 45 mM potassium ion, about 290
ng/mL IL-7,
and about 20 ng/mL IL-21. In some aspects, the MRM comprises at least about 50
mM potassium
ion, about 290 ng/mL IL-7, and about 20 ng/mL IL-21. In some aspects, the MRM
comprises at
least about 55 mM potassium ion, about 290 ng/mL IL-7, and about 20 ng/mL IL-
21. In some
aspects, the MRM comprises at least about 60 mM potassium ion, about 290 ng/mL
IL-7, and about
20 ng/mL IL-21. In some aspects, the MRM comprises at least about 65 mM
potassium ion, about
290 ng/mL IL-7, and about 20 ng/mL IL-21. In some aspects, the MR_IVI
comprises at least about
70 mM potassium ion, about 290 ng/mL IL-7, and about 20 ng/mL IL-21. In some
aspects, the
MR1VI comprises at least about 75 mM potassium ion, about 290 ng/mL IL-7, and
about 20 ng/mL
IL-21. In some aspects, the MR_I\4 comprises at least about 80 mM potassium
ion, about 290 ng/mL
IL-7, and about 20 ng/mL IL-21. In some aspects, the MRM comprises at least
about 85 mM
potassium ion, about 290 ng/mL IL-7, and about 20 ng/mL IL-21. In some
aspects, the MRM
comprises at least about 90 mM potassium ion, about 290 ng/mL IL-7, and about
20 ng/mL IL-21.
In some aspects, the MRM comprises (i) at least about 70 mM potassium ion,
(ii) about 60 mM
sodium, (iii) about 1.4 mM calcium, (iv) about 16 mM glucose, (v) about 290
ng/mL IL-7, and (vi)
about 20 ng/mL IL-21.
[0331] In some aspects, the MRM comprises at least about 30 mM
to at least about 100
mM potassium ion, about 0.4 ng/mL IL-15, and about 20 ng/mL IL-21. In some
aspects, the MRM
comprises more than 40 mM potassium ion, about 0.4 ng/mL IL-15, and about 20
ng/mL IL-21. In
some aspects, the MRM comprises at least about 45 mM potassium ion, about 0.4
ng/mL IL-15,
and about 20 ng/mL IL-21. In some aspects, the MRM comprises at least about 50
mM potassium
ion, about 0.4 ng/mL IL-15, and about 20 ng/mL IL-21. In some aspects, the MRM
comprises at
least about 55 mM potassium ion, about 0.4 ng/mL IL-15, and about 20 ng/mL IL-
21. In some
aspects, the MRM comprises at least about 60 mM potassium ion, about 0.4 ng/mL
IL-15, and
about 20 ng/mL IL-21. In some aspects, the MRM comprises at least about 65 mM
potassium ion,
about 0.4 ng/mL IL-15, and about 20 ng/mL IL-21. In some aspects, the MRM
comprises at least
about 70 mM potassium ion, about 0.4 ng/mL IL-15, and about 20 ng/mL IL-21. In
some aspects,
the MRM comprises at least about 75 mM potassium ion, about 0.4 ng/mL IL-15,
and about 20
ng/mL IL-21. In some aspects, the MR_M comprises at least about 80 mM
potassium ion, about 0.4
ng/mL IL-15, and about 20 ng/mL IL-21. In some aspects, the MRM comprises at
least about 85
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mM potassium ion, about 0.4 ng/mL IL-15, and about 20 ng/mL IL-21. In some
aspects, the MRM
comprises at least about 90 mM potassium ion, about 0.4 ng/mL IL-15, and about
20 ng/mL IL-
21. In some aspects, the MRM comprises (i) at least about 70 mM potassium ion,
(ii) about 60 mM
sodium, (iii) about 1.4 mM calcium, (iv) about 16 mM glucose, (v) about 0.4
ng/mL 1L-15, and
(vi) about 20 ng/mL IL-21.
[0332] In some aspects, the MRM comprises at least about 30 mM
to at least about 100
mM potassium ion, NaCl, and about 300 ng/mL IL-2; wherein the total
concentration of potassium
ion and NaCl is from 110 mM to 140 mM. In some aspects, the MRM comprises more
than 40
mM potassium ion, NaCl, and about 300 ng/mL IL-2; wherein the total
concentration of potassium
ion and NaCl is from 110 mM to 140 mM. In some aspects, the MRM comprises at
least about 45
mM potassium ion, NaCl, and about 300 ng/mL IL-2; wherein the total
concentration of potassium
ion and NaCl is from 110 mM to 140 mM. In some aspects, the MRM comprises at
least about 50
mM potassium ion, NaCl, and about 300 ng/mL IL-2; wherein the total
concentration of potassium
ion and NaCl is from 110 mM to 140 mM. In some aspects, the MRM comprises at
least about 55
mM potassium ion, NaCl, and about 300 ng/mL IL-2; wherein the total
concentration of potassium
ion and NaCl is from 110 mM to 140 mM. In some aspects, the MRM comprises at
least about 60
mM potassium ion, NaCl, and about 300 ng/mL IL-2; wherein the total
concentration of potassium
ion and NaCl is from 110 mM to 140 mM. In some aspects, the MRM comprises at
least about 65
mM potassium ion, NaCl, and about 300 ng/mL IL-2; wherein the total
concentration of potassium
ion and NaCl is from 110 mM to 140 mM. In some aspects, the MRM comprises at
least about 70
mM potassium ion, NaCl, and about 300 ng/mL IL-2; wherein the total
concentration of potassium
ion and NaCl is from 110 mM to 140 mM. In some aspects, the MRM comprises at
least about 75
mM potassium ion, NaCl, and about 300 ng/mL IL-2; wherein the total
concentration of potassium
ion and NaCl is from 110 mM to 140 mM. In some aspects, the MRM comprises at
least about 80
mM potassium ion, NaCl, and about 300 ng/mL IL-2; wherein the total
concentration of potassium
ion and NaCl is from 110 mM to 140 mM. In some aspects, the MRM comprises at
least about 85
mM potassium ion, NaC1, and about 300 ng/mL IL-2; wherein the total
concentration of potassium
ion and NaCl is from 110 mM to 140 mM. In some aspects, the MRM comprises at
least about 90
mM potassium ion, NaCl, and about 300 ng/mL IL-2; wherein the total
concentration of potassium
ion and NaC1 is from 110 mM to 140 mM. In some aspects, the MRM comprises (i)
at least about
70 mM potassium ion, (ii) about 60 mM sodium (e. g. , NaC1), (iii) about 1. 4
mM calcium, (iv)
about 16 mM glucose, and (v) about 10 ng/mL IL-2.
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[0333] In some aspects, the MRM comprises at least about 30 mM
to at least about 100
mM potassium ion, NaC1, about 300 ng/mL IL-2, and about 290 ng/mL IL-7;
wherein the total
concentration of potassium ion and NaCl is from 110 mM to 140 mM. In some
aspects, the MRM
comprises more than 40 mM potassium ion, NaCl, and about 300 ng/mL IL-2 and
about 290 ng/mL
IL-7; wherein the total concentration of potassium ion and NaC1 is from 110 mM
to 140 mM. In
some aspects, the MRM comprises at least about 45 mM potassium ion, NaCl,
about 300 ng/mL
IL-2, and about 290 ng/mL IL-7; wherein the total concentration of potassium
ion and NaCl is
from 110 mM to 140 mM. In some aspects, the MRM comprisess at least about 40
mM potassium
ion, NaCl, about 300 ng/mL IL-2, and about 290 ng/mL IL-7; wherein the total
concentration of
potassium ion and NaCl is from 110 mM to 140 mM. In some aspects, the MRM
comprises at least
about 55 mM potassium ion, NaCl, about 300 ng/mL IL-2, and about 290 ng/mL IL-
7; wherein the
total concentration of potassium ion and NaCl is from 110 mM to 140 mM. In
some aspects, the
MRM comprises at least about 60 mM potassium ion, NaCl, about 300 ng/mL IL-2,
and about 290
ng/mL IL-7; wherein the total concentration of potassium ion and NaCl is from
110 mM to 140
mM. In some aspects, the MRM comprises at least about 65 mM potassium ion,
NaCl, about 300
ng/mL IL-2, and about 290 ng/mL IL-7; wherein the total concentration of
potassium ion and NaCl
is from 110 mM to 140 mM. In some aspects, the MRM comprises at least about 70
mM potassium
ion, NaCl, about 300 ng/mL IL-2, and about 290 ng/mL IL-7; wherein the total
concentration of
potassium ion and NaCl is from 110 mM to 140 mM. In some aspects, the MRM
comprises at least
about 75 mM potassium ion, NaCl, about 300 ng/mL IL-2, and about 290 ng/mL IL-
7; wherein the
total concentration of potassium ion and NaCl is from 110 mM to 140 mM. In
some aspects, the
MRM comprises at least about 80 mM potassium ion, NaCl, about 300 ng/mL IL-2,
and about 290
ng/mL IL-7; wherein the total concentration of potassium ion and NaCl is from
110 mM to 140
mM. In some aspects, the MRM comprises at least about 85 mM potassium ion,
NaCl, about 300
ng/mL IL-2, and about 290 ng/mL IL-7; wherein the total concentration of
potassium ion and NaCl
is from 110 mM to 140 mM. In some aspects, the MRM comprises at least about 90
mM potassium
ion, NaC1, about 300 ng/mL IL-2, and about 290 ng/mL IL-7; wherein the total
concentration of
potassium ion and NaC1 is from 110 mM to 140 mM. In some aspects, the MRM
comprises (i) at
least about 70 mM potassium ion, (ii) about 60 mM sodium (e. g. , NaCl), (iii)
about 1. 4 mM
calcium, (iv) about 16 mM glucose, (v) about 300 ng/mL IL-2, and (vi) about
290 ng/mL IL-7.
[0334] In some aspects, the MRM comprises at least about 30 mM
to at least about 100
mM potassium ion, NaCl, about 300 ng/mL IL-2, and about 0. 4 ng/mL IL-15;
wherein the total
concentration of potassium ion and NaCl is from 110 mM to 140 mM. In some
aspects, the MRM
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comprises more than 40 mM potassium ion, NaCl, about 300 ng/mL IL-2, and about
0. 4 ng/mL
IL-15; wherein the total concentration of potassium ion and NaCl is from 110
mM to 140 mM. In
some aspects, the MRM comprises at least about 45 mM potassium ion, NaCl,
about 300 ng/mL
IL-2, and about 0. 4 ng/mL IL-15; wherein the total concentration of potassium
ion and NaCl is
from 110 mM to 140 mM. In some aspects, the MRM comprises at least about 50 mM
potassium
ion, NaC1, about 300 ng/mL IL-2, and about 0. 4 ng/mL IL-15; wherein the total
concentration of
potassium ion and NaCl is from 110 mM to 140 mM. In some aspects, the MRM
comprises at least
about 55 mM potassium ion, NaCl, about 300 ng/mL 1L-2, and about 0. 4 ng/mL IL-
15; wherein
the total concentration of potassium ion and NaCl is from 110 mM to 140 mM. In
some aspects,
the MRM comprises at least about 60 mM potassium ion, NaCl, about 300 ng/mL IL-
2, and about
0. 4 ng/mL IL-15; wherein the total concentration of potassium ion and NaCl is
from 110 mM to
140 mM. In some aspects, the MRM comprises at least about 65 mM potassium ion,
NaCl, about
300 ng/mL IL-2, and about 0. 4 ng/mL IL-15; wherein the total concentration of
potassium ion and
NaCl is from 110 mM to 140 mM. In some aspects, the 1VIR1VI comprises at least
about 70 mM
potassium ion, NaCl, about 300 ng/mL IL-2, and about 0. 4 ng/mL IL-15; wherein
the total
concentration of potassium ion and NaCl is from 110 mM to 140 mM. In some
aspects, the MRM
comprises at least about 75 mM potassium ion, NaCl, about 300 ng/mL IL-2, and
about 0. 4 ng/mL
IL-15; wherein the total concentration of potassium ion and NaCl is from 110
mM to 140 mM. In
some aspects, the MRM comprises at least about 80 mM potassium ion, NaCl,
about 300 ng/mL
IL-2, and about 0. 4 ng/mL IL-15; wherein the total concentration of potassium
ion and NaCl is
from 110 mM to 140 mM. In some aspects, the MRM comprises at least about 85 mM
potassium
ion, NaC1, about 300 ng/mL IL-2, and about 0. 4 ng/mL IL-15; wherein the total
concentration of
potassium ion and NaCl is from 110 mM to 140 mM. In some aspects, the MRM
comprises at least
about 90 mM potassium ion, NaCl, about 300 ng/mL IL-2, and about 0. 4 ng/mL IL-
15; wherein
the total concentration of potassium ion and NaCl is from 110 mM to 140 mM. In
some aspects,
the MRM comprises (i) at least about 70 mM potassium ion, (ii) about 60 mM
sodium (e. g. ,
NaC1), (iii) about 1. 4 mM calcium, (iv) about 16 mM glucose, (v) about 300
ng/mL 1L-2, and (vi)
about 0. 4 ng/mL IL-15.
[0335] In some aspects, the MRM comprises at least about 30 mM
to at least about 100
mM potassium ion, NaC1, about 300 ng/mL IL-2, about 290 ng/mL IL-7, and about
0. 4 ng/mL IL-
15; wherein the total concentration of potassium ion and NaC1 is from 110 mM
to 140 mM. In
some aspects, the MRM comprises more than 40 mM potassium ion, NaCl, about 300
ng/mL IL-
2, about 290 ng/mL IL-7, and about 0. 4 ng/mL IL-15; wherein the total
concentration of potassium
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ion and NaCl is from 110 mM to 140 mM. In some aspects, the MRM comprises at
least about 45
mM potassium ion, NaCl, about 300 ng/mL IL-2, about 290 ng/mL IL-7, and about
0. 4 ng/mL IL-
15; wherein the total concentration of potassium ion and NaCl is from 110 mM
to 140 mM. In
some aspects, the MRM comprises at least about 50 mM potassium ion, NaCl,
about 300 ng/mL
IL-2, about 290 ng/mL IL-7, and about 0. 4 ng/mL IL-15; wherein the total
concentration of
potassium ion and NaCl is from 110 mM to 140 mM. In some aspects, the MRM
comprises at least
about 55 mM potassium ion, NaCl, about 300 ng/mL IL-2, about 290 ng/mL IL-7,
and about 0. 4
ng/mL IL-15; wherein the total concentration of potassium ion and NaC1 is from
110 mM to 140
mM. In some aspects, the MRM comprises at least about 60 mM potassium ion,
NaCl, about 300
ng/mL IL-2, about 290 ng/mL IL-7, and about 0. 4 ng/mL IL-15; wherein the
total concentration
of potassium ion and NaCl is from 110 mM to 140 mM. In some aspects, the MR1VI
comprises at
least about 65 mM potassium ion, NaC1, about 300 ng/mL IL-2, about 290 ng/mL
IL-7, and about
0. 4 ng/mL IL-15; wherein the total concentration of potassium ion and NaCl is
from 110 mM to
140 mM. In some aspects, the MRM comprises at least about 70 mM potassium ion,
NaCl, about
300 ng/mL IL-2, about 290 ng/mL IL-7, and about 0. 4 ng/mL IL-15; wherein the
total
concentration of potassium ion and NaCl is from 110 mM to 140 mM. In some
aspects, the MRM
comprises at least about 75 mM potassium ion, NaCl, and about 10 ng/mL IL-2,
about 1 ng/mL
IL-7, and about 1 ng/mL IL-15; wherein the total concentration of potassium
ion and NaCl is from
110 mM to 140 mM. In some aspects, the MRM comprises at least about 80 mM
potassium ion,
NaCl, about 300 ng/mL IL-2, about 290 ng/mL IL-7, and about 0. 4 ng/mL IL-15;
wherein the total
concentration of potassium ion and NaCl is from 110 mM to 140 mM. In some
aspects, the MRM
comprises at least about 85 mM potassium ion, NaCl, about 300 ng/mL IL-2,
about 290 ng/mL IL-
7, and about 0. 4 ng/mL IL-15; wherein the total concentration of potassium
ion and NaCl is from
110 mM to 140 mM. In some aspects, the MRM comprises at least about 90 mM
potassium ion,
NaCl, about 300 ng/mL IL-2, about 290 ng/mL IL-7, and about 0. 4 ng/mL IL-15;
wherein the total
concentration of potassium ion and NaCl is from 110 mM to 140 mM. In some
aspects, the MRM
comprises (i) at least about 70 mM potassium ion, (ii) about 60 mM sodium (e.
g. , NaC1), (iii)
about 1. 4 mM calcium, (iv) about 16 mM glucose, (v) about 300 ng/mL IL-2,
(vi) about 290 ng/mL
IL-7, and (vii) about 0. 4 ng/mL IL-15.
[0336] In some aspects, the MRM comprises at least about 30 mM
to at least about 100
mM potassium ion, NaC1, about 300 ng/mL IL-2, and about 30 ng/mL IL-21;
wherein the total
concentration of potassium ion and NaCl is from 110 mM to 140 mM. In some
aspects, the MRM
comprises more than 40 mM potassium ion, NaCl, about 300 ng/mL IL-2, and about
30 ng/mL IL-
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21; wherein the total concentration of potassium ion and NaCl is from 110 mM
to 140 mM. In
some aspects, the MRM comprises at least about 45 mM potassium ion, NaCl,
about 300 ng/mL
IL-2, and about 30 ng/mL IL-21; wherein the total concentration of potassium
ion and NaCl is
from 110 mM to 140 mM. In some aspects, the MRM comprises at least about 50 mM
potassium
ion, NaCl, about 300 ng/mL IL-2, and about 30 ng/mL IL-21; wherein the total
concentration of
potassium ion and NaCl is from 110 mM to 140 mM. In some aspects, the MRM
comprises at least
about 55 mM potassium ion, NaCl, about 300 ng/mL IL-2, and about 30 ng/mL IL-
21; wherein the
total concentration of potassium ion and NaCl is from 110 mM to 140 mM In some
aspects, the
MR_M comprises at least about 60 mM potassium ion, NaC1, about 300 ng/mL IL-2,
and about 30
ng/mL IL-21; wherein the total concentration of potassium ion and NaC1 is from
110 mM to 140
mM. In some aspects, the MRM comprises at least about 65 mM potassium ion,
NaCl, about 300
ng/mL IL-2, and about 30 ng/mL IL-21; wherein the total concentration of
potassium ion and NaCl
is from 110 mM to 140 mM. In some aspects, the MRM comprises at least about 70
mM potassium
ion, NaCl, about 300 ng/mL IL-2, and about 30 ng/mL IL-21; wherein the total
concentration of
potassium ion and NaCl is from 110 mM to 140 mM. In some aspects, the MRM
comprises at least
about 75 mM potassium ion, NaCl, about 300 ng/mL IL-2, and about 30 ng/mL IL-
21; wherein the
total concentration of potassium ion and NaCl is from 110 mM to 140 mM. In
some aspects, the
MRM comprises at least about 80 mM potassium ion, NaC1, about 300 ng/mL IL-2,
and about 30
ng/mL IL-21; wherein the total concentration of potassium ion and NaC1 is from
110 mM to 140
mM. In some aspects, the MRM comprises at least about 85 mM potassium ion,
NaCl, about 300
ng/mL IL-2, and about 30 ng/mL IL-21; wherein the total concentration of
potassium ion and NaCl
is from 110 mM to 140 mM. In some aspects, the MRM comprises at least about 90
mM potassium
ion, NaCl, about 300 ng/mL IL-2, and about 30 ng/mL IL-21; wherein the total
concentration of
potassium ion and NaC1 is from 110 mM to 140 mM. In some aspects, the MRM
comprises (i) at
least about 70 mM potassium ion, (ii) about 60 mM sodium (e. g. , NaCl), (iii)
about 1. 4 mM
calcium, (iv) about 16 mM glucose, (v) about 300 ng/mL IL-2, and (vi) about 30
ng/mL IL-21.
[0337] In some aspects, the MRM comprises at least about 30 mM
to at least about 100
mM potassium ion, NaC1, about 290 ng/mL IL-7, and about 20 ng/mL IL-21;
wherein the total
concentration of potassium ion and NaCl is from 110 mM to 140 mM. In some
aspects, the MRM
comprises more than 40 mM potassium ion, NaC1, about 290 ng/mL IL-7, and about
20 ng/mL IL-
21; wherein the total concentration of potassium ion and NaC1 is from 110 mM
to 140 mM. In
some aspects, the MRM comprises at least about 45 mM potassium ion, NaCl,
about 290 ng/mL
IL-7, and about 20 ng/mL IL-21; wherein the total concentration of potassium
ion and NaCl is
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from 110 mM to 140 mM. In some aspects, the MRM comprises at least about 50 mM
potassium
ion, NaCl, about 290 ng/mL IL-7, and about 20 ng/mL IL-21; wherein the total
concentration of
potassium ion and NaCl is from 110 mM to 140 mM. In some aspects, the MRI\4
comprises at least
about 55 mM potassium ion, NaCl, about 290 ng/mL IL-7, and about 20 ng/mL 1L-
21; wherein the
total concentration of potassium ion and NaCl is from 110 mM to 140 mM. In
some aspects, the
MRM comprises at least about 60 mM potassium ion, NaC1, about 290 ng/mL IL-7,
and about 20
ng/mL IL-21; wherein the total concentration of potassium ion and NaC1 is from
110 mM to 140
mM. In some aspects, the MRM comprises at least about 65 mM potassium ion,
NaCl, about 290
ng/mL IL-7, and about 20 ng/mL IL-21; wherein the total concentration of
potassium ion and NaCl
is from 110 mM to 140 mM. In some aspects, the MRNI comprises at least about
70 mM potassium
ion, NaCl, about 290 ng/mL IL-7, and about 20 ng/mL IL-21; wherein the total
concentration of
potassium ion and NaCl is from 110 mM to 140 mM. In some aspects, the MRM
comprises at least
about 75 mM potassium ion, NaCl, about 290 ng/mL IL-7, and about 20 ng/mL IL-
21; wherein the
total concentration of potassium ion and NaCl is from 110 mM to 140 mM. In
some aspects, the
MR.1\'1 comprises at least about 80 mM potassium ion, NaC1, about 290 ng/mL IL-
7, and about 20
ng/mL IL-21; wherein the total concentration of potassium ion and NaC1 is from
110 mM to 140
mM. In some aspects, the MRM comprises at least about 85 mM potassium ion,
NaCl, about 290
ng/mL IL-7, and about 20 ng/mL IL-21; wherein the total concentration of
potassium ion and NaCl
is from 110 mM to 140 mM. In some aspects, the MRM comprises at least about 90
mM potassium
ion, NaCl, about 290 ng/mL IL-7, and about 20 ng/mL IL-21; wherein the total
concentration of
potassium ion and NaC1 is from 110 mM to 140 mM. In some aspects, the MRM
comprises (i) at
least about 70 mM potassium ion, (ii) about 60 mM sodium (e. g. , NaCl), (iii)
about 1. 4 mM
calcium, (iv) about 16 mM glucose, (v) about 290 ng/mL IL-7, and (vi) about 20
ng/mL IL-21.
[0338] In some aspects, the MRM comprises at least about 30 mM
to at least about 100
mM potassium ion, NaCl, about 0. 4 ng/mL IL-15, and about 20 ng/mL IL-21;
wherein the total
concentration of potassium ion and NaCl is from 110 mM to 140 mM. In some
aspects, the MRM
comprises more than 40 mM potassium ion, NaC1, about 0. 4 ng/mL IL-15, and
about 20 ng/mL
IL-21; wherein the total concentration of potassium ion and NaCl is from 110
mM to 140 mM. In
some aspects, the MRIV1 comprises at least about 45 mM potassium ion, NaCl,
about 0. 4 ng/mL
IL-15, and about 20 ng/mL IL-21; wherein the total concentration of potassium
ion and NaCl is
from 110 mM to 140 mM. In some aspects, the MRM comprises at least about 50 mM
potassium
ion, NaC1, about 0. 4 ng/mL IL-15, and about 20 ng/mL IL-21; wherein the total
concentration of
potassium ion and NaCl is from 110 mM to 140 mM. In some aspects, the MRM
comprises at least
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about 55 mM potassium ion, NaCl, about 0. 4 ng/mL IL-15, and about 20 ng/mL IL-
21; wherein
the total concentration of potassium ion and NaCl is from 110 mM to 140 mM. In
some aspects,
the MRM comprises at least about 60 mM potassium ion, NaCl, about 0. 4 ng/mL
IL-15, and about
20 ng/mL IL-21; wherein the total concentration of potassium ion and NaCl is
from 110 mM to
140 mM. In some aspects, the MRM comprises at least about 65 mM potassium ion,
NaCl, about
0. 4 ng/mL IL-15, and about 20 ng/mL IL-21; wherein the total concentration of
potassium ion and
NaCl is from 110 mM to 140 mM. In some aspects, the 1VIR1VI comprises at least
about 70 mM
potassium ion, NaC1, about 0. 4 ng/mL IL-15, and about 20 ng/mL IL-21; wherein
the total
concentration of potassium ion and NaCl is from 110 mM to 140 mM. In some
aspects, the MRM
comprises at least about 75 mM potassium ion, NaCl, about 0. 4 ng/mL IL-15,
and about 20 ng/mL
IL-21; wherein the total concentration of potassium ion and NaCl is from 110
mM to 140 mM. In
some aspects, the MRM comprises at least about 80 mM potassium ion, NaC1,
about 0. 4 ng/mL
IL-15, and about 20 ng/mL IL-21; wherein the total concentration of potassium
ion and NaCl is
from 110 mM to 140 mM. In some aspects, the MRM comprises at least about 85 mM
potassium
ion, NaCl, about 0. 4 ng/mL IL-15, and about 20 ng/mL IL-21; wherein the total
concentration of
potassium ion and NaCl is from 110 mM to 140 mM. In some aspects, the MRM
comprises at least
about 90 mM potassium ion, NaCl, about 0. 4 ng/mL IL-15, and about 20 ng/mL IL-
21; wherein
the total concentration of potassium ion and NaCl is from 110 mM to 140 mM. In
some aspects,
the MRM comprises (i) at least about 70 mM potassium ion, (ii) about 60 mM
sodium (e. g. ,
NaCl), (iii) about 1. 4 mM calcium, (iv) about 16 mM glucose, (v) about 0. 4
ng/mL IL-15, and
(vi) about 20 ng/mL IL-21.
II.G. T Cell Culture Media (e.g., Metabolic Reprograming Media)
[0339] In some aspects, the MRM is prepared by adding potassium
ion to a basal medium.
Any basal medium that is used to culture immune cells, (e.g., T cells, NK
cells, and/or TILs), can
be used.
[0340] In some aspects, the MRM further comprises one or more
essential amino acids. In
some aspects, the basal media comprises one or more essential amino acids
selected form L-
arginine, L-cy stifle, L-isoleucine, L-leucine, L-lysine, L-methionine, L-
phenylalanine, L-
threonine, L-tryptophan, L-histidine, L-tyrosine, L-valine, and L-glutamine,
or any combination
thereof. In some aspects, the basal media comprises L-glutamine.
[0341] In some aspects, the 1VIRI\4 comprises at least about
0.01 mM of one or more
essential amino acids. In some aspects, the MRM comprises about 0.01 mM to
about 10 mM of
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one or more essential amino acids. In some aspects, the MRM comprises about
0.01 mM to about
mM, about 0.01 mM to about 9 mM, about 0.01 mM to about 8 mM, about 0.01 mM to
about
7 mM, about 0.01 mM to about 6 mM, about 0.01 mM to about 5 mM, about 0.01 mM
to about 4
mM, about 0.01 mM to about 3 mM, about 0.01 mM to about 2 mM, about 0.01 mM to
about 1
mM, about 0.1 mM to about 10 mM, about 0.1 mM to about 9 mM, about 0.1 mM to
about 8 mM,
about 0.1 mM to about 7 mM, about 0.1 mM to about 6 mM, about 0.1 mM to about
5 mM, about
0.1 mM to about 4 mM, about 0.1 mM to about 3 mM, about 0.1 mM to about 2 mM,
about 0.1
mM to about 1 mM, about 1 mM to about 10 mM, about 1 mM to about 9 mM, about 1
mM to
about 8 mM, about 1 mM to about 7 mM, about 1 mM to about 6 mM, about 1 mM to
about 5 mM,
about 1 mM to about 4 mM, about 1 mM to about 3 mM, or about 1 mM to about 2
mM of one or
more essential amino acids.
[0342] In some aspects, the MRM comprises at least about 0.01
mM, at least about 0.1
mM, at least about 0.5 mM, at least about 1.0 mM, at least about 2 mM, at
least about 3 mM, at
least about 4 mM, at least about 5 mM, at least about 6 mM, at least about 7
mM, at least about 8
mM, at least about 9 mM, at least about 10 mM, at least about 11 mM, at least
about 12 mM, at
least about 13 mM, at least about 14 mM, or at least about 15 mM or at least
about 50 mM of one
or more essential amino acids.
[0343] In some aspects, the MRM comprises about 0.01 mM, about
0.05 mM, about 0.1
mM, about 0.2 mM, about 0.3 mM, about 0.4 mM, about 0.5 mM, about 0.6 mM,
about 0.7 mM,
about 0.8 mM, about 0.9 mM, about 1 mM, about 1.1 mM, about 1.2 mM, about 1.3
mM, about
1.4 mM, about 1.5 mM, about 1.6 mM, about 1.7 mM, about 1.8 mM, about 1.9 mM,
about 2.0
mM, about 2.1 mM, about 2.2 mM, about 2.3 mM, about 2.4 mM, about 2.5 mM,
about 2.6 mM,
about 2.7 mM, about 2.8 mM, about 2.9 mM, about 3.0 mM, about 3.1 mM, about
3.2 mM, about
3.3 mM, about 3.4 mM, about 3.5 mM, about 3.6 mM, about 3.7 mM, about 3.8 mM,
about 3.9
mM, about 4.0 mM, about 4.1 mM, about 4.2 mM, about 4.3 mM, about 4.4 mM,
about 4.5 mM,
about 4.6 mM, about 4.7 mM, about 4.8 mM, about 4.9 mM, about 5.0 mM, about
5.1 mM, about
5.2 mM, about 5.3 mM, about 5.4 mM, about 5.5 mM, about 5.6 mM, about 5.7 mM,
about 5.8
mM, about 5.9 mM, about 6.0 mM, about 6.1 mM, about 6.2 mM, about 6.3 mM,
about 6.4 mM,
about 6.5 mM, about 6.6 mM, about 6.7 mM, about 6.8 mM, about 6.9 mM, or about
7.0 mM of
one or more essential amino acids.
[0344] In some aspects, the MRM comprises L-glutamine. In some
aspects, MRM
comprises at least about 0.01 mM L-glutamine. In some aspects, the MRM
comprises about 0.01
mM to about 10 mM L-glutamine. In some aspects, the MRM comprises about 0.01
mM to about
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-114-
mM, about 0.01 mM to about 9 mM, about 0.01 mM to about 8 mM, about 0.01 mM to
about
7 mM, about 0.01 mM to about 6 mM, about 0.01 mM to about 5 mM, about 0.01 mM
to about 4
mM, about 0.01 mM to about 3 mM, about 0.01 mM to about 2 mM, about 0.01 mM to
about 1
mM, about 0.1 mM to about 10 mM, about 0.1 mM to about 9 mM, about 0.1 mM to
about 8 mM,
about 0.1 mM to about 7 mM, about 0.1 mM to about 6 mM, about 0.1 mM to about
5 mM, about
0.1 mM to about 4 mM, about 0.1 mM to about 3 mM, about 0.1 mM to about 2 mM,
about 0.1
mM to about 1 mM, about 1 mM to about 10 mM, about 1 mM to about 9 mM, about 1
mM to
about 8 mM, about 1 mM to about 7 mM, about 1 mM to about 6 mM, about 1 mM to
about 5 mM,
about 1 mM to about 4 mM, about 1 mM to about 3 mM, or about 1 mM to about 2
mM L-
glutamine.
[0345] In some aspects, the MRM comprises at least about 0.01
mM, at least about 0.1
mM, at least about 0.5 mM, at least about 1.0 mM, at least about 2 mM, at
least about 3 mM, at
least about 4 mM, at least about 5 mM, at least about 6 mM, at least about 7
mM, at least about 8
mM, at least about 9 mM, at least about 10 mM, at least about 11 mM, at least
about 12 mM, at
least about 13 mM, at least about 14 mM, or at least about 15 mM or at least
about 50 mM L-
gl utamine.
[0346] In some aspects, the MRM comprises about 0.01 mM, about
0.05 mM, about 0.1
mM, about 0.2 mM, about 0.3 mM, about 0.4 mM, about 0.5 mM, about 0.6 mM,
about 0.7 mM,
about 0.8 mM, about 0.9 mM, about 1 mM, about 1.1 mM, about 1.2 mM, about 1.3
mM, about
1.4 mM, about 1.5 mM, about 1.6 mM, about 1.7 mM, about 1.8 mM, about 1.9 mM,
about 2.0
mM, about 2.1 mM, about 2.2 mM, about 2.3 mM, about 2.4 mM, about 2.5 mM,
about 2.6 mM,
about 2.7 mM, about 2.8 mM, about 2.9 mM, about 3.0 mM, about 3.1 mM, about
3.2 mM, about
3.3 mM, about 3.4 mM, about 3.5 mM, about 3.6 mM, about 3.7 mM, about 3.8 mM,
about 3.9
mM, about 4.0 mM, about 4.1 mM, about 4.2 mM, about 4.3 mM, about 4.4 mM,
about 4.5 mM,
about 4.6 mM, about 4.7 mM, about 4.8 mM, about 4.9 mM, about 5.0 mM, about
5.1 mM, about
5.2 mM, about 5.3 mM, about 5.4 mM, about 5.5 mM, about 5.6 mM, about 5.7 mM,
about 5.8
mM, about 5.9 mM, about 6.0 mM, about 6.1 mM, about 6.2 mM, about 6.3 mM,
about 6.4 mM,
about 6.5 mM, about 6.6 mM, about 6.7 mM, about 6.8 mM, about 6.9 mM, or about
7.0 mM L-
glutamine. In some aspects, the MRM comprises about 1.7 mM L-glutamine. In
some aspects, the
MRM comprises about 1.68 mM L-glutamine.
[0347] In some aspects, the MRM comprises about 0.14 mM L-
glutamine. In some aspects,
the 1V1R_M comprises about 0.15 mM L-glutamine. In some aspects, the MRM
comprises about
1.76 mM L-glutamine. In some aspects, the MRM comprises about 1.83 mM L-
glutamine. In some
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aspects, the MRM comprises about 1.84 mM L-glutamine. In some aspects, the MRM
comprises
about L97 mM L-glutamine. In some aspects, the MRM comprises about 2.05 mM L-
glutamine.
In some aspects, the MRM comprises about 2.11 mM L-glutamine. In some aspects,
the MRM
comprises about 2.18 mM L-glutamine. In some aspects, the M_RM comprises about
5.41 mM L-
glutamine. In some aspects, the MRM comprises about 5.47 mM L-glutamine. In
some aspects,
the MRM comprises about < 0.10 mM L-glutamine.
[0348] In some aspects, the MRM comprises L-glutamic acid. In
some aspects, the MRM
comprises at least about 0.01 mM L-glutamic acid. In some aspects, the MRM
comprises about
0.01 mM to about 10 mM L-glutamic acid. In some aspects, the MRM comprises
about 0.01 mM
to about 10 mM, about 0.01 mM to about 9 mM, about 0.01 mM to about 8 mM,
about 0.01 mM
to about 7 mM, about 0.01 mM to about 6 mM, about 0.01 mM to about 5 mM, about
0.01 mM to
about 4 mM, about 0.01 mM to about 3 mM, about 0.01 mM to about 2 mM, about
0.01 mM to
about 1 mM, about 0.1 mM to about 10 mM, about 0.1 mM to about 9 mM, about 0.1
mM to about
8 mM, about 0.1 mM to about 7 mM, about 0.1 mM to about 6 mM, about 0.1 mM to
about 5 mM,
about 0.1 mM to about 4 mM, about 0.1 mM to about 3 mM, about 0.1 mM to about
2 mM, about
0.1 mM to about 1 mM, about 1 mM to about 10 mM, about 1 mM to about 9 mM,
about 1 mM to
about 8 mM, about 1 mM to about 7 mM, about 1 mM to about 6 mM, about 1 mM to
about 5 mM,
about 1 mM to about 4 mM, about 1 mM to about 3 mM, or about 1 mM to about 2
mM L-glutamic
acid.
[0349] In some aspects, the MRIVI comprises at least about 0.01
mM, at least about 0.1
mM, at least about 0.5 mM, at least about 1.0 mM, at least about 2 mM, at
least about 3 mM, at
least about 4 mM, at least about 5 mM, at least about 6 mM, at least about 7
mM, at least about 8
mM, at least about 9 mM, at least about 10 mM, at least about 11 mM, at least
about 12 mM, at
least about 13 mM, at least about 14 mM, or at least about 15 mM or at least
about 50 mM L-
glutamic acid.
[0350] In some aspects, the MRM comprises about 0.01 mM, about
0.05 mM, about 0.1
mM, about 0.2 mM, about 0.3 mM, about 0.4 mM, about 0.5 mM, about 0.6 mM,
about 0.7 mM,
about 0.8 mM, about 0.9 mM, about 1 mM, about 1.1 mM, about 1.2 mM, about 1.3
mM, about
1.4 mM, about 1.5 mM, about 1.6 mM, about 1.7 mM, about 1.8 mM, about 1.9 mM,
about 2.0
mM, about 2.1 mM, about 2.2 mM, about 2.3 mM, about 2.4 mM, about 2.5 mM,
about 2.6 mM,
about 2.7 mM, about 2.8 mM, about 2.9 mM, about 3.0 mM, about 3.1 mM, about
3.2 mM, about
3.3 mM, about 3.4 mM, about 3.5 mM, about 3.6 mM, about 3.7 mM, about 3.8 mM,
about 3.9
mM, about 4.0 mM, about 4.1 mM, about 4.2 mM, about 4.3 mM, about 4.4 mM,
about 4.5 mM,
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about 4.6 mM, about 4.7 mM, about 4.8 mM, about 4.9 mM, about 5.0 mM, about
5.1 mM, about
5.2 mM, about 5.3 mM, about 5.4 mM, about 5.5 mM, about 5.6 mM, about 5.7 mM,
about 5.8
mM, about 5.9 mM, about 6.0 mM, about 6.1 mM, about 6.2 mM, about 6.3 mM,
about 6.4 mM,
about 6.5 mM, about 6.6 mM, about 6.7 mM, about 6.8 mM, about 6.9 mM, or about
7.0 mM L-
glutamic acid.
[0351] In some aspects, the MRM comprises about 0.15 mM L-
glutamic acid. In some
aspects, the MRM comprises about 0.17 mM L-glutamic acid. In some aspects, the
MRM
comprises about 0.18 mM L-glutamic acid. In some aspects, the MR_M comprises
about 0.19 mM
L-glutamic acid. In some aspects, the MR_M comprises about 0.85 mM L-glutamic
acid. In some
aspects, the MR_I\4 comprises about 0.86 mM L-glutamic acid. In some aspects,
the MRM
comprises about 0.9 mM L-glutamic acid. In some aspects, the MRM comprises
about 0.95 mM
L-glutamic acid. In some aspects, the MRM comprises about 1.06 mM L-glutamic
acid. In some
aspects, the MRI\4 comprises about 1.09 mM L-glutamic acid. In some aspects,
the MRM
comprises about < 0.10 mM L-glutamic acid.
[0352] In some aspects, the 1VIRI\4 comprises a dipeptide. In
some aspects, the MRM
comprises glutamine-glutamine (Gin-Gin). In some aspects, the MRM comprises
alanyl-glutamine
(Ala-Gin).
[0353] In some aspects, the MRM comprises at least about 0.1 mM
dipeptide (e.g., Ala-
Gin). In some aspects, the MR1V1 comprises about 0.1 mM to about 50 mM
dipeptide (e.g., Ala-
Gin). In some aspects, the MRM comprises about 0.1 mM to about 40 mM, about
0.1 mM to about
35 mM, about 0.1 mM to about 30 mM, about 0.1 mM to about 25 mM, about 0.1 mM
to about 20
mM, about 1 mM to about 20 mM, about 2 mM to about 20 mM, about 3 mM to about
20 mM,
about 4 mM to about 20 mM, about 5 mM to about 20 mM, about 6 mM to about 20
mM, about 7
mM to about 20 mM, about 8 mM to about 20 mM, about 9 mM to about 20 mM, about
10 mM to
about 20 mM, about 1 mM to about 10 mM, about 2 mM to about 10 mM, about 3 mM
to about
mM, about 4 mM to about 10 mM, about 5 mM to about 10 mM, about 6 mM to about
10 mM,
about 7 mM to about 10 mM, about 8 mM to about 10 mM, or about 9 mM to about
10 mM
dipeptide (e.g., Ala-Gin).
[0354] In some aspects, the MRI\4 comprises at least about 0.1
mM, at least about 1.0 mM,
at least about 2 mM, at least about 3 mM, at least about 4 mM, at least about
5 mM, at least about
6 mM, at least about 7 mM, at least about 8 mM, at least about 9 mM, at least
about 10 mM, at
least about 11 mM, at least about 12 mM, at least about 13 mM, at least about
14 mM, at least
about 15 mM, at least about 16 mM, at least about 17 mM, at least about 18 mM,
at least about 19
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mM, at least about 20 mM, at least about 25 mM, at least about 30 mM, or at
least about 50 mM
dipeptide (e.g., Ala-Gin).
[0355] In some aspects, the MRM comprises about 1 mM, about 1.1
mM, about 1.2 mM,
about 1.3 mM, about 1.4 mM, about 1.5 mM, about 1.6 mM, about 1.7 mM, about
1.8 mM, about
1.9 mM, or about 2.0 mM dipeptide (e.g., Ala-Gin). In some aspects, the basal
medium comprises
about 1.7 mM dipeptide (e.g., Ala-Gln). In some aspects, the MRM comprises
about 1.68 mM
dipeptide (e.g., Ala-Gin).
[0356] In some aspects, the MRM comprises about 6 mM, about 6.1
mM, about 6.2 mM,
about 6.3 mM, about 6.4 mM, about 6.5 mM, about 6.6 mM, about 6.7 mM, about
6.8 mM, about
6.9 mM, about 7.0 mM, about 7.1 mM, or about 7.2 mM dipeptide (e.g., Ala-Gin).
In some aspects,
the MRM comprises about 6.8 mM dipeptide (e.g., Ala-Gin). In some aspects, the
MR_M comprises
about 6.81 mM dipeptide (e.g., Ala-Gin). In some aspects, the MRIVI comprises
about 6.9 mM
dipeptide (e.g., Ala-Gin). In some aspects, the MRIV1 comprises about 6.96 mM
dipeptide (e.g.,
Ala-Gin). In some aspects, the MRIV1 comprises about 7.0 mM dipeptide (e.g.,
Ala-Gin).
[0357] In some aspects, the MRM comprises less than about 5 mM
ammonia (NH3). In
some aspects, the MR1VI comprises less than about 4 mM, less than about 3.5
mM, less than about
3 mM, less than about 2.5 mM, less than about 2 mM, less than about 1.5 mM,
less than about 1
mM, less than about 0.5 mM, less than about 0.4 mM, less than about 0.3 mM,
less than about 0.2
mM, or less than about 0.1 mM ammonia. In some aspects, the MRM comprises
about 0.01 mM
ammonia to less than about 2 mM ammonia, about 0.01 mM ammonia to less than
about 1.9 mM
ammonia, about 0.01 mM ammonia to less than about 1.8 mM ammonia, about 0.01
mM ammonia
to less than about 1.7 mM ammonia, about 0.01 mM ammonia to less than about
1.6 mM ammonia,
about 0.01 mM ammonia to less than about 1.5 mM ammonia, about 0.01 mM ammonia
to less
than about 1.4 mM ammonia, about 0.01 mM ammonia to less than about 1.3 mM
ammonia, about
0.01 mM ammonia to less than about 1.2 mM ammonia, about 0.01 mM ammonia to
less than
about 1.1 mM ammonia, about 0.01 mM ammonia to less than about 1 mM ammonia,
about 0.01
mM ammonia to less than about 0.9 mM ammonia, about 0.01 mM ammonia to less
than about 0.8
mM ammonia, about 0.01 mM ammonia to less than about 0.7 mM ammonia, about
0.01 mM
ammonia to less than about 0.6 mM ammonia, about 0.01 mM ammonia to less than
about 0.5 mM
ammonia, about 0.01 mM ammonia to less than about 0.4 mM ammonia, about 0.01
mM ammonia
to less than about 0.3 mM ammonia, about 0.01 mM ammonia to less than about
0.2 mM ammonia,
or about 0.01 mM ammonia to less than about 0.1 mM ammonia. In some aspects,
the MRM
comprises about 1.2 mM ammonia. In some aspects, the MRM comprises about 1.25
mM
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ammonia. In some aspects, the MRM comprises about 1.259 mM ammonia. In some
aspects, the
MRIVI comprises about 1.28 mM ammonia. In some aspects, the MRM comprises
about 1.3 mM
ammonia. In some aspects, the 1VIRI\4 comprises about 0.3 mM ammonia. In some
aspects, the
MR1\4 comprises about 0.34 mM ammonia. In some aspects, the MRM comprises
about 0.35 mM
ammonia. In some aspects, the MRM comprises about 0.36 mM ammonia. In some
aspects, the
MRM comprises about 0.37 mM ammonia. In some aspects, the MRM comprises less
than about
0.3 mM ammonia. In some aspects, the MRIVI comprises less than about 0.29 mM
ammonia. In
some aspects, thelVIRM comprises less than about 0.28 mM ammonia. In some
aspects, the MRM
comprise less than about 0.278 mM ammonia. In some aspects, the MRM do not
comprise
ammonia.
[0358] In some aspects, the MRM comprises lactate. In some
aspects, the MRM does not
comprise lactate.
[0359] In some aspects, the MR1VI, e.g., secondary TIL expansion
medium and/or third (or
final) TIL expansion medium, further comprises a CD3 agonist and/or a CD28
agonist. The CD3
agonist and/or the CD28 agonist can stimulate TILs that are being cultured in
the media. In some
aspects, a CD3 agonist can be any molecule that is capable of binding to CD3
complex and
activating CD3. In some aspects, a CD3 agonist is a small molecule. In some
aspects, a CD3 agonist
is a protein. In some aspects, a CD3 agonist is an anti-CD3 antibody. The term
"anti-CD3 antibody"
as used herein 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 complex in
T cells. In some aspects, an anti-CD3 antibody comprises OKT-3, also known as
muromonab, and
UCHT-1. Other anti-CD3 antibodies include, for example, visilizumab
otelixizumab, and
teplizumab.
[0360] The term "OKT-3" or "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 OK T-3 (30 ng/mL, MACS GMT' CD3 pure, Miltenyi Biotech, Inc.,
San Diego,
Calif., USA) and muromonab or variants, conservative amino acid substitutions,
glycoforms, or
biosimilars thereof. A hybridoma capable of producing OKT-3 is deposited with
European
Collection of Authenticated Cell Cultures (ECACC) and assigned Catalogue No
86022706. A
hybridoma capable of producing OKT-3 is also deposited with the American Type
Culture
Collection and assigned the ATCC accession number CRL 8001.
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[0361] In some aspects, a CD28 agonist can be any molecule that
is capable of activating
CD28 or its downstream pathway. In some aspects, a CD28 agonist is a small
molecule. In some
aspects, a CD28 agonist is a protein. In some aspects, a CD28 agonist is an
anti- CD28 antibody.
The term "anti- CD28 antibody" as used herein refers to an antibody or variant
thereof, e.g., a
monoclonal antibody and including human, humanized, chimeric or murine
antibodies which are
directed against CD28 and activate T cells. In some aspects, an anti-CD28
antibody comprises
Catalog #100182-1 (BPS Bioscicence), Catalog #100186-1 (BPS Bioscience).
[0362] In some aspects, the CD3 agonist and the CD28 agonist are
added in the MRM
together. In some aspects, the CD3 agonist and the CD28 agonist are added in
the 1VIR_M
concurrently in one composition. In some aspects, the CD3 agonist and the CD28
agonist are added
in sequence. In some aspects, the MRM, e.g., secondary TIL expansion media
and/or third (or
final) TIL expansion media, comprises and/or is supplemented with a
substituent comprising both
a CD3 agonist and a CD28 agonist, e.g., TRANSACTTm. In some aspects, the MIRM
comprises at
least about 1:100 TRANSACTTm. In some aspects, the MR_Mcomprises at least
about 1:150
TRANSACTTm. In some aspects, the MRNIcomprises at least about 1:200
TRANSACTTm. In some
aspects, the MRM comprises at least about 1:250 TRANSACTTm. In some aspects,
the MRM
comprises at least about 1:300 TRANSACTTm. In some aspects, the MRM comprises
at least about
1:350 TRANSACTTm. In some aspects, the MRM comprises at least about 1:400
TRANSACTTm.
In some aspects, the MRNI comprises at least about 1:450 TRANSACTTm. In some
aspects, the
MRN1 comprises at least about 1:500 TRAN SAC TTm
[0363] In some aspects, the MRM, e.g., secondary TIL expansion
media and/or third (or
final) TIL expansion media, comprises and/or is supplemented with a TRANSACTTm
alternative.
Artificial antigen presenting cells (aAPCs) such as genetically engineered
human K562 aAPCs can
be used for rapid expansion of TILs. In some aspects, the aAPC is generated by
transducing K562
cells with a polycistronic lentiviral vector comprising genes encoding CD70,
CD80, CD86, 41BB
ligand, and 0X40 ligand. K562 cells do not express HLA-A, I-11,A-B, or 1-11A-
DR molecules,
which makes them a powerful tool for T cell expansion when transduced with the
above mentioned
co-stimulatory ligands (See, e.g., Suhoski et al., Molecular therapy, 2007).
In some aspects,
secondary TIL expansion and/or third TIL expansion comprises co-culturing the
TILs with aAPCs
+ OKT3. In some aspects, secondary TIL expansion and/or third TIL expansion
comprises co-
culturing the TILs with irradiated APCs (e.g., PBMC) in the presense of OKT3
(e.g., at least about
30 ng/mL OKT3) instead of TRANSACTTm. In some aspects, the ratio of immune
cells (e.g., TILs)
to feeder cells (e.g. , aAPCs) is at least about 1:50, at least about 1:100,
at least about 1:150, at least
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about 1:200, at least about 1:250, at least about 1:300, at least about 1:350,
at least about 1:400, at
least about 1:450, or at least about 1:500. In some aspects, the ratio of
immune cells (e.g., TILs) to
feeder cells (e.g., aAPCs) is at least about 1:100. In some aspects, the ratio
of immune cells (e.g.,
TILs) to feeder cells (e.g., aAPCs) is at least about 1:200.
[0364] In some aspects, the MRM, e.g., secondary TIL expansion
media and/or third (or
final) TIL expansion media, comprise and/or are supplemented with a CD27
ligand (CD27L).
CD27 ligand (CD70) is capable of binding to its receptor, and then upon
binding, the receptor is
capable of generating and long-term maintenance of T cell immunity. CD27 is a
member of the
TNF-receptor superfamily. CD27, a transmembrane homodimeric
phosphoglycoprotein of 120
lcDa, also appears to have a costimulatory role. CD27L, CD70, is a
transmembrane glycoprotein
expressed on T and B cells in response to antigen stimulation; it is thus
considered a marker of the
early stages of activation. In vitro, the interaction of CD27 on a T cell and
CD70 on a B cell
enhances T cell activation in terms of proliferation but only relatively low
amounts of IL-2 are
secreted. Studies of knockout mice have shown that CD27 plays a minor part in
naive T cell
activation but is crucial for the generation of T cell memory.
[0365] In some aspects, the MRM, e.g., secondary TIL expansion
media and/or third (or
final) TIL expansion media, comprises about 0.1 g/m1 to about 50 gg/m1CD27L.
In some aspects,
the MRM comprises and/or is supplemented with about 0.1 gg/ml to about 40
ps/ml, about 0.1
jig/ml to about 30 jig/ml, about 0.1 jig/ml to about 20 jig/ml, about 0.1
gg/m1 to about 10 jig/ml,
about 0.1 gg/m1 to about 5 pg/ml, about 1 jig/ml to about 10 jig/ml, about 2
jig/ml to about 10
jig/ml, about 3 jig/ml to about 10 pg/ml, about 4 gs/m1 to about 10 jig/ml,
about 5 jig/ml to about
jig/ml, about 1 jig/ml to about 9 jig/ml, about 1 jig/m1 to about 8 ps/ml,
about 1 ps/m1 to about
7 g/ml, about 1 gg/m1 to about 6 ps/ml, about 1 gg/m1 to about 5 jig/ml,
about 3 jig/ml to about
7 ps/ml, about 4 jig/m1 to about 6 jig/ml, about 3 jig/ml to about 6 ps/ml, or
about 4 gg/ml to about
7 g/m1 CD27L.
[0366] In some aspects, the MRM, e.g., secondary TIL expansion
media and/or third (or
final) TIL expansion media, comprises and/or is supplemented with at least
about 0.1 jig/ml, at
least about 1 jig/ml, at least about 2 jig/ml, at least about 3 jig/ml, at
least about 4 jig/ml, at least
about 5 jig/ml, at least about 6 jig/ml, at least about 7 jig/ml, at least
about 8 jig/ml, at least about
9 jig/ml, at least about 10 s/ml, at least about 11 jig/ml, at least about 12
jig/ml, at least about 13
jig/ml, at least about 14 jig/ml, at least about 15 pg/ml, at least about 16
jig/ml, at least about 17
jig/ml, at least about 18 jig/ml, at least about 19 pg/ml, at least about 20
gg/ml, at least about 25
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g/ml, at least about 30 jig/ml, or at least about 50 g/m1 CD27L. In some
aspects, the MRM
comprises and/or is supplemented with at least about 5 g/m1 CD27L.
[0367] In some aspects, the MR.1\4, e.g., secondary TIL
expansion medium and/or third (or
final) TIL expansion medium, comprises and/or is supplemented with 4-1BB
ligand (4-1BBL). 4-
1BBL (4-1BB ligand, CD137L) is found on APCs (antigen presenting cells) and
binds to 4-1BB
(also known as CD137), a type 2 transmembrane glycoprotein receptor belonging
to the TNF
superfamily, which is expressed on activated T Lymphocytes. 4-1BB ligand can
be used to activate
T cells in vitro. In some aspects, the MRM, e.g., secondary T1L expansion
media and/or third (or
final) TIL expansion media, comprise about 0.1 s/m1 to about 50 tig/m1CD27L.
In some aspects,
the MRM comprises and/or is supplemented with about 0.1 g/m1 to about 10
g/ml, about 0.1
jig/m1 to about 9 gg/ml, about 0.1 jig/ml to about 8 g/ml, about 0.1 g/m1 to
about 7 g/ml, about
0.1 g/m1 to about 6 g/ml, about 0.1 g/m1 to about 5 g/ml, about 0.1 g/m1
to about 4 g/ml,
about 0.1 jig/m1 to about 3 g/ml, about 0.1 g/m1 to about 2 g/ml, about 0.1
g/m1 to about 1
g/ml, 1 s/m1 to about 10 g/ml, about 1 g/m1 to about 5 jig/ml, about 1
jig/m1 to about 4 g/ml,
about 1 g/m1 to about 3 g/ml, or about 1 g/m1 to about 2 g/m1 4-1BBL.
[0368] In some aspects, the MRM, e.g., secondary TIL expansion
media and/or third (or
final) TIL expansion media, comprise and/or are supplemented with at least
about 0.1 g/ml, at
least about 0.2 g/ml, at least about 0.3 g/ml, at least about 0.4 g/ml, at
least about 0.5 g/ml,
at least about 0.6 g/ml, at least about 0.7 g/ml, at least about 0.8 g/ml,
at least about 0.9 g/ml,
at least about 1 g/ml, at least about 1.1 g/ml, at least about 1.2 g/ml, at
least about 1.3 g/ml,
at least about 1.4 g/ml, at least about 1.5 g/ml, at least about 1.6 g/ml,
at least about 1.7 g/ml,
at least about 1.8 g/ml, at least about 1.9 g/ml, at least about jig/ml, at
least about 2 g/ml, at
least about 3 g/ml, at least about 4 jig/ml, at least about 5 g/ml, or at
least about 10 g/m1 4-
1BBL. In some aspects, the MRM comprises and/or is supplemented with at least
about 1 p.g/m1
4-1BBL.
[0369] In some aspects, a 4-1BBL is added in the MRM together
with a CD27L. In some
aspects, a 4-1BBL is added in the MRM concurrently with a CD27L. In some
aspects, a 4-1BBL
is added in the MRM with a CD27L in sequence. In some aspects, the MRIVI used
during an
expansion process (e.g., a secondary expansion and/or a final expansion)
comprises
TRANSACTTm, 4-1 BBL, and CD27L. In some aspects, the MRM comprises at least
about 1:100
TRANSACTTm, at least about 1 s/m1 4-1BBL, and at least about 5 g/ml CD27L.
In some
aspects, the 1V1R_M used during an expansion process (e.g., a secondary
expansion and/or a final
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expansion) comprises at least about 1:100 TRANSACTTm, at least about 1 p..g/m1
4-1BBL, and at
least about 5 mg/m1 CD27L.
[0370] In some aspects, the MRM, e.g., initial TIL culture
medium, secondary TIL
expansion medium and/or third (or final) TIL expansion medium, is modified
from a basal medium
selected from a balanced salt solution (e.g., PBS, DPBS, HBSS, EBSS),
Dulbecco's Modified
Eagle's Medium (DMEM), Click's medium, Minimal Essential Medium (MEM), Basal
Medium
Eagle (BME), F-10, F-12, RPMI 1640, Glasgow Minimal Essential Medium (GMEM),
alpha
Minimal Essential Medium (alpha MEM), Iscove's Modified Dulbecco's Medium
(IMDM), M199,
OpTmizerTm CTSTm T-Cell Expansion Basal Medium (ThermoFisher), OPTMIZERTm
Complete,
IM1VIUNOCULTTm XF (STEMCELLTm Technologies), IMMUNOCULTTm XF, AIM V,
TEXMACSTm medium, and any combination thereof. In some aspects, the basal
medium is serum
free. In some aspects, the basal medium further comprises immune cell serum
replacement (ICSR).
For example, in some aspects, the basal medium comprises OPTMIZERTm Complete
supplemented with ICSR, AIM V supplemented with ICSR, INIMUNOCULTTm XF
supplemented
with ICSR, RPMI supplemented with ICSR, TEXMACSTm supplemented with ICSR, or
any
combination thereof. In particular aspects, the basal media comprises
OPTMIZERTm complete. In
some aspects, suitable basal medium includes Click's medium, OpTimizer (CTS )
medium,
Stemline T cell expansion medium (Sigma-Aldrich), AIM V medium (CTS ),
TexMAC S
medium (Miltenyi Biotech), ImmunoCult medium (Stem Cell Technologies), PRIME-
XV T-
Cell Expansion XSFM (Irvine Scientific), Iscoves medium, and/or RPMI-1640
medium.
[0371] The present disclosure comprises a MRNI comprising basal
media, NaCl, KC1,
calcium, and glucose, wherein the concentration of NaCl is between about 40 mM
and about
80mM, the concentration of KC1 is between 40 and 90mM, the concentration of
calcium is between
about 0.5mM and about 2.8mM, and the concentration of glucose between about 10
mM and about
24mM.
[0372] In some aspects, the MRNI further comprises immune cells.
In some aspects, the
immune cells comprises TILs.
[0373] In some aspects, the MRI\4 further comprises IL-2, IL-7,
IL-15, IL-21, or any
combination thereof In some aspects, the MRM further comprises IL-2 and IL-21.
In some aspects,
the concentration of IL-2 is about 200 ng/ml to about 400 ng/ml (e.g., about
200 ng/ml, about 300
ng/ml, or about 400 ng/ml). In some aspects, the concentration of IL-21 is
about 20 ng/ml to about
40 ng/ml, (e.g., about 20 ng/ml, about 30ng/ml, or about 40 ng/ml).
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[0374] In some aspects, the MRM further comprises about 2.5%
serum supplement (CTSTm
Immune Cell SR, Thermo Fisher), 2 mM L-glutamine, 2 mM L-glutamax, MEM Non-
Essential
Amino Acids Solution, Pen-strep, 20 g/m1 FUNGINTM, Sodium pyruvate, or any
combination
thereof. In some aspects, the MRM further comprises O-Acetyl-L-carnitine
hydrochloride. In some
aspects, the MRM further comprises a kinase inhibitor.
[0375] In some aspects, the MRIVI further comprises a CD3
agonist. In some aspects, the
CD3 agonist is an anti-CD3 antibody. In some aspects, the anti-CD3 antibody
comprises OKT-3.
[0376] In some aspects, the MRM further comprises a CD28
agonist. In some aspects, the
CD28 agonist is an anti-CD28 antibody. In some aspects, the MRM further
comprises a CD27
ligand (CD27L). In some aspects, the MRM further comprises a 4-1BB ligand (4-
1BBL).
[0377] In some aspects, the present disclosure includes a cell
culture comprising the MRM
disclosed herein, a cell bag comprising the MRM disclosed herein, or a
bioreactor comprising the
MRM disclosed herein.
11.11. Cells
[0378] Some aspects of the present disclosure are directed to
methods of culturing TILs,
comprising placing the TILs in a medium comprising potassium ion at a
concentration of greater
than 5 mM, as disclosed herein. Some aspects of the present disclosure are
directed to methods of
culturing TILs, comprising placing the Tits in a medium comprising potassium
ion at a
concentration higher than 40 mM, as disclosed herein. Some aspects of the
present disclosure are
directed to methods of culturing TILs, comprising placing the TILs in a medium
comprising
potassium ion at a concentration of at least about 50 mM, as disclosed herein.
Some aspects of the
present disclosure are directed to methods of culturing TILs, comprising
placing the TILs in a
medium comprising potassium ion at a concentration of at least about 40 mM to
at least about 90
mM, as disclosed herein.
[0379] Some aspects of the present disclosure are directed to
methods of culturing TILs,
comprising placing the TILs in a medium comprising potassium ion at a
concentration higher than
40 mM and NaCl at a concentration of less than 100 mM, as disclosed herein.
Some aspects of the
present disclosure are directed to methods of culturing TILs, comprising
placing the TILs in a
medium comprising potassium ion at a concentration of at least about 50 mM and
NaCl at a
concentration of less than 90 mM, as disclosed herein. Some aspects of the
present disclosure are
directed to methods of culturing Tits, comprising placing the TILs in a medium
comprising
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potassium ion at a concentration of at least about 40 mM to at least about 90
mM and NaCl at a
concentration of less than 100 mM to 50 mM, as disclosed herein.
[0380] The TILs that are placed in the MRM can be TILs that are
collected and/or isolated
from a subject in need of a therapy. In some aspects, the TILs that are placed
in the medium have
been expanded prior to being placed in a MRM disclosed herein. The TILs that
are placed in the
medium can be referred to as starting (initial, i.e., patient sample,
apheresis sample, buffy coat)
TILs. The TILs that result from culturing them in the media disclosed herein
can be referred to as
resulting (cultured) TILs.
[0381] In some aspects, the Tits are present in a tumor sample
obtained from a subject.
Accordingly, in some aspects, the method comprises placing a tumor sample into
an MRM
disclosed herein. During standard TIL culture, tumor samples, e.g., a tumor
biopsy or a fragment
thereof, is plated in an initial TIL culture medium, and cultured for at least
about 14-19 days. In
some aspects, the tumor sample, e.g., the tumor biopsy, is cultured in an MRM
in an initial TIL
culture for at least about 7 days, at least about 8 days, at least about 9
days, at least about 10 days,
at least about 11 days, at least about 12 days, at least about 13 days, at
least about 14 days, at least
about 15 days, at least about 16 days, at least about 17 days, at least about
18 days, at least about
19 days, at least about 20 days, at least about 21 day. In some aspects, the
initial TIL culture lasts
about 14 days. In some aspects the initial TIL culture lasts sufficient number
of days until a cell
yield of about 2x106 to about 10x106 cells are produced.
[0382] In some aspects, the proportion of CD8+ TILs to non-CD8+
TILs (e.g., the
proportion of CD8+ TILs to CD4+ TILs) is increased following the initial TIL
culture, as compared
to the proportion of CD8+ Tits to non-CD8+ TILs prior to the initial TIL
culture. In some aspects,
the proportion of CD8+ TILs to non-CDS TILs (e.g, the proportion of CD8+ TILs
to CD4+ TILs)
is increased following the initial TIL culture, as compared to the proportion
of CDS+ TILs to non-
CD8+ TILs following an initial TIL culture in a basal medium or a medium that
does not comprise
an increased concentration of potassium ion (control medium). In some aspects,
the proportion of
CD8+ TILs is increased by at least about 1.5-fold, at least about 2-fold, at
least about 2.5-fold, at
least about 3-fold, at least about 3.5-fold, at least about 4-fold, at least
about 4.5-fold, at least about
5-fold, at least about 6-fold, at least about 7-fold, at least about 8-fold,
at least about 9-fold, at least
about 10-fold, at least about 15-fold, at least about 20-fold, at least about
25-fold, at least about
30-fold, at least about 45-fold, or at least about 50-fold. In some aspects,
the proportion of CDS+
TILs is increased by at least about 40-fold. In some aspects, the proportion
of CDS+ Tits is
increased by at least about 50-fold.
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[0383] In some aspects, the proportion of CD8 TILs is increased
by at least about 20%, at
least about 25%, at least about 30%, at least about 35%, at least about 40%,
at least about 45%, at
least about 50%, at least about 60%, at least about 70%, at least about 75%,
at least about 80%, at
least about 90%, at least about 100%, at least about 125%, at least about
150%, at least about 175%,
at least about 200%, at least about 250%, at least about 300%, at least about
350%, at least about
400%, at least about 450%, or at least about 500%. In some aspects, the
proportion of CDS+ TILs
is increased by at least about 20%. In some aspects, the proportion of CDS+
TILs is increased by
at least about 40%. In some aspects, the proportion of CD8+ TILs is increased
by at least about
60%. In some aspects, the proportion of CD8+ TILs is increased by at least
about 80%. In some
aspects, the proportion of CD8+ TILs is increased by at least about 100%.
[0384] In some aspects, the proportion of CD8+ TILs is increased
to at least about 20%, at
least about 25%, at least about 30%, at least about 35%, at least about 40%,
at least about 45%, at
least about 50%, at least about 55%, at least about 60%, at least about 65%,
at least about 70%, at
least about 75%, at least about 80%, at least about 85%, at least about 90%,
at least about 95%, at
least about 96%, at least about 97%, at least about 98%, or at least about 99%
of the total number
of TILs in the culture. In some aspects, the proportion of CD8' TILs is
increased to at least about
20% of the total number of TILs in the culture. In some aspects, the
proportion of CD8+ TILs is
increased to at least about 30% of the total number of TILs in the culture. In
some aspects, the
proportion of CD8+ TILs is increased to at least about 40% of the total number
of TILs in the
culture. In some aspects, the proportion of CD8+ TILs is increased to at least
about 50% of the total
number of TILs in the culture. In some aspects, the proportion of CDS+ TILs is
increased to at least
about 60% of the total number of TILs in the culture. In some aspects, the
proportion of CD8+ TILs
is increased to at least about 70% of the total number of TILs in the culture.
In some aspects, the
proportion of CDS+ TILs is increased to at least about 75% of the total number
of TILs in the
culture. In some aspects, the proportion of CDS+ TILs is increased to at least
about 80% of the total
number of TILs in the culture. In some aspects, the proportion of CD8+ Tits is
increased to at least
about 90% of the total number of TILs in the culture.
[0385] In some aspects, the number of tumor-reactive cells in
the culture is increased by
about 2-fold to about 500-fold, about 2-fold to about 250-fold, about 2-fold
to about 200-fold,
about 2-fold to about 150-fold, about 2-fold to about 100-fold, about 2-fold
to about 90-fold, about
2-fold to about 80-fold, about 2-fold to about 70-fold, about 2-fold to about
60-fold, about 2-fold
to about 50-fold, about 2-fold to about 40-fold, about 2-fold to about 30-
fold, about 2-fold to about
20-fold, about 2-fold to about 10-fold, about 5-fold to about 200-fold, about
5-fold to about 150-
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fold, about 5-fold to about 100-fold, about 5-fold to about 90-fold, about 5-
fold to about 80-fold,
about 5-fold to about 70-fold, about 5-fold to about 60-fold, about 5-fold to
about 50-fold, about
5-fold to about 40-fold, about 5-fold to about 30-fold, about 5-fold to about
20-fold, about 5-fold
to about 10-fold, about 10-fold to about 150-fold, about 10-fold to about 100-
fold, about 10-fold
to about 90-fold, about 10-fold to about 80-fold, about 10-fold to about 70-
fold, about 10-fold to
about 60-fold, about 10-fold to about 50-fold, about 10-fold to about 40-fold,
about 10-fold to
about 30-fold, or about 10-fold to about 20-fold following the culture methods
disclosed herein, as
compared to the number of tumor-reactive cells prior to the initial Tit
culture. In some aspects,
the number of tumor-reactive cells in the culture is increased by at least
about 2-fold, at least about
2-fold, at least about 3-fold, at least about 4-fold, at least about 5-fold,
at least about 6-fold, at least
about 7-fold, at least about 8-fold, at least about 9-fold, at least about 10-
fold, at least about 15-
fold, at least about 20-fold, at least about 25-fold, at least about 30-fold,
at least about 35-fold, at
least about 40-fold, at least about 45-fold, at least about 50-fold, at least
about 60-fold, at least
about 70-fold, at least about 80-fold, at least about 90-fold, at least about
100-fold, at least about
125-fold, at least about 150-fold, at least about 175-fold, at least about 200-
fold, at least about 250-
fold, at least about 300-fold, at least about 350-fold, at least about 400-
fold, at least about 450-
fold, or at least about 500-fold following the culture methods disclosed
herein, as compared to the
number of tumor-reactive cells prior to the initial TIL culture. In some
aspects, the number of
tumor-reactive cells in the culture is increased by at least about 2-fold
following the culture
methods disclosed herein, as compared to the number of tumor-reactive cells
prior to the initial
TIL culture. In some aspects, the number of tumor-reactive cells in the
culture is increased by at
least about 3-fold following the culture methods disclosed herein, as compared
to the number of
tumor-reactive cells prior to the initial TIL culture. In some aspects, the
number of tumor-reactive
cells in the culture is increased by at least about 4-fold following the
culture methods disclosed
herein, as compared to the number of tumor-reactive cells prior to the initial
TIL culture. In some
aspects, the number of tumor-reactive cells in the culture is increased by at
least about 5-fold
following the culture methods disclosed herein, as compared to the number of
tumor-reactive cells
prior to the initial TIL culture. In some aspects, the number of tumor-
reactive cells in the culture
is increased by at least about 10-fold following the culture methods disclosed
herein, as compared
to the number of tumor-reactive cells prior to the initial TIL culture
[0386] In some aspects, the number of tumor-reactive cells in
the culture is increased by
about 2-fold to about 500-fold, about 2-fold to about 250-fold, about 2-fold
to about 200-fold,
about 2-fold to about 150-fold, about 2-fold to about 100-fold, about 2-fold
to about 90-fold, about
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2-fold to about 80-fold, about 2-fold to about 70-fold, about 2-fold to about
60-fold, about 2-fold
to about 50-fold, about 2-fold to about 40-fold, about 2-fold to about 30-
fold, about 2-fold to about
20-fold, about 2-fold to about 10-fold, about 5-fold to about 200-fold, about
5-fold to about 150-
fold, about 5-fold to about 100-fold, about 5-fold to about 90-fold, about 5-
fold to about 80-fold,
about 5-fold to about 70-fold, about 5-fold to about 60-fold, about 5-fold to
about 50-fold, about
5-fold to about 40-fold, about 5-fold to about 30-fold, about 5-fold to about
20-fold, about 5-fold
to about 10-fold, about 10-fold to about 150-fold, about 10-fold to about 100-
fold, about 10-fold
to about 90-fold, about 10-fold to about 80-fold, about 10-fold to about 70-
fold, about 10-fold to
about 60-fold, about 10-fold to about 50-fold, about 10-fold to about 40-fold,
about 10-fold to
about 30-fold, or about 10-fold to about 20-fold following the culture methods
disclosed herein, as
compared to the number of tumor-reactive cells following expansion using
control methods (e.g.,
in medium comprising less than 40 mM potassium ion, e.g., 4 mM potassium ion).
In some aspects,
the number of tumor-reactive cells in the culture is increased by at least
about 2-fold, at least about
2-fold, at least about 3-fold, at least about 4-fold, at least about 5-fold,
at least about 6-fold, at least
about 7-fold, at least about 8-fold, at least about 9-fold, at least about 10-
fold, at least about 15-
fold, at least about 20-fold, at least about 25-fold, at least about 30-fold,
at least about 35-fold, at
least about 40-fold, at least about 45-fold, at least about 50-fold, at least
about 60-fold, at least
about 70-fold, at least about 80-fold, at least about 90-fold, at least about
100-fold, at least about
125-fold, at least about 150-fold, at least about 175-fold, at least about 200-
fold, at least about 250-
fold, at least about 300-fold, at least about 350-fold, at least about 400-
fold, at least about 450-
fold, or at least about 500-fold following the culture methods disclosed
herein, as compared to the
number of tumor-reactive cells following expansion using control methods
(e.g., in medium
comprising less than 40 mM potassium ion, e.g., 4 mM potassium ion). In some
aspects, the number
of tumor-reactive cells in the culture is increased by at least about 2-fold
following the culture
methods disclosed herein, as compared to the number of tumor-reactive cells
following expansion
using control methods (e.g., in medium comprising less than 40 mM potassium
ion, e.g., 4 mM
potassium ion). In some aspects, the number of tumor-reactive cells in the
culture is increased by
at least about 3-fold following the culture methods disclosed herein, as
compared to the number of
tumor-reactive cells following expansion using control methods (e.g., in
medium comprising less
than 40 mM potassium ion, e.g., 4 mM potassium ion). In some aspects, the
number of tumor-
reactive cells in the culture is increased by at least about 4-fold following
the culture methods
disclosed herein, as compared to the number of tumor-reactive cells following
expansion using
control methods (e.g., in medium comprising less than 40 mM potassium ion,
e.g., 4 mM potassium
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ion). In some aspects, the number of tumor-reactive cells in the culture is
increased by at least
about 5-fold following the culture methods disclosed herein, as compared to
the number of tumor-
reactive cells following expansion using control methods (e.g., in medium
comprising less than 40
mM potassium ion, e.g., 4 mM potassium ion). In some aspects, the number of
tumor-reactive cells
in the culture is increased by at least about 10-fold following the culture
methods disclosed herein,
as compared to the number of tumor-reactive cells following expansion using
control methods
(e.g., in medium comprising less than 40 mM potassium ion, e.g., 4 mM
potassium ion).
[0387] In some aspects, the tumor sample is isolated from a
human subject. In some
aspects, the starting tumor sample isolated from a human subject, and the TILs
therein are
expanded for an allogeneic cell therapy. In some aspects, the tumor sample is
isolated from a
human subject, and the TILs therein are expanded for an autologous cell
therapy.
ILL TIL Isolation, Expansion, and Harvest
[0388] Any method of TIL isolation, culture, and/or expansion
can be modified according
to the methods disclosed herein, e.g., by culturing and/or expanding the TILs
in a culture medium
described herein.
ILIA_ Initial Expansion
[0389] In general, TILs are obtained from a tumor sample
obtained from a human subject.
Any methods for obtaining a tumor biopsy from a subject can be used in the
methods disclosed
herein, so long as the tumor sample contains a mixture of tumor and TILs. In
some aspects, the
tumor sample is isolated through a tumor resection. In some aspects, the tumor
sample is isolated
by a needle biopsy (see, e.g., US Publication No. US 2020/0277573, which is
incorporated by
reference herein in its entirety). In some aspects, the tumor sample comprises
a solid tumor,
including a primary tumor, invasive tumor or metastatic tumor. In other
aspects, the tumor sample
comprises a liquid tumor, such as a tumor obtained from a hematological
malignancy. The tumor
may be of any cancer type, including, but not limited to, breast, pancreatic,
prostate, colorectal,
cervical, lung, brain, renal, stomach, liver (including but not limited to
hepatocellular carcinoma)
and skin (including but not limited to squamous cell carcinoma, basal cell
carcinoma, and
melanoma). In some aspects, the tumor comprises a melanoma. In some aspects,
the tumor
comprises a colorectal cancer. In some aspects, the tumor comprises a
pancreatic cancer. In some
aspects, the tumor comprises a head and neck cancer. In some aspects, the
tumor comprises a
cervical cancer. In some aspects, the tumor comprises an ovarian cancer. In
some aspects, the tumor
comprises an non-small cell lung cancer. In some aspects, the tumor comprises
a breast cancer. In
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some aspects, the tumor sample is cryopreserved after the tumor sample is
dissociated. In some
aspects, the tumor sample is dissociated in MRNI prior to cryopreservation. In
some aspects, the
tumor sample is cryopreserved prior to TIL isolation/expansion. In some
aspects, the tumor sample
is cryopreserved after initial TIL expansion. In some aspects the tumor sample
is fresh, e.g., not
cryopreserved. In some aspects, the tumor sample is placed directly into MRM
media.
[0390] In some aspects, the donor patient (e.g., the subject
from which the tumor is
obtained) is treatment naive (i.e., the patient has not received a prior
therapy for the treatment of
the tumor). In some aspects, the donor patient has received one or more prior
therapy for the
treatment of the tumor. In some aspects, the subject has received at least one
prior therapy, at least
two prior therapies, at least three prior therapies, or at least four prior
therapies. In some aspects,
the subject is relapsed or refractory to one or more prior therapy.
[0391] In some aspects, the subject has received one or more
prior anticancer therapy. In
some aspects, the prior anticancer therapy comprises a standard of care
therapy. In some aspects,
the prior anticancer therapy comprises an immunotherapy. In some aspects, the
prior therapy
comprises an immunotherapy comprising a checkpoint inhibitor. In some aspects,
the prior therapy
comprises an immunotherapy comprising an anti-PD-1 antibody, an anti-CTLA-4
antibody, an
anti-LAG-3 antibody, or any combination thereof
[0392] In some aspects, the subject is administered one or more
therapy that enhances the
isolation and/or expansion of TILs prior to resection of the tumor sample. In
some aspects, the
subject is administered a kinase inhibitor or an ITK inhibitor. Examples of
kinase inhibitors and/or
ITK inhibitors can be found, for example, in Int'l Publication No.
W02019217753, which is
incorporated by reference herein in its entirety. In some aspects, the kinase
inhibitor and/or the
ITK inhibitor is added to the culture medium during the initial expansion
and/or the second
expansion. In some aspects, the ITK inhibitor is selected from the group
consisting of
aminothiazole-based ITK inhibitors, benzimidazole-based ITK inhibitors,
aminopyrimi dine-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 some aspects, the ITK inhibitor is selected from the
group consisting of
ibrutinib, dasatinib, bosutinib, nilotinib, erlotinib, BMS509744, CTA056,
GSK2250665A,
PF06465469, and any combination thereof.
[0393] In some aspects, the tumor sample is cut into smaller
fragments. In some aspects,
the one or more of the smaller fragments is at least about 1 mm2, at least
about 1.5 mm2, at least
about 2 mm2, at least about 2.5 mm2, at least about 3 mm2, at least about 3.5
mm2, at least about 4
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mm2, at least about 4.5 mm2, at least about 5 mm2, at least about 5.5 mm2, at
least about 6 mm2, or
at least about 6.5 mm2. In some aspects, the one or more of the smaller
fragments is at least about
1 mm3, at least about 1.5 mm3, at least about 2 mm3, at least about 2.5 mm3,
at least about 3 mm3,
at least about 3.5 mm3, at least about 4 mm3, at least about 4.5 mm3, at least
about 5 mm3, at least
about 5.5 mm3, at least about 6 mm3, at least about 6.5 mm3, at least about 7
mm3, at least about
7.5 mm3, at least about 8 mm3, at least about 8.5 mm3, at least about 9 mm3,
at least about 9.5 mm3,
or at least about 10 mm3. In some aspects, the tumor samples are subjected to
an enzymatic digest,
by culturing the tumor samples in an enzymatic media (e.g., RPMI 1640 buffer
or MRM
supplemented with glutamate (e.g., about 2 mM), gentamicine (e.g., about 10
mcg/mL), DNase
(e.g., about 30 units/mL), and collagenase (e.g., about 1.0 mg/mL)). In some
aspects, the tumor
digests are produced by placing the tumor in the enzymatic media and/or
mechanically dissociating
(i.e., di saggregating) the tumor (e.g., for about 1 minute), followed by
incubation at 37 C. in 5%
CO2 (e.g., for 30 minutes), 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 can be performed
to remove these
cells. The mechanical and/or enzymatic dissociation can be performed in any
medium. In some
aspects, the mechanical and/or enzymatic dissociation is performed in an MRM
medium disclosed
herein.
[0394] In some aspects, the mechanical dissociation comprises
applying a physical
pressure to the resected tumor. In some aspects, the mechanical dissociation
comprises repeated
physical pressure. In some aspects, the repeated physical pressure is applied
at least about 50 times,
at least about 60 times, at least about 70 times, at least about 80 times, at
least about 90 times, at
least about 100 times, at least about 110 times, at least about 120 times, at
least about 130 times,
at least about 140 times, at least about 150 times, at least about 160 times,
at least about 170 times,
at least about 180 times, at least about 190 times, at least about 200 times,
at least about 210 times,
at least about 220 times, at least about 230 times, at least about 240 times,
at least about 250 times,
at least about 260 times, at least about 270 times, at least about 280 times,
at least about 290 times,
at least about 300 times, at least about 310 times, at least about 320 times,
at least about 330 times,
at least about 340 times, at least about 350 times, or at least about 360
times per minute. In some
aspects, the repeated physical pressure is applied at least about 120 to 260
times per minute. In
some aspects, the repeated physical pressure is applied up to about 6N/cm2, up
to about 5.5 N/cm2,
up to about 5.0 N/cm2, up to about 4.5 N/cm2, up to about 4.0 N/cm2, up to
about 3.5 N/cm2, up to
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about 3.0 N/cm2. In some aspects, the mechanical dissociation proceeds for
about 90 minutes or
less, about 85 minutes or less, about 80 minutes or less, about 75 minutes or
less, about 70 minutes
or less, about 65 minutes or less, about 60 minutes or less, about 55 minutes
or less, or about 50
minutes or less. In some aspects, the mechanical dissociation is applied at
room temperature. In
some aspects, the mechanical dissociation is applied at less than room
temperature. In some
aspects, the mechanical dissociation is applied according to the methods
disclosed in and/or using
a device disclosed in Int'l Publication No. WO 2021/123832, which is
incorporated by reference
herein in its entirety.
[0395] In some aspects, the tumor sample (i.e., the resected
tumor tissue sampl or the
dissociated tumor sample) or the fragments thereof is placed into a culture
medium, e.g., a culture
medium disclosed herein, wherein the culture medium further comprises IL-2. In
some aspects, the
culture medium comprises at least about 4000 IU/ml IL-2, at least about 4500
IU/ml IL-2, at least
about 5500 IU/m1 IL-2, at least about 6000 IU/ml IL-2, or at least about 6500
IU/ml IL-2. In some
aspects, the culture medium comprises at least about 600 IU/ml IL-2. In some
aspects, the culture
medium comprises at least about 100 ng/mL IL-2. In some aspects, the culture
medium comprises
at least about 200 ng/mL IL-2. In some aspects, the culture medium comprises
at least about 300
ng/mL IL-2. In some aspects, the culture medium comprises at least about 400
ng/mL IL-2. In
some aspects, the culture medium comprises at least about 500 ng/mL IL-2. In
some aspects, the
culture medium comprises at least about 600 ng/mL IL-2.
[0396] In other aspects, the tumor sample or the fragments
thereof is placed into a culture
medium, e.g., a culture medium disclosed herein, wherein the culture medium
further comprises
IL-21. In some aspects, the culture medium comprises at least about 1.0 ng/mL
IL-21. In some
aspects, the culture medium comprises at least about 2.0 ng/mL IL-21. In some
aspects, the culture
medium comprises at least about 3.0 ng/mL IL-21. In some aspects, the culture
medium comprises
at least about 4.0 ng/mL IL-21. In some aspects, the culture medium comprises
at least about 5.0
ng/mL IL-21. In some aspects, the culture medium comprises at least about 6.0
ng/mL IL-21. In
some aspects, the culture medium comprises at least about 7.0 ng/mL IL-21. In
some aspects, the
culture medium comprises at least about 8.0 ng/mL 1L-21. In some aspects, the
culture medium
comprises at least about 9.0 ng/mL IL-21. In some aspects, the culture medium
comprises at least
about 10 ng/mL IL-21. In some aspects, the culture medium comprises at least
about 15 ng/mL IL-
21. In some aspects, the culture medium comprises at least about 20 ng/mL IL-
21. In some aspects,
the culture medium comprises at least about 30 ng/mL IL-21.
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[0397] Individual tumor fragments can be cultured together in a
single culture chamber,
e.g., well, or individual tumor fragments can be cultured in separate culture
chambers, e.g., wells.
A standard culture medium for promoting TIL evasion from cultured tumor
samples comprises
RPMI 1640 supplemented with Glutamax (Gibco/Tnvitrogen; Carlsbad, Calif),
1xPen-Strep
(Gibco/Invitrogen; Carlsbad, Calif.), 50 pm 2-mercaptoethanol
(Gibco/Invitrogen; Carlsbad,
Calif.), 20 pg/ml Gentamicin (Gibco/Invitrogen; Carlsbad, Calif.), and 1 mM
pyruvate
(Gibco/Invitrogen; Carlsbad, Calif). In some aspects, a standard culture
medium is modified
according to the present disclosure In some aspects, a standard culture medium
comprises CTSTm
OpTimizerTm supplemented with serum supplement (CT STm Immune Cell SR, Thermo
Fisher), L-
glutamine (Gibco), L-glutamax (Gibco), MEM Non-Essential Amino Acids Solution
(Gibco), Pen-
strep (Gibco), funginTM (InvivoGen), Sodium pyruvate (Gibco), IL-2, IL-21, O-
Acetyl-L-carnitine
hydrochloride (Sigma), or any combination thereof In some aspects, a standard
culture medium
comprises CTSTm OpTimizerTm supplemented with about 2.5% serum supplement
(CTSTm
Immune Cell SR, Thermo Fisher), about 2 mM L-glutamine (Gibco), about 2 mM L-
glutamax
(Gibco), MEM Non-Essential Amino Acids Solution (Gibco), Pen-strep (Gibco),
about 20pg/m1
funginTM (InvivoGen), Sodium pyruvate (Gibco), about IL-2 (300ng/mL), about IL-
21 (30ng/m1),
and about 1mM of O-Acetyl-L-carnitine hydrochloride (Sigma).
[0398] In some aspects, tumor samples or fragments thereof are
cultured in an initial
culture for at least about 1 week, at least about 2 weeks, or at least about 3
weeks. In some aspects,
tumor samples or fragments thereof are cultured for at least about 2 weeks. As
used herein, "tumor
samples" refers to tumor tissue and/or disaggregated tumor tissue (i.e., a
cell suspension resulting
from mechanical and/or chemical di saggregati on of tumor tissue). In some
aspects, the tumor
samples or fragments are cultured in an initial culture for 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.
[0399] In some aspects, the initial culture further comprises
contacting the tumor samples
or fragments with a tumor necrosis factor receptor superfamily (TNFRSF)
agonist. In some aspects,
the TNFRSF agonist comprises a 4-1BB agonist, an 0X40 agonist, a CD27 agonist,
a GITR
agonist, a HVEM agonist, a CD95 agonist, or any combination thereof. In some
aspects, the
TNFRSF agonist is any TNFRSF agonist disclosed in U.S. Publication No. US
2020/0121719 Al,
which is incorporated by reference herein in its entirety. In some aspects,
the initial culture further
comprises contacting the tumor samples or fragments thereof with about 10-500
ng/ml 4-1BB
ligand. In some aspects, initial culture further comprises contacting the
tumor samples or fragments
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thereof with about 50 ng/ml, about 60 ng/ml, about 70 ng/ml, about 75 ng/ml,
about 80 ng/ml,
about 90 ng/ml, about 100 ng/ml, about 125 ng/ml, about 150 ng/ml, about 175
ng/ml, about 200
ng/ml, about 250 ng/ml, about 300 ng/ml, about 350 ng/ml, about 400 ng/ml,
about 450 ng/ml,
about 500 ng/ml, about 550 ng/ml, about 600 ng/ml, about 650 ng/ml, about 700
ng/ml, about 750
ng/ml, about 800 ng/ml, about 850 ng/ml, about 900 ng/ml, about 950 ng/ml,
about 1000 ng/ml, or
about 1100 ng/ml 4-1BB ligand. In some aspects, initial culture further
comprises contacting the
tumor samples or fragments thereof with about 100 ng/ml 4-1BB ligand. In some
aspects, the tumor
samples or fragments thereof are contacted with the 4-1BB ligand on about day
3 of the initial
culture, on about day 4 of the initial culture, on about day 5 of the initial
culture, on about day 6 of
the initial culture, or on about day 7 of the initial culture. In some
aspects, the tumor samples or
fragments thereof are contacted with the 4-1BB ligand on about day 5 of the
initial culture.
[0400] In some aspects, the initial culture further comprises
contacting the tumor samples
or fragments thereof with TRANSACTTm. In some aspects, initial culture further
comprises
contacting the tumor samples or fragments thereof with TRANSACTTm (e.g., about
1:50, about
1:100, about 1:150, about 1:200, about 1:250, about 1:300, about 1:350, or
about 1:400). In some
aspects, the tumor samples or fragments thereof are contacted with the
TRANSACTTm on about
day 4 of the initial culture, on about day 5 of the initial culture, on about
day 6 of the initial culture,
or on about day 7 of the initial culture. In some aspects, the tumor samples
or fragments thereof
are contacted with the TRANSACTTm on about day 5 of the initial culture. In
some aspects, the
initial culture further comprises contacting the tumor samples or fragments
thereof with both 4-
1BB ligand and TRANSACTTm. In some aspects, the tumor samples or fragments
thereof are
contacted with both 4-1BB ligand and TRANSACTTm on about day 3 of the initial
culture. In some
aspects, the tumor samples or fragments thereof are contacted with both 4-1BB
ligand and
TRANSACTTm on about day 4 of the initial culture. In some aspects, the tumor
samples or
fragments thereof are contacted with both 4-1BB ligand and TRANSACTTm on about
day 5 of the
initial culture. In some aspects, the tumor samples or fragments thereof are
contacted with both 4-
1BB ligand and TRANSACTTm on about day 6 of the initial culture In some
aspects, the tumor
samples or fragments thereof are contacted with both 4-1BB ligand and
TRANSACTTm on about
day 7 of the initial culture. In some aspects, the tumor samples or fragments
thereof are contacted
with both 4-1BB ligand and TRANSACTTm on about day 8 of the initial culture.
[0401] In some aspects, tumor samples or fragments thereof are
cultured in an initial
culture until cell yield in the initial culture reaches at least about 1x105
to at least about 1x108, at
least about 5x105 to at least about 1x108, at least about 1x106 to at least
about 1x108, at least about
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2x106 to at least about 1x108, at least about 3x106 to at least about 1x108,
at least about 4x106 to at
least about 1x108, at least about 5x106 to at least about 1x108, at least
about 1x105 to at least about
5x107, at least about 5xl0 to at least about 10x106, at least about 1x106 to
at least about 10x106,
at least about 2x106 to at least about 10x106, at least about 3x106 to at
least about 10x106, at least
about 4x106 to at least about 10x106, or at least about 5x106 to at least
about 10x106 cells per
cultured fragment. In some aspects, tumor samples or fragments thereof are
cultured in an initial
culture until cell yield in the initial culture reaches at least about 2x106-
10x106 cells per fragment.
In some aspects, tumor samples or fragments thereof are cultured in an initial
culture until cell
yield in the initial culture reaches at least about 10x106-50x106. In some
aspects, tumor samples or
fragments thereof are cultured in an initial culture until cell yield in the
initial culture reaches at
least about 10x106, at least about 15x106, at least about 20x106, at least
about 25x106, at least about
30x106, at least about 35x106, at least about 40x106, at least about 45x106,
or at least about 50 x106'
In some aspects, tumor samples or fragments thereof are cultured in an initial
culture until cell
yield in the initial culture reaches at least about 20x106. In some aspects,
tumor samples or
fragments thereof are cultured in an initial culture until cell yield in the
initial culture reaches at
least about 25x106. In some aspects, tumor samples or fragments thereof are
cultured in an initial
culture until cell yield in the initial culture reaches at least about 30x106.
In some aspects, tumor
samples or gragments thereof are cultured in an initial culture until cell
yield in the initial culture
reaches at least about 35x106. In some aspects, tumor samples or gragments
thereof are cultured in
an initial culture until cell yield in the initial culture reaches at least
about 40x106.
[0402] In some aspects, tumor samples or fragments thereof are
cultured in an initial
culture until cell yield in the initial culture reaches at least about 1x105,
at least about 2x105, at
least about 3x105, at least about 4x105, at least about 5x105, at least about
6x105, at least about
7x105, at least about 8x105, at least about 9x105, at least about 1x106, at
least about 2x106, at least
about 3x106, at least about 4x106, at least about 5x106, at least about 6x106,
at least about 7x106,
at least about 8x106, at least about 9x106, or at least about 10x106 cells per
fragment. In some
aspects, tumor samples or fragments thereof are cultured in an initial culture
until cell yield in the
initial culture reaches at least about 2x106 cells per fragment. In some
aspects, tumor samples or
fragments thereof are cultured in an initial culture until cell yield in the
initial culture reaches at
least about 3x106 cells per fragment. In some aspects, tumor samples or
fragments thereof are
cultured in an initial culture until cell yield in the initial culture reaches
at least about 4x106 cells
per fragment. In some aspects, tumor samples or fragments thereof are cultured
in an initial culture
until cell yield in the initial culture reaches at least about 5x106 cells per
fragment. In some aspects,
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tumor samples or fragments thereof are cultured in an initial culture until
cell yield in the initial
culture reaches at least about 6x106 cells per fragment. In some aspects,
tumor samples or
fragments thereof are cultured in an initial culture until cell yield in the
initial culture reaches at
least about 7x106 cells per fragment. In some aspects, tumor samples or
fragments thereof are
cultured in an initial culture until cell yield in the initial culture reaches
at least about 8x106 cells
per fragment. In some aspects, tumor samples or fragments thereof are cultured
in an initial culture
until cell yield in the initial culture reaches at least about 9x106 cells per
fragment. In some aspects,
tumor samples or fragments thereof are cultured in an initial culture until
cell yield in the initial
culture reaches at least about 10x106 cells per fragment.
[0403]
In some aspects, the cells (e.g., TILs) are passed through a strainer
following the
initial culture_ In some aspects, the cells (e.g., TILs) are passed through an
at least about 10 !_tm,
an at least about 15 pm, an at least about 20 p.m, an at least about 25 pm, an
at least about 30 p.m,
an at least about 35 m, an at least about 40
an at least about 45 p.m, an at least about 50 !Am
strainer following the initial culture. In some aspects, the cells (e.g.,
TILs) are passed through an
about 40 p.m strainer following the initial culture.
[0404]
In some aspects, the initial expansion step is carried out in one or
more gas
permeable flasks (e.g., GREX flasks). In some aspects, the initial expansion
step is carried out in
static GREX. In some aspects, the initial expansion is carried out in a
stirred tank. In some aspects
the initial expansion step is carried out in a bioreactor. In some aspects,
the initial expansion is
carried out in a closed system (e.g., using a GREX closed system).
[0405]
In some aspects, the initial expansion comprises contacting the tumor
samples or
fragments/cells thereof with TRANSACTTm (or other suitable T cell activation
reagent) and 4-1BB
ligand. In some aspects, the initial expansion comprises contacting the tumor
samples or fragments
thereof with TRANSACTTm and 4-1BB ligand on about day 3, about day 4, about
day 5, about day
6, or about day 7 of the initial culture; wherein initial expansion is
performed for about 11 days. In
some aspects, the initial expansion comprises contacting the tumor samples or
fragments thereof
with TRANSACTTm and 4-1BB ligand on about day 3, about day 4, about day 5,
about day 6, or
about day 7 of the initial culture; wherein initial expansion is performed for
about 12 daysin some
aspects, the initial expansion comprises contacting the tumor samples or
fragments thereof with
TRANSACTTm and 4-1BB ligand on about day 3, about day 4, about day 5, about
day 6, or about
day 7 of the initial culture; wherein initial expansion is performed for about
13 days. In some
aspects, the initial expansion comprises contacting the tumor samples or
fragments thereof with
TRANSACTTm and 4-1BB ligand on about day 3, about day 4, about day 5, about
day 6, or about
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day 7 of the initial culture; wherein initial expansion is performed for about
14 days.In some
aspects, the initial expansion comprises contacting the tumor samples or
fragments thereof with
TRANSACTTm and 4-1BB ligand on about day 3, about day 4, about day 5, about
day 6, or about
day 7 of the initial culture; wherein initial expansion is performed for about
15 days.
[0406] In some aspects, the TILs are cryopreserved following the
initial expansion. In some
aspects, the TILs from the initial expansion are inoculated directly into a
secondary expansion,
e.g., as described herein, following initial expansion.
11.1.2. Secondary Expansion
[0407] In some aspects, the TILs are subjected to a secondary
expansion. In some aspects,
the secondary expansion step is carried out in one or more gas permeable
flasks (e.g., GREX
flasks). In some aspects, the TILs are transitioned to the secondary expansion
without opening the
closed system. In some aspects, the TILs from the first expansion are screened
for tumor-specific
cytolytic acitivty prior to advancing the TILs to the secondary expansion. In
some aspects, the TILs
are screened for expression of one or more biomarkers prior to advancing to
secondary expansion.
In some aspects, the biomarker comprises expression of one or more gene
typically expressed by
more naive TILs, e.g., CD8+, CD27+, CD3+, CD957, CD45RA7, CCR77, CD62L7,
TCF77, or any
combination thereof. In some aspects, the TILs are screened for expression of
PD-1 prior to
advancing to secondary expansion. In some aspects, the TILs from the first
expansion are not
screened prior to advancing the TILs to the secondary expansion. In some
aspects, all TILs
obtained in the initial expansion are subjected to the secondary expansion. In
some aspects, the
TILs from the first expansion are pooled prior to advancement to secondary
expansion.
ILL2.a. Secondary Expansion by REP
[0408] In some aspects, the TILs are subjected to a secondary
expansion using a Rapid
Expansion Protocol (REP). See, e.g., Dudley, et al., Science 298:850-54
(2002); Dudley, et al.,
Clin. Oncol. 23:2346-57 (2005); Dudley, et al., .I. Chit. Oncol. 26:5233-39
(2008); Riddell, et al.,
Science 257:238-41 (1992); and Dudley, et al., I. Immunother. 26:332-42
(2003), each of which is
incorporated by reference herein in its entirety. In some aspects, TILs are
rapidly expanded using
non-specific T-cell receptor stimulation in the presence of feeder lymphocytes
and interleukin-2
(IL-2), IL-7, IL-15, IL-21, or combinations thereof. In certain aspects, the
TILs are rapidly
expanded in the presence of IL-2, IL-15, and IL-21. In some aspects, the
concentration of IL-2 in
the media during rapid expansion is lower than the concentration of IL-2 in
the media during the
initial culture. In some aspects, the concentration of IL-2 during rapid
expansion is less than 300
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ng/ml. In some aspects, the concentration of IL-2 during rapid expansion is
about 50 ng/ml, about
55 ng/ml, about 60 ng/ml, about 65 ng/ml, about 70 ng/ml, about 73.6 ng/ml,
about 75 ng/ml, about
80 ng/ml, about 85 ng/ml, about 90 ng/ml, about 95 ng/ml, about 100 ng/ml,
about 105 ng/ml,
about 110 ng/ml, about 115 ng/ml, about 120 ng/ml, about 125 ng/ml, about 130
ng/ml, about 135
ng/ml, about 140 ng/ml, about 145 ng/ml, about 150 ng/ml, about 175 ng/ml,
about 200 ng/ml,
about 225 ng/ml, about 250 ng/ml, or about 275 ng/ml. In some aspects, the
concentration of IL-2
during rapid expansion is about 50 ng/ml. In some aspects, the concentration
of IL-2 during rapid
expansion is about 55 ng/ml. In some aspects, the concentration of IL-2 during
rapid expansion is
about 60 ng/ml. In some aspects, the concentration of IL-2 during rapid
expansion is about 65
ng/ml. In some aspects, the concentration of IL-2 during rapid expansion is
about 70 ng/ml. In
some aspects, the concentration of IL-2 during rapid expansion is about 73.6
ng/ml. In some
aspects, the concentration of IL-2 during rapid expansion is about 75 ng/ml.
In some aspects, the
concentration of IL-2 during rapid expansion is about 80 ng/ml. In some
aspects, the concentration
of IL-2 during rapid expansion is about 85 ng/ml. In some aspects, the
concentration of IL-2 during
rapid expansion is about 90 ng/ml. In some aspects, the concentration of IL-2
during rapid
expansion is about 95 ng/ml. In some aspects, the concentration of IL-2 during
rapid expansion is
about 100 ng/ml.
[0409] In some aspects, the concentration of IL-21 in the media
during rapid expansion is
lower than the concentration of IL-21 in the media during the initial culture.
In some aspects, the
concentration of IL-21 during rapid expansion is less than 30 ng/ml. In some
aspects, the
concentration of IL-21 during rapid expansion is about 1 ng/ml, about 2 ng/ml,
about 3 ng/ml,
about 4 ng/ml, about 5 ng/ml, about 6 ng/ml, about 7 ng/ml, about 8 ng/ml,
about 9 ng/ml, about
ng/ml, about 11 ng/ml, about 12 ng/ml, about 13 ng/ml, about 14 ng/ml, about
15 ng/ml, about
16 ng/ml, about 17 ng/ml, about 18 ng/ml, about 19 ng/ml, about 20 ng/ml,
about 21 ng/ml, about
22 ng/ml, about 23 ng/ml, about 24 ng/ml, about 25 ng/ml, about 26 ng/ml,
about 27 ng/ml, about
28 ng/ml, or about 29 ng/ml. In some aspects, the concentration of IL-21
during rapid expansion
is about 5 ng/ml. In some aspects, the concentration of IL-21 during rapid
expansion is about 6
ng/ml. In some aspects, the concentration of 1L-21 during rapid expansion is
about 7 ng/ml. In
some aspects, the concentration of IL-21 during rapid expansion is about 8
ng/ml. In some aspects,
the concentration of IL-21 during rapid expansion is about 9 ng/ml. In some
aspects, the
concentration of IL-21 during rapid expansion is about 10 ng/ml. In some
aspects, the
concentration of IL-21 during rapid expansion is about 11 ng/ml. In some
aspects, the
concentration of IL-21 during rapid expansion is about 12 ng/ml. In some
aspects, the
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concentration of IL-21 during rapid expansion is about 13 ng/ml. In some
aspects, the
concentration of IL-21 during rapid expansion is about 14 ng/ml. In some
aspects, the
concentration of IL-21 during rapid expansion is about 15 ng/ml.
[0410] In some aspects, the concentration of IL-15 in the media
during rapid expansion is
about 0.1 ng/ml, about 0.2 ng/ml, about 0.3 ng/ml, about 0.4 ng/ml, about 0.5
ng/ml, about 0.6
ng/ml, about 0.7 ng/ml, about 0.8 ng/ml, about 0.9 ng/ml, about 1.0 ng/ml,
about 1.1 ng/ml, about
1.2 ng/ml, about 1.3 ng/ml, about 1.4 ng/ml, about 1.5 ng/ml, about 1.6 ng/ml,
about 1.7 ng/ml,
about 1.8 ng/ml, about 1.9 ng/ml, about 2.0 ng/ml, about 2.25 ng/ml, about 2.5
ng/ml, about 2.75
ng/ml, about 3.0 ng/ml, about 3.5 ng/ml, about 4.0 ng/ml, about 4.5 ng/ml, or
about 5.0 ng/ml. In
some aspects, the concentration of IL-15 during rapid expansion is about 0.1
ng/ml. In some
aspects, the concentration of IL-15 during rapid expansion is about 0.2 ng/ml.
In some aspects, the
concentration of IL-15 during rapid expansion is about 0.3 ng/ml. In some
aspects, the
concentration of IL-15 during rapid expansion is about 0.4 ng/ml. In some
aspects, the
concentration of IL-15 during rapid expansion is about 0.5 ng/ml. In some
aspects, the
concentration of IL-15 during rapid expansion is about 0.6 ng/ml. In some
aspects, the
concentration of IL-15 during rapid expansion is about 0.7 ng/ml. In some
aspects, the
concentration of IL-15 during rapid expansion is about 0.8 ng/ml. In some
aspects, the
concentration of IL-15 during rapid expansion is about 0.9 ng/ml. In some
aspects, the
concentration of IL-15 during rapid expansion is about 1.0 ng/ml.
[0411] The non-specific T-cell receptor stimulus can include,
e.g., OKT3 (e.g., about 30
ng/ml), a mouse monoclonal anti-CD3 antibody (available from Ortho-McNeil ,
Raritan, N.J. or
Miltenyi Biotec, Bergisch Gladbach, Germany). In some aspects, TILs are
rapidly expanded by
stimulation of peripheral blood mononuclear cells (PBMC) in vitro with one or
more antigens
(including antigenic portions thereof, such as epitope(s), or a cell of the
cancer, which can be
optionally expressed from a vector, such as an human leukocyte antigen A2 (HLA-
A2) binding
peptide, e.g., approximately 0.3 pM MART-1:26-35 (27 L) or gp100:209-217
(210M)), in the
presence of a T-cell growth factor, such as around 200-400 111/m1 of a T-cell
growth factor, such
as 300 IU/m1 IL-2 or IL-15. In some aspects, TILs are expanded by stimulation
using
TRANSACTTm. In some aspects, the in vitro-induced TILs are rapidly expanded by
stimulation
with the same antigen(s) of the cancer pulsed onto FILA-A2-expressing antigen-
presenting cells.
In some aspects, the TILs can be stimulated with irradiated, autologous
lymphocytes or with
irradiated 1-11A-A2+ allogeneic lymphocytes and IL-2.
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[0412] In some aspects, the TILs are stimulated during the
second expansion by culturing
the cells in a medium comprising TRANSACTTm and optionally 4-1BBL and/or
CD27L. In some
aspects, the TILs are stimulated during the second expansion by culturing the
cells in a medium
comprising TRANSACTTm, 4-1BBL, and CD27L. In some aspects, the TILs are
stimulated during
the second expansion by culturing the cells in a medium comprising at least
about 1:100
TRANSACTTm, at least about 1 lig/m1 4-1BBL, and at least about 5 [tg/m1 CD27L.
[0413] In some aspects, one or more TILs are genetically
modified before, during, or after
T1L expansion. Genetic modification of the Tits can be achieved using any
methods known in the
art. In some aspects, one or more TILs are modified using a Cas9 endonuclease
(CRISPR; see, e.g.,
US2017067021A1, which is incorporated by reference herein in its entirety),
TALEN, a zing-
finger endonuclease, site directed mutagenesis, or any combination thereof In
some aspects, one
or more TILs are genetically modified to disrupt or ablate expression of human
cytokine inducible
SH2-containing protein (CISH; see, e.g., US10406177B2, which is incorporated
by reference
herein in its entirety). In some aspects, one or more TILs is modified using
an AAV, e.g., one or
more of the TILs comprise an AAV. In some aspects one or more TILs is modified
using a
lentivirus or a retrovirus. In some aspects, one or more TILs are genetically
modified to express an
exogenous modified or engineered T cell receptor (TCR). In some aspects, one
or more TILs are
genetically modified to express chimeric antigen receptor (CAR). In some
aspects, one or more
TILs are genetically modified to express CD86. In some aspects, one or more
TILs are genetically
modified to express OX4OL. In some aspects, one or more TILs are genetically
modified to express
4-1BBL. In some aspects, one or more TILs are genetically modified to express
an anti-PD1
antibody.
[0414] In some aspects, the TILs are expanded in a culture
medium that further comprises
a tumor necrosis factor receptor superfamily (TNFRSF) agonist. Any TNFRSF
agonist can be used
in the methods disclosed herein. Non-limiting examples of TNFRSF agonists can
be found, for
example, in US20200121719A1, which is incorporated by reference herein in its
entirety. In some
aspects, the TNFRSF agonist is added after the initial culture. In some
aspects, the TNFRSF agonist
is added during the second and/or or final expansion.
[0415] In some aspects, the TILs are expanded in a culture
medium that further comprises
a 4-1BB agonist. Any 4-1BB agonist can be used in the methods disclosed
herein. In some aspects,
the 4-1BB agonist comprises a 4-1BB antibody. Non-limiting examples of 4-1BB
agonists can be
found, for example, in US20200032209A1, which is incorporated by reference
herein in its
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entirety. In some aspects, the 4-1BB agonist is added after the initial
culture. In some aspects, the
4-1BB agonist is added during the second or final expansion.
[0416] In some aspects, the TILs are stimulated during the
second expansion by culturing
the cells in a medium comprising TRANSACTTm and optionally 4-1BBL and/or
CD27L. In some
aspects, the TILs are stimulated during the second expansion by culturing the
cells in a medium
comprising TRANSACTTm, 4-1BBL, and CD27L. In some aspects, the TILs are
stimulated during
the second expansion by culturing the cells in a medium comprising at least
about 1:100
TRANSACTTm, at least about 1 ng/ml 4-1BBL, and at least about 5 ing/m1 CD27L.
[0417] In some aspects, the TILs are expanded in a culture
medium that further comprises
an adenosine a2a receptor antagonist. Any adenosine a2a receptor antagonist
can be used in the
methods disclosed herein Non-limiting examples of adenosine a2a receptor
antagonist can be
found, for example, in US20210137930A1, which is incorporated by reference
herein in its
entirety. In some aspects, the adenosine a2a receptor antagonist is selected
from the group
consisting of vipadenant, CPI-444 (ciforadenant), SCH58261, ZM241385,
SCH420814, SYN115,
8-CSC, KW-6002, A2A receptor antagonist 1, ADZ4635, ST4206, KF21213,
SCH412348, and
71VIMG-49, or pharmaceutically acceptable salts, solvates, hydrates,
cocrystals, or prodrugs
thereof, and combinations thereof. In some aspects, the adenosine a2a receptor
antagonist is added
during the initial culture. In some aspects, the adenosine a2a receptor
antagonist is added during
the second and/or or final expansion.
[0418] In some aspects, the TILs are expanded in a culture
medium that further comprises
an AKT pathway inhibitor (AKTi). Any AKTi can be used in the methods disclosed
herein. Non-
limiting examples of AKTi that can be used in the present disclosure can be
found, for example, in
W02020096927, which is incorporated by reference herein in its entirety. In
some aspects, the
AKTi is selected from the group consisting of afuresertib, uprosertib,
ipatasertib, AT7867,
AT13148, and pharmaceutically acceptable salts, solvates, hydrates,
cocrystals, or prodrugs
thereof. In some aspects, the AKTi is an mTOR inhibitor, e.g., AZD8055 or
pharmaceutically
acceptable salts, solvates, hydrates, cocrystals, or prodrugs thereof. In some
aspects, the AKTi is
an PI3K inhibitor, e.g., LY294002 or pharmaceutically acceptable salts,
solvates, hydrates,
cocrystals, or prodrugs thereof In some aspects, the AKTi is added during the
initial culture. In
some aspects, the AKTi is added during the second and/or or final expansion.
[0419] In some aspects, the expanded cells are reactivated or
stimulated by contacting the
expanded TILs with one or more antigen presenting cell. Any antigen presenting
cell can be used
in the methods disclosed herein. In some aspects, the antigen presenting cell
is a genetically
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modified cell. In some aspects, the antigen presenting cell comprises a tumor
antigen or a fragment
thereof on the cell surface. In some aspects, the expanded TILs are contacted
with antigen
presenting cells which comprises more than one tumor antigen or a fragment
thereof on the cell
surface.
[0420] In some aspects, the antigen presenting cell (APC) is
genetically engineered. In
some aspects, the APC is genetically engineered for tunable expression of one
or more transgene,
e.g., an antigen or a stimulatory signal. In some aspects, the APC is
genetically engineered
according to a method disclosed in W02020/086742, which is incorporated by
reference herein in
its entirety. In some aspects, the APC is genetically engineered to express
one or more stimulatory
molecule. In some aspects, the APC is genetically engineered to express CD86,
0C4OL, 4-1BBL,
or any combination thereof. In some aspects, the APC is an APC disclosed in US
Patent No. US
10,415,015, which is incorporated by reference herein in its entirety.
[0421] In some aspects, the TILs are cultured in a secondary TIL
media until cell yield in
the secondary expansion reaches at least about 1x107 to at least about 50x107,
at least about 2x107
to at least about 40x107, at least about 3x107 to at least about 30x107, at
least about 4x107 to at least
about 25x107, at least about 5x107 to at least about 20x107, at least about
1x107 to at least about
20x107, at least about 2x107 to at least about 20x107, at least about 3x107 to
at least about 20x107,
or at least about 4x107 to at least about 20x107 cells. In some aspects, the
TILs are cultured in a
secondary TIL media until cell yield in the secondary expansion reaches at
least about 5x107 to at
least about 20x107 cells. In some aspects, the TILs are cultured in a
secondary TIL media until cell
yield in the secondary expansion reaches at least about 1x107, at least about
2x107, at least about
3x107, at least about 4x107, at least about 5x107, at least about 6x107, at
least about 7x107, at least
about 8x107, at least about 9x107, at least about 10x107, at least about
11x107, at least about 12x107,
at least about 13x107, at least about 14x107, at least about 15x107, at least
about 16x107, at least
about 17x1107, at least about 18x107, at least about 19x107, or at least about
20x107 cells. In some
aspects, the TILs are cultured in a secondary TIL media until cell yield in
the secondary expansion
reaches at least about 5x107 cells. In some aspects, the TILs are cultured in
a secondary TIL media
until cell yield in the secondary expansion reaches at least about 6x107
cells. In some aspects, the
TILs are cultured in a secondary TIL media until cell yield in the secondary
expansion reaches at
least about 7x107 cells. In some aspects, the TILs are cultured in a secondary
expansion until cell
yield in the secondary TIL media reaches at least about 8x107 cells. In some
aspects, the TILs are
cultured in a secondary expansion until cell yield in the secondary TIL media
reaches at least about
9x107 cells. In some aspects, the TILs are cultured in a secondary TIL media
until cell yield in the
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secondary expansion reaches at least about 10x107 cells. In some aspects, the
TILs are cultured in
a secondary TIL media until cell yield in the secondary expansion reaches at
least about 15x107
cells. In some aspects, the Tits are cultured in a secondary TIL media until
cell yield in the
secondary expansion reaches at least about 20x107 cells.
[0422] In some aspects, Tits are subjected to a final expansion.
In some aspects, the TILs
are transitioned from the secondary expansion to the final expansion without
opening the closed
system (e.g., the GREX closed system). In some aspects, the final expansion
step is carried out in
one or more gas permeable flasks (e.g., GREX flasks). In some aspects, the
secondary expansion
corresponds with a first phase of the REP protocol (i.e., the REP protocol up
until the cells are
split), and the final expansion corresponds with the second phase of the REP
protocol (i.e., the REP
protocol after the cells are split). As such, in some aspects, the secondary
expansion has a duration
of about 3 to 7 days (e.g., about 5 days, about 6 days, or about 7 days), and
the final expansion has
a duration of about 3 to 7 days (e.g., about 5 days, about 6 days, or about 7
days).
[0423] In some aspects, the media during final expansion
comprises IL-2, IL-7, IL-15, IL-
21, or combinations thereof. In certain aspects, the media during final
expansion comprises IL-2,
IL-15, and IL-21. In some aspects, the concentration of IL-2 in the media
during final expansion
is lower than the concentration of IL-2 in the media during the initial
culture. In some aspects, the
concentration of IL-2 during final expansion is less than 300 ng/ml. In some
aspects, the
concentration of IL-2 during final expansion is about 50 ng/ml, about 55
ng/ml, about 60 ng/ml,
about 65 ng/ml, about 70 ng/ml, about 73.6 ng/ml, about 75 ng/ml, about 80
ng/ml, about 85 ng/ml,
about 90 ng/ml, about 95 ng/ml, about 100 ng/ml, about 105 ng/ml, about 110
ng/ml, about 115
ng/ml, about 120 ng/ml, about 125 ng/ml, about 130 ng/ml, about 135 ng/ml,
about 140 ng/ml,
about 145 ng/ml, about 150 ng/ml, about 175 ng/ml, about 200 ng/ml, about 225
ng/ml, about 250
ng/ml, or about 275 ng/ml. In some aspects, the concentration of IL-2 during
final expansion is
about 50 ng/ml. In some aspects, the concentration of IL-2 during final
expansion is about 55
ng/ml. In some aspects, the concentration of IL-2 during final expansion is
about 60 ng/ml. In some
aspects, the concentration of IL-2 during final expansion is about 65 ng/ml.
In some aspects, the
concentration of IL-2 during final expansion is about 70 ng/ml. In some
aspects, the concentration
of IL-2 during final expansion is about 73.6 ng/ml. In some aspects, the
concentration of IL-2
during final expansion is about 75 ng/ml. In some aspects, the concentration
of IL-2 during final
expansion is about 80 ng/ml. In some aspects, the concentration of IL-2 during
final expansion is
about 85 ng/ml In some aspects, the concentration of IL-2 during final
expansion is about 90
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ng/ml. In some aspects, the concentration of IL-2 during final expansion is
about 95 ng/ml. In some
aspects, the concentration of IL-2 during final expansion is about 100 ng/ml.
[0424] In some aspects, the concentration of IL-21 in the media
during final expansion is
lower than the concentration of IL-21 in the media during the initial culture.
In some aspects, the
concentration of IL-21 during final expansion is less than 30 ng/ml. In some
aspects, the
concentration of IL-21 during final expansion is about 1 ng/ml, about 2 ng/ml,
about 3 ng/ml, about
4 ng/ml, about 5 ng/ml, about 6 ng/ml, about 7 ng/ml, about 8 ng/ml, about 9
ng/ml, about 10
ng/ml, about 11 ng/ml, about 12 ng/ml, about 13 ng/ml, about 14 ng/ml, about
15 ng/ml, about 16
ng/ml, about 17 ng/ml, about 18 ng/ml, about 19 ng/ml, about 20 ng/ml, about
21 ng/ml, about 22
ng/ml, about 23 ng/ml, about 24 ng/ml, about 25 ng/ml, about 26 ng/ml, about
27 ng/ml, about 28
ng/ml, or about 29 ng/ml. In some aspects, the concentration of IL-21 during
final expansion is
about 5 ng/ml. In some aspects, the concentration of IL-21 during final
expansion is about 6 ng/ml.
In some aspects, the concentration of IL-21 during final expansion is about 7
ng/ml. In some
aspects, the concentration of IL-21 during final expansion is about 8 ng/ml.
In some aspects, the
concentration of IL-21 during final expansion is about 9 ng/ml. In some
aspects, the concentration
of IL-21 during final expansion is about 10 ng/ml. In some aspects, the
concentration of IL-21
during final expansion is about 11 ng/ml. In some aspects, the concentration
of IL-21 during final
expansion is about 12 ng/ml. In some aspects, the concentration of IL-21
during final expansion is
about 13 ng/ml. In some aspects, the concentration of IL-21 during final
expansion is about 14
ng/ml. In some aspects, the concentration of IL-21 during final expansion is
about 15 ng/ml.
[0425] In some aspects, the concentration of IL-15 in the media
during final expansion is
about 0.1 ng/ml, about 0.2 ng/ml, about 0.3 ng/ml, about 0.4 ng/ml, about 0.5
ng/ml, about 0.6
ng/ml, about 0.7 ng/ml, about 0.8 ng/ml, about 0.9 ng/ml, about 1.0 ng/ml,
about 1.1 ng/ml, about
1.2 ng/ml, about 1.3 ng/ml, about 1.4 ng/ml, about 1.5 ng/ml, about 1.6 ng/ml,
about 1.7 ng/ml,
about 1.8 ng/ml, about 1.9 ng/ml, about 2.0 ng/ml, about 2.25 ng/ml, about 2.5
ng/ml, about 2.75
ng/ml, about 3.0 ng/ml, about 3.5 ng/ml, about 4.0 ng/ml, about 4.5 ng/ml, or
about 5.0 ng/ml. In
some aspects, the concentration of IL-15 during final expansion is about 0.1
ng/ml. In some
aspects, the concentration of IL-15 during final expansion is about 0.2 ng/ml.
In some aspects, the
concentration of IL-15 during final expansion is about 0.3 ng/ml. In some
aspects, the
concentration of IL-15 during final expansion is about 0.4 ng/ml. In some
aspects, the
concentration of IL-15 during final expansion is about 0.5 ng/ml. In some
aspects, the
concentration of IL-15 during final expansion is about 0.6 ng/ml. In some
aspects, the
concentration of IL-15 during final expansion is about 0.7 ng/ml. In some
aspects, the
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concentration of IL-15 during final expansion is about 0.8 ng/ml. In some
aspects, the
concentration of IL-15 during final expansion is about 0.9 ng/ml. In some
aspects, the
concentration of IL-15 during final expansion is about LO ng/ml.
[0426] In some aspects, the final expansion comprises a
stimulation. In some aspects the
stimulation is the same as the stimulation used during the secondary
expansion. In some aspects,
the TILs are stimulated during the final expansion by culturing the cells in
an MRIVI comprising
TRANSACTTm, 4-1BBL, CD27L, or any combination thereof. In some aspects, the
TILs are
stimulated during the final expansion by culturing the cells in an MRIVI
comprising TRANSACTTm
and optionally 4-1BBL and/or CD27L. In some aspects, the TILs are stimulated
during the final
expansion by culturing the cells in an MRM comprising at least about 1:100
TRANSACTTm, at
least about 1 ng/ml 4-1BBL, and at least about 5 lug/m1 CD27L.
[0427] In some aspects, the final expansion step is carried out
in static GREX. In some
aspects, the final expansion is carried out in a stirred tank. In some aspects
the final expansion step
is carried out in a bioreactor. In some aspects, the final expansion is
continued until the cell yield
in the final TIL media reaches at least about 40x109 to at least about
100x109, at least about 40x109
to at least about 90x109, at least about 40x109 to at least about 80x109, at
least about 40x109 to at
least about 70x109, at least about 40x109 to at least about 60x109, at least
about 40x109 to at least
about 50x109, at least about 10x109 to at least about 100x109, at least about
20x109 to at least about
100x109, at least about 30x109 to at least about 100x109, at least about
30x109 to at least about
50x109, or at least about 35x109 to at least about 45x109 cells. In some
aspects, the final expansion
is continued until the cell yield in the final TIL media reaches at least
about 40x109 to at least about
100x109 cells. In some aspects, the final expansion is continued until the
cell yield in the final TIL
media reaches at least about 40x109, at least about 45x109, at least about
50x109, at least about
55x109, at least about 60x109, at least about 65x109, at least about 70x109,
at least about 75x109,
at least about 80x109, at least about 85x109, at least about 90x109, at least
about 95x109, or at least
about 100x109 cells. In some aspects, the final expansion is continued until
the cell yield in the
final TIL media reaches at least about 40x109 cells. In some aspects, the
final expansion is
continued until the cell yield in the final TIL media reaches at least about
50x109 cells. In some
aspects, the final expansion is continued until the cell yield in the final
TIL media reaches at least
about 60x109 cells. In some aspects, the final expansion is continued until
the cell yield in the final
TIL media reaches at least about 70x109 cells. In some aspects, the final
expansion is continued
until the cell yield in the final T11, media reaches at least about 80x109
cells. In some aspects, the
final expansion is continued until the cell yield in the final TIL media
reaches at least about 90x109
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cells. In some aspects, the final expansion is continued until the cell yield
in the final TIL media
reaches at least about 100x109 cells.
[0428] In some aspects, the final expansion is continued until
the cell yield in the final TIL
media for at least about 7 to at least about 21 days. In some aspects, the
final expansion is continued
until the cell yield in the final TIL media for at least about 7 days. In some
aspects, the final
expansion is continued until the cell yield in the final TIL media for at
least about 8 days. In some
aspects, the final expansion is continued until the cell yield in the final
TIL media for at least about
9 days. In some aspects, the final expansion is continued until the cell yield
in the final T1L media
for at least about 10 days. In some aspects, the final expansion is continued
until the cell yield in
the final TIL media for at least about 11 days. In some aspects, the final
expansion is continued
until the cell yield in the final TIL media for at least about 12 days. In
some aspects, the final
expansion is continued until the cell yield in the final TIL media for at
least about 13 days. In some
aspects, the final expansion is continued until the cell yield in the final
TIL media for at least about
14 days. In some aspects, the final expansion is continued until the cell
yield in the final TIL media
for at least about 15 days. In some aspects, the final expansion is continued
until the cell yield in
the final TIL media for at least about 16 days. In some aspects, the final
expansion is continued
until the cell yield in the final TIL media for at least about 17 days. In
some aspects, the final
expansion is continued until the cell yield in the final TIL media for at
least about 18 days. In some
aspects, the final expansion is continued until the cell yield in the final
TIL media for at least about
19 days. In some aspects, the final expansion is continued until the cell
yield in the final TIL media
for at least about 20 days. In some aspects, the final expansion is continued
until the cell yield in
the final TIL media for at least about 21 days.
[0429] In some aspects, the secondary expansion and the final
expansion are merged into
a single secondary expansion. In some aspects, the single secondary expansion
comprises all
aspects of the secondary expansion and the final expansion. In some aspects,
the single secondary
expansion takes place in a closed system (e.g., a GREX closed system), wherein
the closed system
is not opened for the duration of the single secondary expansion. In some
aspects, the cells are split
during the single secondary expansion once the cells reach high confluence.
[0430] In some aspects, the full duration of the expansion
process (e.g., (i) the initial
expansion process, the secondary expansion process, and the final expansion
process; or (ii) the
initial expansion process and the single secondary expansion process) is 22
days or less. Generation
of young TILs using shorter expansion processes confers various benefits on
the resulting T1L
composition. As such, the culture conditions and methods disclosed herein
confer additional
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benefits, e.g., increased stem-like characteristics, expanded clonal
diversity, improved cytolytic
activity, and/or increased CD8+ cell expansion, on those already identified
for young TILs.
11.1.2. b. Secondary Expansion by Static-REP and Dynamic-REP
[0431] In some aspects, secondary expansion comprises a static-
REP step followed by a
dynamic-REP step. As used herein, "static-REP" refers to an expansion step
wherein TILs
harvested from pre-REP or an initial expansion, e.g., as disclosed herein, are
further expanded in
a medim comprising greater than 4 mM potassium ion, e.g., an MRM disclosed
herein, a CD3
agonist (e.g., OKT3), and antigen presenting cells (e.g., irradiated
allogeneic PBMCs). In some
aspects, the agitation of the culture is not applied during static-REP.
"Dynamic-REP," as used
herein, refers to an expansion step wherein TILs from the static-REP are
further expanded in a
medium comprising greater than 4 mM potassium ion until a therapeutic number
of TILs is
reached, wherein the culture occurs under agitation. In some aspects, the
method comprises
expanding a population of TILs obtained from a human subject comprising (a)
culturing the TILs
in a medium comprising greater than 4 mM potassium ion, a CD3 agonist, and
antigen-presenting
cells (a "static-REP step"); and (b) adding to the TILs from the static-REP
step a medium
comprising greater than 4 mM potassium ion, wherein agitation is applied to
the culture (a
"dynamic-REP step"). In some aspects, no CD3 agonist is added during the
dynamid-REP. In some
aspects, no antigen-presenting cells are added during the dynamic-REP step. In
some aspects, no
CD3 agonist and no antigen-presenting cells are added during the dynamic-REP
step.
[0432] In some aspects, the TILs obtained during static-REP are
added directly to the
dynamic-REP. In some aspects, the TILs are not split between the static-REP
and the dynamic-
REP. In some aspects, the TILs are cryopreserved following the static-REP and
prior to the
dynamic-REP.
[0433] In some aspects, static-REP medium comprising the
expanded TILs is applied
directly to the dynamic-REP As such, in some aspects, day 0 of dynamic-REP
includes TILs
present in a medium that comprises a CD3 agonist and antigen-presenting cells,
which was carried
over from the static-REP to the dynamic-REP with the TILs. However, in some
aspects, no
additional CD3 agonist or antigen-presenting cells are added during the
dynamic-REP step. Over
time, an increasing concentration of CD3 agonist that had carried over from
the static-REP is
degraded or otherwise lost, resulting in a decreasing concentration of CD3
agonist during the
dynamic-REP step.
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[0434] Further, the dynamic-REP step comprises applying
agitation to the culture. In some
aspects, the agitation comprises rocking the culture. Traditional REP culture
includes culturing
TILs in the presences of antigen-presenting cells, such as irradiated PBMCs.
However, PBMCs
have reduced viability in culture when subjected to agitation, such as
rocking. As such, traditional
REP culture is performed without rocking or otherwise agitating the cells.
However, the present
disclosure surprisingly found that the methods disclosed herein, which include
a dynamic-REP
step that comprises agitation, increased the yield and improved the properties
of the resulting TIL
product. This would not have been expected, as the dynamic-REP step disclosed
herein allows for
gradually reducing the presense of both the CD3 agonist and the antigen-
presenting cells during
TIL expansion, two factors which are hallmarks of REP methods.
[0435] In some aspects, the dynamic-REP step comprises
perfusion. As used herein,
"perfusion" refers to a method of culturing cells, e.g., TILs, wherein a
portion of the culture
medium is constantly replaced with fresh medium without removing the cultured
cells, e.g., TILs.
The rate of perfusion can be carefully controlled. In some aspects, the
perfusion comprises
continuous media exchange at a rate of about 10%, about 15%, about 20%, about
25%, about 30%,
about 35%, about 40%, about 45%, about 50%, about 55%, or about 60% of the
working volume
of the culture vessel every 24 hours. In some aspects, the perfusion comprises
continuous media
exchange at a rate of about 5% of the working volume of the culture vessel
every 24 hours.In some
aspects, the perfusion comprises continuous media exchange at a rate of about
10% of the working
volume of the culture vessel every 24 hours.In some aspects, the perfusion
comprises continuous
media exchange at a rate of about 15% of the working volume of the culture
vessel every 24
hours.In some aspects, the perfusion comprises continuous media exchange at a
rate of about 20%
of the working volume of the culture vessel every 24 hours.In some aspects,
the perfusion
comprises continuous media exchange at a rate of about 25% of the working
volume of the culture
vessel every 24 hours. In some aspects, the perfusion comprises continuous
media exchange at a
rate of about 30% of the working volume of the culture vessel every 24 hours.
In some aspects, the
perfusion comprises continuous media exchange at a rate of about 35% of the
working volume of
the culture vessel every 24 hours. In some aspects, the perfusion comprises
continuous media
exchange at a rate of about 40% of the working volume of the culture vessel
every 24 hours. In
some aspects, the perfusion comprises continuous media exchange at a rate of
about 45% of the
working volume of the culture vessel every 24 hours. In some aspects, the
perfusion comprises
continuous media exchange at a rate of about 50% of the working volume of the
culture vessel
every 24 hours. In some aspects, the perfusion comprises continuous media
exchange at a rate of
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about 55% of the working volume of the culture vessel every 24 hours. In some
aspects, the
perfusion comprises continuous media exchange at a rate of about 60% of the
working volume of
the culture vessel every 24 hours.
[0436] In some aspects, the perfusion rate is constant for the
entirety of the dynamic-REP
step. In some aspects, the perfusion rate is varied during the dynamic-REP
step. In some aspects,
the perfusion comprises continuous media exchange at a rate of about 25% of
the working volume
of the culture vessel every 24 hours for the first 48 hours of the dynamic-REP
culture, and wherein
the perfusion comprises continuous media exchange at a rate of about 50% of
the working volume
of the culture vessel every 24 hours for the remainder of the dynamic-REP
culture.
[0437] In some aspects, the volume of the culture medium is held
constant for the entirety
of the dynamic-REP step. In some aspects, the volume of the culture medium is
varied during the
dynamic-REP step. In some aspects, the volume of the culture medium is
increased during the
dynamic-REP step. In some aspects, the perfusion comprises adding more medium
to the culture
than is removed. In some aspects, the volume of the culture medium is
increased from hour 0 to
hour 48 of the dynamic-REP step. In some aspects, the volume of the culture
medium is increased
from hour 0 to hour 48 of the dynamic-REP step, and the volume of the culture
is maintained, i.e.,
neither increased in volume by more than 10% nor decreased in volume by more
than 10%, from
hour 48 until the completion of the dynamic-REP step. In some aspects, the
volume of the medium
is increased by at least about 10%, at least about 15%, at least about 20%, at
least about 25%, at
least about 30%, at least about 35%, at least about 40%, at least about 45%,
or at least about 50%
every 24 hours for the first 48 hours. In some aspects, the volume of the
medium is increased by
at least about 10%, at least about 15%, at least about 20%, at least about
25%, at least about 30%,
at least about 35%, at least about 40%, at least about 45%, at least about
50%, at least about 55%,
at least about 60%, at least about 65%, at least about 70%, or at least about
75% from hour 0 to
hour 48 of the dynamic-REP step. In some aspects, the volume of the medium is
increased by at
least about 25% from hour 0 to hour 48 of the dynamic-REP step. In some
aspects, the volume of
the medium is increased by at least about 33% from hour 0 to hour 48 of the
dynamic-REP step.
In some aspects, the volume of the medium is increased by at least about 50%
from hour 0 to hour
48 of the dynamic-REP step. In some aspects, the volume of the medium is
increased by at least
about 65% from hour 0 to hour 48 of the dynamic-REP step. In some aspects, the
volume of the
medium is increased by at least about 66% from hour 0 to hour 48 of the
dynamic-REP step. In
some aspects, the volume of the medium is increased by at least about 75% from
hour 0 to hour 48
of the dynamic-REP step.
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[0438]
In some aspects, the fresh medium applied during perfusion comprises
greater than
4 mM potassium ion, e.g., an MRNI disclosed herein. In some aspects, the fresh
medium applied
during perfusion comprises greater than 4 mM potassium ion, e.g., an MR1\4
disclosed herein, and
does not comprise a CD3 agonist, e.g., OKT-3. In some aspects, the fresh
medium applied during
perfusion comprises greater than 4 mM potassium ion, e.g., an MRNI disclosed
herein, and does
not comprise antigen-presenting cells, e.g., irradiated PBMCs. In some
aspects, the fresh medium
applied during perfusion comprises greater than 4 mM potassium ion, e.g., an
MRM disclosed
herein, and does not comprise a CD3 agonist
OKT-3) or antigen-presenting cells (e.g.,
irradiated PBMC s).
[0439]
As the perfusion comprises continuously replacing a portion of the
medium with
fresh medium that does not comprise CD3 agonist, the concentration of any CD3
agonist in the
starting medium (i.e., at day 0 of the dynamic-REP step) will decrease
throughout the dynamic-
REP. In some aspects, the concentration of CD3 agonist in the dynamic-REP
culture decreases at
a rate of about 10%, about 15%, about 20%, about 25%, about 30%, about 35%,
about 40%, about
45%, about 50%, about 55%, or about 60% every 24 hours. In some aspects, the
rate at which the
CD3 agonist decreases in the culture will be linked to the rate of perfusion.
In some aspects, the
concentration of CD3 agonist in the dynamic-REP culture is less than about 75%
that of the
concentration of CD3 agonist in the static-REP culture at least about 24 hours
after initiation of the
dynamic-REP. In some aspects, the concentration of CD3 agonist in the dynamic-
REP culture is
less than about 45% that of the concentration of CD3 agonist in the static-REP
culture at least about
48 hours after initiation of the dynamic-REP. In some aspects, the
concentration of CD3 agonist in
the dynamic-REP culture is less than about 35% that of the concentration of
CD3 agonist in the
static-REP culture at least about 72 hours after initiation of the dynamic-
REP. In some aspects, the
concentration of CD3 agonist in the dynamic-REP culture is less than about 27%
that of the
concentration of CD3 agonist in the static-REP culture at least about 96 hours
after initiation of the
dynamic-REP. In some aspects, the concentration of CD3 agonist in the dynamic-
REP culture is
less than about 21% that of the concentration of CD3 agonist in the static-REP
culture at least about
120 hours after initiation of the dynamic-REP.
[0440]
As the perfusion comprises continuously replacing a portion of the
medium with
fresh medium that does not comprise additional antigen-presenting cells (e.g.,
irradiated PBMCs),
the number of viable antigen-presenting cells present in the starting medium
(i.e., at day 0 of the
dynamic-REP step) will decrease throughout the dynamic-REP Further, PBMCs have
low
viability in culture when the culture is subjected to an agitation, e.g.,
rocking. As the dynamic-REP
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comprises subjecting the culture to agitation, any PBMCs carried over from
static-REP will start
to decrease in number during dynamic-REP. In some aspects, the number of
viable antigen-
presenting cells, e.g., irradiated PBMCs, in the dynamic-REP culture is less
than about 90%, less
than about 80%, less than about 70%, less than about 60%, less than about 50%,
less than about
45%, less than about 40%, less than about 35%, less than about 30%, less than
about 25%, less
than about 20%, less than about 15%, less than about 10%, or less than about
5% the number of
antigen-presenting cells in the static-REP at least about 24 hours after
initiation of the dynamic-
REP. In some aspects, the number of viable antigen-presenting cells in the
dynamic-REP is culture
less than about 50%, less than about 45%, less than about 40%, less than about
35%, less than
about 30%, less than about 25%, less than about 20%, less than about 15%, less
than about 10%,
or less than about 5% the number of antigen-presenting cells in the static-REP
at least about 48
hours after initiation of the dynamic-REP. In some aspects, the number of
viable antigen-presenting
cells in the dynamic-REP culture is less than about 25%, less than about 20%,
less than about 15%,
less than about 10%, or less than about 5% the number of antigen-presenting
cells in the static-
REP at least about 72 hours after initiation of the dynamic-REP. In some
aspects, the number of
viable antigen-presenting cells in the dynamic-REP culture is less than about
10% the number of
antigen-presenting cells in the static-REP at least about 96 hours after
initiation of the dynamic-
REP.
[0441] In some aspects, the duration of the static-REP is about
5 days. In some aspects, the
duration of the dynamic-REP is about 9 days to about 13 days. In some aspects,
the duration of the
dynamic-REP is about 9 days. In some aspects, the duration of the dynamic-REP
is about 10 days.
In some aspects, the duration of the dynamic-REP is about 11 days. In some
aspects, the duration
of the dynamic-REP is about 12 days. In some aspects, the duration of the
dynamic-REP is about
13 days. In some aspects, duration of the static-REP is about 5 days, and the
duration of the
dynamic-REP is about 9 days. In some aspects, duration of the static-REP is
about 5 days, and the
duration of the dynamic-REP is about 10 days. In some aspects, duration of the
static-REP is about
days, and the duration of the dynamic-REP is about 11 days. In some aspects,
duration of the
static-REP is about 5 days, and the duration of the dynamic-REP is about 12
days. In some aspects,
the TILs are cryopreserved following conclusion of the dynamic-REP step.
[0442] In some aspects, the medium of the static-REP further
comprises IL-2., e.g.,
recombinant human IL-2. In some aspects, the medium of the static-REP culture
comprises at least
about 1000 IU, at least about 1100 IU, at least about 1200 IU, at least about
1300 IU, at least about
1400 IU, at least about 1500 IU, at least about 1600 IU, at least about 1700
IU, at least about 1800
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IU, at least about 1900 IU, or at least about 2000 IU IL-2. In some aspects,
the medium of the
static-REP culture comprises about 1000 IU IL-2. In some aspects, the medium
of the static-REP
culture comprises about 1100 IU IL-2. In some aspects, the medium of the
static-REP culture
comprises about 1200 IU IL-2. In some aspects, the medium of the static-REP
culture comprises
about 1300 IU IL-2. In some aspects, the medium of the static-REP culture
comprises about 1400
IU IL-2. In some aspects, the medium of the static-REP culture comprises about
1500 IU IL-2. In
some aspects, the medium of the static-REP culture comprises about 1600 IU IL-
2. In some
aspects, the medium of the static-REP culture comprises about 1700 IU IL-2. In
some aspects, the
medium of the static-REP culture comprises about 1800 IU IL-2. In some
aspects, the medium of
the static-REP culture comprises about 1900 IU IL-2. In some aspects, the
medium of the static-
REP culture comprises about 2000 IU IL-2.
[0443] In some aspects, the medium of the static-REP further
comprises IL-21, e.g.,
recombinant human IL-21. In some aspects, the medium of the static-REP culture
comprises at
least about 5 ng/mL, at least about 6 ng/mL, at least about 7 ng/mL, at least
about 8 ng/mL, at least
about 9 ng/mL, at least about 10 ng/mL, at least about 11 ng/mL, at least
about 12 ng/mL, at least
about 13 ng/mL, at least about 14 ng/mL, or at least about 15 ng/mL IL-21. In
some aspects, the
medium of the static-REP culture comprises about 5 ng/mL IL-21. In some
aspects, the medium
of the static-REP culture comprises about 6 ng/mL IL-21. In some aspects, the
medium of the
static-REP culture comprises about 7 ng/mL IL-21. In some aspects, the medium
of the static-REP
culture comprises about 8 ng/mL IL-21. In some aspects, the medium of the
static-REP culture
comprises about 9 ng/mL IL-21. In some aspects, the medium of the static-REP
culture comprises
about 10 ng/mL IL-21. In some aspects, the medium of the static-REP culture
comprises about 11
ng/mL IL-21. In some aspects, the medium of the static-REP culture comprises
about 12 ng/mL
IL-21. In some aspects, the medium of the static-REP culture comprises about
13 ng/mL IL-21. In
some aspects, the medium of the static-REP culture comprises about 14 ng/mL IL-
21. In some
aspects, the medium of the static-REP culture comprises about 15 ng/mL IL-21.
[0444] In some aspects, the medium of the static-REP culture
comprises IL-15, e.g.,
recombinant human 1L-15. In some aspects, the medium of the static-REP culture
comprises at
least about 0.1 ng/mL, at least about 0.2 ng/mL, at least about 0.3 ng/mL, at
least about 0.4 ng/mL,
at least about 0.5 ng/mL, at least about 0.6 ng/mL, at least about 0.7 ng/mL,
at least about 0.8
ng/mL, at least about 0.9 ng/mL, or at least about 1 ng/mL IL-15. In some
aspects, the medium of
the static-REP culture comprises about 0.1 ng/mL IL-15. In some aspects, the
medium of the static-
REP culture comprises about 0.2 ng/mL IL-15. In some aspects, the medium of
the static-REP
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culture comprises about 0.3 ng/mL IL-15. In some aspects, the medium of the
static-REP culture
comprises about 0.4 ng/mL IL-15. In some aspects, the medium of the static-REP
culture comprises
about 0.5 ng/mL IL-I5. In some aspects, the medium of the static-REP culture
comprises about 0.6
ng/mL IL-15. In some aspects, the medium of the static-REP culture comprises
about 0.7 ng/mL
IL-15. In some aspects, the medium of the static-REP culture comprises about
0.8 ng/mL IL-15.
In some aspects, the medium of the static-REP culture comprises about 0.9
ng/mL IL-15. In some
aspects, the medium of the static-REP culture comprises about 1 ng/mL IL-15.
[0445] In some aspects, the medium of the static-REP culture
comprises IL-2 and IL-21.
In some aspects, the static-REP culture comprises about 1500 IU IL-2 and about
10 ng/mL
[0446] In some aspects, the medium of the static-REP culture
comprises IL-2 and IL-15.
In some aspects, the medium of the static-REP culture comprises about 1500 IU
IL-2 and about
0.4 ng/mL IL-15.
[0447] In some aspects, the medium of the static-REP culture
comprises IL-2, IL-21, and
IL-15. In some aspects, the medium of the static-REP culture comprises about
1500 IU IL-2, about
ng/mL IL-21, and about 0.4 ng/mL IL-15.
[0448] In some aspects, the perfused fresh medium of the dynamic-
REP further comprises
IL-2., e.g., recombinant human IL-2. In some aspects, the perfused fresh
medium of the dynamic-
REP culture comprises at least about 1000 IU, at least about 1100 IU, at least
about 1200 IU, at
least about 1300 IU, at least about 1400 IU, at least about 1500 IU, at least
about 1600 IU, at least
about 1700 IU, at least about 1800 IU, at least about 1900 IU, or at least
about 2000 IU IL-2. In
some aspects, the perfused fresh medium of the dynamic-REP culture comprises
about 1000 IU
IL-2. In some aspects, the perfused fresh medium of the dynamic-REP culture
comprises about
1100 IU IL-2. In some aspects, the perfused fresh medium of the dynamic-REP
culture comprises
about 1200 IU IL-2. In some aspects, the perfused fresh medium of the dynamic-
REP culture
comprises about 1300 IU IL-2. In some aspects, the perfused fresh medium of
the dynamic-REP
culture comprises about 1400 IU IL-2. In some aspects, the perfused fresh
medium of the dynamic-
REP culture comprises about 1500 -CU IL-2. In some aspects, the perfused fresh
medium of the
dynamic-REP culture comprises about 1600 IU IL-2. In some aspects, the
perfused fresh medium
of the dynamic-REP culture comprises about 1700 IU IL-2. In some aspects, the
perfused fresh
medium of the dynamic-REP culture comprises about 1800 IU IL-2 In some
aspects, the perfused
fresh medium of the dynamic-REP culture comprises about 1900 IU IL-2. In some
aspects, the
perfused fresh medium of the dynamic-REP culture comprises about 2000 IU IL-2.
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[0449] In some aspects, the perfused fresh medium of the dynamic-
REP further comprises
IL-21, e.g., recombinant human IL-2L In some aspects, the perfused fresh
medium of the dynamic-
REP culture comprises at least about 5 ng/mL, at least about 6 ng/mL, at least
about 7 ng/mL, at
least about 8 ng/mL, at least about 9 ng/mL, at least about 10 ng/mL, at least
about 11 ng/mL, at
least about 12 ng/mL, at least about 13 ng/mL, at least about 14 ng/mL, or at
least about 15 ng/mL
IL-21. In some aspects, the perfused fresh medium of the dynamic-REP culture
comprises about 5
ng/mL IL-21. In some aspects, the perfused fresh medium of the dynamic-REP
culture comprises
about 6 ng/mL IL-21. In some aspects, the perfused fresh medium of the dynamic-
REP culture
comprises about 7 ng/mL IL-21. In some aspects, the perfused fresh medium of
the dynamic-REP
culture comprises about 8 ng/mL IL-21. In some aspects, the perfused fresh
medium of the
dynamic-REP culture comprises about 9 ng/mL IL-21. In some aspects, the
perfused fresh medium
of the dynamic-REP culture comprises about 10 ng/mL IL-21. In some aspects,
the perfused fresh
medium of the dynamic-REP culture comprises about 11 ng/mL IL-21. In some
aspects, the
perfused fresh medium of the dynamic-REP culture comprises about 12 ng/mL IL-
21. In some
aspects, the perfused fresh medium of the dynamic-REP culture comprises about
13 ng/mL IL-21.
In some aspects, the perfused fresh medium of the dynamic-REP culture
comprises about 14 ng/mL
IL-21. In some aspects, the perfused fresh medium of the dynamic-REP culture
comprises about
15 ng/mL IL-21.
[0450] In some aspects, the perfused fresh medium of the dynamic-
REP culture comprises
IL-15, e.g., recombinant human IL-15. In some aspects, the perfused fresh
medium of the dynamic-
REP culture comprises at least about 0.1 ng/mL, at least about 0.2 ng/mL, at
least about 0.3 ng/mL,
at least about 0.4 ng/mL, at least about 0.5 ng/mL, at least about 0.6 ng/mL,
at least about 0.7
ng/mL, at least about 0.8 ng/mL, at least about 0.9 ng/mL, or at least about 1
ng/mL IL-15. In some
aspects, the perfused fresh medium of the dynamic-REP culture comprises about
0.1 ng/mL IL-15.
In some aspects, the perfused fresh medium of the dynamic-REP culture
comprises about 0.2
ng/mL IL-15. In some aspects, the perfused fresh medium of the dynamic-REP
culture comprises
about 0.3 ng/mL IL-15. In some aspects, the perfused fresh medium of the
dynamic-REP culture
comprises about 0.4 ng/mL IL-15. In some aspects, the perfused fresh medium of
the dynamic-
REP culture comprises about 0.5 ng/mL IL-15. In some aspects, the perfused
fresh medium of the
dynamic-REP culture comprises about 0.6 ng/mL IL-15. In some aspects, the
perfused fresh
medium of the dynamic-REP culture comprises about 0.7 ng/mL IL-15. In some
aspects, the
perfused fresh medium of the dynamic-REP culture comprises about 0.8 ng/mL IL-
15. In some
aspects, the perfused fresh medium of the dynamic-REP culture comprises about
0.9 ng/mL IL-15.
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In some aspects, the perfused fresh medium of the dynamic-REP culture
comprises about 1 ng/mL
IL-15.
[0451] In some aspects, the perfused fresh medium of the dynamic-
REP culture comprises
IL-2 and IL-21. In some aspects, the static-REP culture comprises about 1500
IU IL-2 and about
ng/mL IL-21.
[0452] In some aspects, the perfused fresh medium of the dynamic-
REP culture comprises
IL-2 and IL-15. In some aspects, the perfused fresh medium of the dynamic-REP
culture comprises
about 1500 IU IL-2 and about 0.4 ng/mL IL-15.
[0453] In some aspects, the perfused fresh medium of the dynamic-
REP culture comprises
IL-2, IL-21, and IL-15. In some aspects, the perfused fresh medium of the
dynamic-REP culture
comprises about 1500 IU IL-2, about 10 ng/mL IL-21, and about 0.4 ng/mL IL-15.
11.1.3. Harvest and Cryopreservation
[0454] In some aspects, the expanded TILs are harvested. TILs
can be harvested using any
method, including by centrifugation. In some aspects, TILs are harvest using
an automated system.
Cell harvesters and/or cell processing systems are commercially available from
a variety of
sources, and any cell-based harvester can be used in the methods disclosed
herein. In some aspects,
the cell harvester and/or cell processing systems is a membrane-based cell
harvester. In some
aspects, the cell harvesting is conducted using a cell processing system,
e.g., the LOVO system
(Fresenius Kabi). In some aspects, the cell harvester and/or cell processing
system can perform
cell separation, washing, fluid-exchange, concentration, and/or other cell
processing steps in a
closed, sterile system.
[0455] In some aspects, the harvest is performed from a closed
system bioreactor. In some
aspects, a closed system is employed for the TIL expansion. in some aspects, a
single bioreactor is
employed. In some aspects, the closed system bioreactor is a single
bioreactor. Examples of
methods of expanding TILs ex vivo in open and closed systems can be found, for
example, in US
Patent No. 10,166,257, which is incorporated by reference herein in its
entirety.
[0456] In some aspects, the expanded TILs are cryopreserved. The
TILs can be
cryopreserved using any methods. Various methods of cryopreserving mammalian
cells, including
TILs, have been described, e.g-., by (i) General Protocol for the
Cryopreservation of Mammalian
Cells, UNC (2007), available at unclineberger. org/ti s sueculture/protocol
s/general-proto col-for-
the-cryopreservation-of-mammalian-cells/; and (ii) Clarke et al., Improved
post-thaw recovery of
peripheral blood stem/progenitor cells using a novel intracellular-like
cryopreservation solution,
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Cytotherapy 2009-6-6, available at sigmaaldrich.com/catalog/papers/19499402;
each of which is
incorporated by reference herein in its entirety.
[0457] In some aspects, the TILs are cultured according to the
following:
(1) Tumor samples are isolated from a subject, and tumors are cut into
fragments and/or
mechanically or chemically disaggregated.
(2) The resulting tumor samples or fragments thereof are then cultured in an
initial culture
comprising a metabolic reprogramming media disclosed herein further
supplemented with 300
ng/mL or 6000 IU/ml IL-2 and 30 ng/ml IL-21.
(3) Optionally, on day 5 following the start of the initial culture, the TILs
are contacted with
TRANSACTTm (1:200) and 100 ng/mL 4-1BB ligand, and the TILs are then cultured
for an
additional 5-9 days or until about 10 x106 to about 200 x 106 cells are
reached. TILs are then
pooled.
(4) At least 0.5 x 106 TILs from step 3 are then mixed with 100-200 times
excess of irradiated
PBMC feeder cells and cultured in media (e.g., a metabolic reprogramming media
disclosed
herein) supplemented with 30 ng/ml anti-CD3 antibody (e.g., OKT3), 75 ng/mL IL-
2, 10 ng/mL
IL-21, and 0.4 ng/mL IL-15. This secondary (REP) culture is continued until a
therapeutically
effective amount of TILs is obtained, as described herein.
III. Compositions of the Disclosure
[0458] Some aspects of the present disclosure are directed to a
composition comprising a
population of TILs, which is enriched in CD8 TILs. In some aspects, the
composition comprises
a population of TILs cultured according to any method disclosed herein. In
some aspects, at least
about 10%, at least about 15%, at least about 20%, at least about 25%, at
least about 30%, at least
about 35%, at least about 40%, at least about 45%, at least about 50%, at
least about 55%, at least
about 60%, at least about 65%, at least about 70%, at least about 75%, or at
least about 80% of the
TILs are CD8+ TILs. In some aspects, at least about 20% of the TILs are CD8+
TILs. In some
aspects, at least about 30% of Tits are CD8+ Tits. In some aspects, at least
about 40% of the TILs
are CDS' TILs. In some aspects, at least about 50% of the TILs are CD8+ TILs.
In some aspects,
at least about 60% of the TILs are CDS+ TILs. In some aspects, at least about
70% of the Tits are
CDS+ TILs. In some aspects, at least about 80% of the TILs are CDS+ TILs. In
some aspects, at
least about 90% of the Tits are CD8+ TILs. In some aspects, at least about 95%
of TILs are CD8+
TILs.
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[0459] Some aspects of the present disclosure are directed to a
composition comprising a
population of expanded Tits, wherein the population of expanded TILs has an
increased clonal
diversity, as compared to the clonal diversity of a population of TILs
expanded using control
methods (e.g., cultured in a medium comprising potassium ion at a
concentration of less than about
mM). In some aspects, the population of expanded TILs has a clonal diversity
that is the same as
the clonal diversity of TILs in a tumor sample. In some aspects, the
population of expanded TILs
has a clonal diversity that is at least about 99% to about 100%, at least
about 98% to about 100%,
at least about 97% to about 100%, at least about 96% to about 100%, at least
about 95% to about
100%, at least about 94% to about 100%, at least about 93% to about 100%, at
least about 92% to
about 100%, at least about 91% to about 100%, at least about 90% to about
100%, at least about
85% to about 100%, at least about 80% to about 100%, at least about 75% to
about 100%, at least
about 70% to about 100%, at least about 65% to about 100%, at least about 60%
to about 100%,
at least about 55% to about 100%, at least about 50% to about 100%, at least
about 45% to about
100%, or at least about 40% to about 100% of the clonal diversity of TILs in a
tumor sample. In
certain aspects, the population of expanded TILs has a clonal diversity that
is at least about 95%
of the clonal diversity of TILs in a tumor sample. In certain aspects, the
population of expanded
TILs has a clonal diversity that is at least about 90% of the clonal diversity
of TILs in a tumor
sample. In certain aspects, the population of expanded TILs has a clonal
diversity that is at least
about 85% of the clonal diversity of TILs in a tumor sample. In certain
aspects, the population of
expanded TILs has a clonal diversity that is at least about 80% of the clonal
diversity of TILs in a
tumor sample. In certain aspects, the population of expanded TILs has a clonal
diversity that is at
least about 75% of the clonal diversity of TILs in a tumor sample. In certain
aspects, the population
of expanded TILs has a clonal diversity that is at least about 70% of the
clonal diversity of TILs in
a tumor sample. In certain aspects, the population of expanded TILs has a
clonal diversity that is
at least about 60% of the clonal diversity of TILs in a tumor sample. In
certain aspects, the
population of expanded TILs has a clonal diversity that is at least about 50%
of the clonal diversity
of TILs in a tumor sample. In certain aspects, the population of expanded TILs
has a clonal
diversity that is at least about 40% of the clonal diversity of TILs in a
tumor sample.
[0460] In some aspects, the population of expanded TILs has a
clonal diversity score of
less than about 0.5, less than about 0.45, less than about 0.4, less than
about 0.35, less than about
0.3, less than about 0.275, less than about 0.25, less than about 0.225, less
than about 0.2, less than
about 0.175, less than about 0.15, less than about 0.125, less than about 0.1,
less than about 0.075,
less than about 0.07, less than about 0.06, or less than about 0.05 as
measured by Simpsons
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clonality. In some aspects, the population of expanded TILs has a clonal
diversity score of less
than about 0.5, as measured by Simpsons clonality. In some aspects, the
population of expanded
TILs has a clonal diversity score of less than about 0.4, as measured by
Simpsons clonality. In
some aspects, the population of expanded TILs has a clonal diversity score of
less than about 0.3,
as measured by Simpsons clonality. In some aspects, the population of expanded
TILs has a clonal
diversity score of less than about 0.275, as measured by Simpsons clonality.
In some aspects, the
population of expanded TILs has a clonal diversity score of less than about
0.25, as measured by
Simpsons clonality. In some aspects, the population of expanded TILs has a
clonal diversity score
of less than about 0.24, as measured by Simpsons clonality. In some aspects,
the population of
expanded TILs has a clonal diversity score of less than about 0.23, as
measured by Simpsons
clonality. In some aspects, the population of expanded TILs has a clonal
diversity score of less
than about 0.22, as measured by Simpsons clonality. In some aspects, the
population of expanded
TILs has a clonal diversity score of less than about 0.21, as measured by
Simpsons clonality. In
some aspects, the population of expanded TILs has a clonal diversity score of
less than about 0.2,
as measured by Simpsons clonality. In some aspects, the population of expanded
TILs has a clonal
diversity score of less than about 0.19, as measured by Simpsons clonality. In
some aspects, the
population of expanded TILs has a clonal diversity score of less than about
0.18, as measured by
Simpsons clonality. In some aspects, the population of expanded TILs has a
clonal diversity score
of less than about 0.17, as measured by Simpsons clonality. In some aspects,
the population of
expanded TILs has a clonal diversity score of less than about 0.16, as
measured by Simpsons
clonality. In some aspects, the population of expanded TILs has a clonal
diversity score of less
than about 0.15, as measured by Simpsons clonality. In some aspects, the
population of expanded
TILs has a clonal diversity score of less than about 0.14, as measured by
Simpsons clonality. In
some aspects, the population of expanded TILs has a clonal diversity score of
less than about 0.13,
as measured by Simpsons clonality. In some aspects, the population of expanded
TILs has a clonal
diversity score of less than about 0.12, as measured by Simpsons clonality. In
some aspects, the
population of expanded TILs has a clonal diversity score of less than about
0.11, as measured by
Simpsons clonality. In some aspects, the population of expanded TILs has a
clonal diversity score
of less than about 0.1, as measured by Simpsons clonality. In some aspects,
the population of
expanded TILs has a clonal diversity score of less than about 0.09, as
measured by Simpsons
clonality. In some aspects, the population of expanded TILs has a clonal
diversity score of less
than about 0.08, as measured by Simpsons clonality. In some aspects, the
population of expanded
TILs has a clonal diversity score of less than about 0.07, as measured by
Simpsons clonality. In
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some aspects, the population of expanded TILs has a clonal diversity score of
less than about 0.06,
as measured by Simpsons clonality. In some aspects, the population of expanded
TILs has a clonal
diversity score of less than about 0.05, as measured by Simpsons clonality.
[0461] In some aspects, the methods described herein selectively
increase the expansion of
tumor-reactive TIL clones by about 2-fold to about 500-fold, about 2-fold to
about 250-fold, about
2-fold to about 200-fold, about 2-fold to about 150-fold, about 2-fold to
about 100-fold, about 2-
fold to about 90-fold, about 2-fold to about 80-fold, about 2-fold to about 70-
fold, about 2-fold to
about 60-fold, about 2-fold to about 50-fold, about 2-fold to about 40-fold,
about 2-fold to about
30-fold, about 2-fold to about 20-fold, about 2-fold to about 10-fold, about 5-
fold to about 200-
fold, about 5-fold to about 150-fold, about 5-fold to about 100-fold, about 5-
fold to about 90-fold,
about 5-fold to about 80-fold, about 5-fold to about 70-fold, about 5-fold to
about 60-fold, about
5-fold to about 50-fold, about 5-fold to about 40-fold, about 5-fold to about
30-fold, about 5-fold
to about 20-fold, about 5-fold to about 10-fold, about 10-fold to about 150-
fold, about 10-fold to
about 100-fold, about 10-fold to about 90-fold, about 10-fold to about 80-
fold, about 10-fold to
about 70-fold, about 10-fold to about 60-fold, about 10-fold to about 50-fold,
about 10-fold to
about 40-fold, about 10-fold to about 30-fold, or about 10-fold to about 20-
fold, as compared to
selective expansion of tumor-reactive TIL clones expanded or cultured using
control methods (e.g.,
in medium comprising less than 40 mM potassium ion, e.g., 4 mM potassium ion).
In some aspects,
the methods described herein selectively increase the expansion of tumor
reactive TIL clones by
about 2-fold. In some aspects, the methods described herein selectively
increase the expansion of
tumor reactive TIL clones by about 5-fold. In some aspects, the methods
described herein
selectively increase the expansion of tumor reactive TIL clones by about 10-
fold.
[0462] In some aspects, the methods described herein selectively
increase the expansion of
tumor-reactive TIL clones, wherein clonal diversity is maintained. In some
aspects, the methods
described herein selectively increase the expansion of tumor-reactive TIL
clones by about 2-fold
to about 50-fold, wherein clonal diversity is maintained by about 70% to about
100%. In some
aspects, the methods described herein selectively increase the expansion of
tumor-reactive TIL
clones by about 2-fold, wherein clonal diversity is maintained by about 70% to
about 100%. In
some aspects, the methods described herein selectively increase the expansion
of tumor-reactive
TIL clones by about 5-fold, wherein clonal diversity is maintained by about
70% to about 100%.
In some aspects, the methods described herein selectively increase the
expansion of tumor-reactive
TlL clones by about 10-fold, wherein clonal diversity is maintained by about
70% to about 100%.
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[0463] In some aspects, the TILs exhibit increased expression of
one or more biomarker
indicative of a less-differentiated phenotype. In some aspects, the TILs
exhibit increased
expression of TCF7. In some aspects, the TILs (e.g., the CD8+ TILs) cultured
according to the
methods disclosed herein exhibit an at least about 2-fold, at least about 3-
fold, at least about 4-
fold, at least about 5-fold, at least about 10-fold, at least about 15-fold,
at least about 20-fold, at
least about 25-fold, at least about 30-fold, at least about 35-fold, at least
about 40-fold, at least
about 45-fold, or at least about 50-fold increase in the expression of TCF7.
In some aspects, the
TILs (e.g., the CDS+ TILs) cultured according to the methods disclosed herein
exhibit an at least
about 40-fold increase in the expression of TCF7. In some aspects, at least
about 5%, at least about
10%, at least about 15%, at least about 20%, at least about 25%, at least
about 30%, at least about
35%, at least about 40%, at least about 45%, at least about 50%, at least
about 55%, at least about
60%, at least about 65%, at least about 70%, or at least about 75% of the
immune cells are CD8+
TCF7 + TILs. In some aspects, at least about 5%, at least about 10%, at least
about 15%, at least
about 20%, at least about 25%, at least about 30%, at least about 35%, at
least about 40%, at least
about 45%, at least about 50% the CD8+ TILs are TCF7.
[0464] In some aspects, the TILs exhibit increased expression of
CD45RO. In some
aspects, the TILs (e.g, the CD8+ TILs) cultured according to the methods
disclosed herein exhibit
an at least about 2-fold, at least about 3-fold, at least about 4-fold, at
least about 5-fold, at least
about 6-fold, at least about 7-fold, at least about 8-fold, at least about 9-
fold, at least about 10-fold,
at least about 11-fold, at least about 12-fold, at least about 13-fold, at
least about 14-fold, or at least
about 15-fold increase in the expression of CD45RO. In some aspects, the TILs
(e.g., the CD8+
TILs) cultured according to the methods disclosed herein exhibit an at least
about 10-fold increase
in the expression of CD45RO. In some aspects, at least about 5%, at least
about 10%, at least about
15%, at least about 20%, at least about 25%, at least about 30%, at least
about 35%, at least about
40%, at least about 45%, at least about 50%, at least about 55%, at least
about 60%, at least about
65%, at least about 70%, or at least about 75% of the immune cells are CD8+
CD45R0- Tits. In
some aspects, at least about 5%, at least about 10%, at least about 15%, at
least about 20%, at least
about 25%, at least about 30%, at least about 35%, at least about 40%, at
least about 45%, at least
about 50% the CD8+ TILs are CD45R0 .
[0465] In some aspects, the TILs exhibit increased expression of
CD62L. In some aspects,
the TILs (e.g., the CDS+ TILs) cultured according to the methods disclosed
herein exhibit an at
least about 2-fold, at least about 3-fold, at least about 4-fold, at least
about 5-fold, at least about 6-
fold, at least about 7-fold, at least about 8-fold, at least about 9-fold, at
least about 10-fold, at least
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about 11-fold, at least about 12-fold, at least about 13-fold, at least about
14-fold, or at least about
15-fold increase in the expression of CD62L. In some aspects, the TILs (e.g.,
the CD8+ TILs)
cultured according to the methods disclosed herein exhibit an at least about
10-fold increase in the
expression of CD62L. In some aspects, at least about 5%, at least about 10%,
at least about 15%,
at least about 20%, at least about 25%, at least about 30%, at least about
35%, at least about 40%,
at least about 45%, at least about 50%, at least about 55%, at least about
60%, at least about 65%,
at least about 70%, or at least about 75% of the immune cells are CD8 CD62L'
TILs. In some
aspects, at least about 5%, at least about 10%, at least about 15%, at least
about 20%, at least about
25%, at least about 30%, at least about 35%, at least about 40%, at least
about 45%, at least about
50% the CD8+ TILs are CD62L.
[0466] In some aspects, the TILs exhibit increased expression of
CD27. In some aspects,
the TILs (e.g., the CD8+ TILs) cultured according to the methods disclosed
herein, exhibit an at
least about 2-fold, at least about 3-fold, at least about 4-fold, at least
about 5-fold, at least about 6-
fold, at least about 7-fold, at least about 8-fold, at least about 9-fold, at
least about 10-fold, at least
about 11-fold, at least about 12-fold, at least about 13-fold, at least about
14-fold, or at least about
15-fold increase in the expression of CD27. In some aspects, the TILs (e.g.,
the CD8' TILs)
cultured according to the methods disclosed herein exhibit an at least about
10-fold increase in the
expression of CD27. In some aspects, at least about 5%, at least about 10%, at
least about 15%, at
least about 20%, at least about 25%, at least about 30%, at least about 35%,
at least about 40%, at
least about 45%, at least about 50%, at least about 55%, at least about 60%,
at least about 65%, at
least about 70%, or at least about 75% of the TILs are CD8+ CD27 + TILs. In
some aspects, at least
about 5%, at least about 10%, at least about 15%, at least about 20%, at least
about 25%, at least
about 30%, at least about 35%, at least about 40%, at least about 45%, at
least about 50% the CD8+
TILs are CD27 .
[0467] In some aspects, the TILs exhibit increased expression of
CD62L and CD27. In
some aspects, the Tits (e.g., the CD8+ TILs) cultured according to the methods
disclosed herein
exhibit an at least about 2-fold, at least about 3-fold, at least about 4-
fold, at least about 5-fold, at
least about 6-fold, at least about 7-fold, at least about 8-fold, at least
about 9-fold, at least about
10-fold, at least about 11-fold, at least about 12-fold, at least about 13-
fold, at least about 14-fold,
or at least about 15-fold increase in the expression of CD62L and CD27. In
some aspects, the TILs
(e.g., the CDS+ TILs) cultured according to the methods disclosed herein
exhibit an at least about
10-fold increase in the expression of CD27. In some aspects, at least about
5%, at least about 10%,
at least about 15%, at least about 20%, at least about 25%, at least about
30%, at least about 35%,
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at least about 40%, at least about 45%, at least about 50%, at least about
55%, at least about 60%,
at least about 65%, at least about 70%, or at least about 75% of the TILs are
CD8-7 CD62L+/CD27+
TILs. In some aspects, at least about 5%, at least about 10%, at least about
15%, at least about
20%, at least about 25%, at least about 30%, at least about 35%, at least
about 40%, at least about
45%, at least about 50% the CD8+ TILs are CD62L+ CD27.
[0468] In some aspects, the TILs exhibit increased expression of
CD28. In some aspects,
the TILs (e.g., the CD8+ TILs) cultured according to the methods disclosed
herein exhibit an at
least about 2-fold, at least about 3-fold, at least about 4-fold, at least
about 5-fold, at least about 6-
fold, at least about 7-fold, at least about 8-fold, at least about 9-fold, at
least about 10-fold, at least
about 11-fold, at least about 12-fold, at least about 13-fold, at least about
14-fold, or at least about
15-fold increase in the expression of CD28. In some aspects, the TILs (e.g.,
the CD8+ TILs)
cultured according to the methods disclosed herein exhibit an at least about
10-fold increase in the
expression of CD28. In some aspects, at least about 5%, at least about 10%, at
least about 15%, at
least about 20%, at least about 25%, at least about 30%, at least about 35%,
at least about 40%, at
least about 45%, at least about 50%, at least about 55%, at least about 60%,
at least about 65%, at
least about 70%, or at least about 75% of the immune cells are CD8-VCD28+
TILs. In some aspects,
at least about 5%, at least about 10%, at least about 15%, at least about 20%,
at least about 25%,
at least about 30%, at least about 35%, at least about 40%, at least about
45%, at least about 50%
the CD8+ TILs are CD28+.
[0469] In some aspects, the Tits exhibit increased expression of
CD27 and CD28. In some
aspects, the TILs (e.g., the CD8+ TILs) cultured according to the methods
disclosed herein exhibit
an at least about 2-fold, at least about 3-fold, at least about 4-fold, at
least about 5-fold, at least
about 6-fold, at least about 7-fold, at least about 8-fold, at least about 9-
fold, at least about 10-fold,
at least about 11-fold, at least about 12-fold, at least about 13-fold, at
least about 14-fold, or at least
about 15-fold increase in the expression of CD27 and CD28. In some aspects,
the TILs (e.g., the
CDS+ TILs) cultured according to the methods disclosed herein exhibit an at
least about 10-fold
increase in the expression of CD27 and CD28 In some aspects, at least about
5%, at least about
10%, at least about 15%, at least about 20%, at least about 25%, at least
about 30%, at least about
35%, at least about 40%, at least about 45%, at least about 50%, at least
about 55%, at least about
60%, at least about 65%, at least about 70%, or at least about 75% of the TILs
are CD8 CD27+
CD28+ TILs. In some aspects, at least about 5%, at least about 10%, at least
about 15%, at least
about 20%, at least about 25%, at least about 30%, at least about 35%, at
least about 40%, at least
about 45%, at least about 50% the CD8' TILs are CD27' CD28'.
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[0470] In some aspects, the TILs exhibit increased expression of
CD27, CD28, PD1, and
CD103. In some aspects, the TILs (e.g., the CD8+ TILs) cultured according to
the methods
disclosed herein exhibit an at least about 2-fold, at least about 3-fold, at
least about 4-fold, at least
about 5-fold, at least about 6-fold, at least about 7-fold, at least about 8-
fold, at least about 9-fold,
at least about 10-fold, at least about 11-fold, at least about 12-fold, at
least about 13-fold, at least
about 14-fold, or at least about 15-fold increase in the expression of CD27,
CD28, PD1, and
CD103. In some aspects, the TILs (e.g., the CD8+ TILs) cultured according to
the methods
disclosed herein exhibit an at least about 10-fold increase in the expression
of CD27, CD28, PD1,
and CD103. In some aspects, at least about 5%, at least about 10%, at least
about 15%, at least
about 20%, at least about 25%, at least about 30%, at least about 35%, at
least about 40%, at least
about 45%, at least about 50%, at least about 55%, at least about 60%, at
least about 65%, at least
about 70%, or at least about 75% of the TILs are CD8+ CD27 + CD28 PD1 '
CD103+ TILs. In some
aspects, at least about 5%, at least about 10%, at least about 15%, at least
about 20%, at least about
25%, at least about 30%, at least about 35%, at least about 40%, at least
about 45%, at least about
50% the CD8+ TILs are CD27 + CD28 + PDT' CD103+.
[0471] In some aspects, the TILs exhibit increased expression of
CD27, CD28, PD1, and
TCF7. In some aspects, the TILs (e.g, the CD8+ TILs) cultured according to the
methods disclosed
herein exhibit an at least about 2-fold, at least about 3-fold, at least about
4-fold, at least about 5-
fold, at least about 6-fold, at least about 7-fold, at least about 8-fold, at
least about 9-fold, at least
about 10-fold, at least about 11-fold, at least about 12-fold, at least about
13-fold, at least about
14-fold, or at least about 15-fold increase in the expression of CD27, CD28,
PD1, and TCF7. In
some aspects, the TILs (e.g., the CD8+ TILs) cultured according to the methods
disclosed herein
exhibit an at least about 10-fold increase in the expression of CD27, CD28,
PD1, and TCF7. In
some aspects, at least about 5%, at least about 10%, at least about 15%, at
least about 20%, at least
about 25%, at least about 30%, at least about 35%, at least ab out 40%, at
least about 45%, at least
about 50%, at least about 55%, at least about 60%, at least about 65%, at
least about 70%, or at
least about 75% of the TILs are CD8+ CD27 CD28 PD1' TCF7 TILs. In some
aspects, at least
about 5%, at least about 10%, at least about 15%, at least about 20%, at least
about 25%, at least
about 30%, at least about 35%, at least about 40%, at least about 45%, at
least about 50% the CD8+
TILs are CD27' CD28 PD1' TCF7'.
[0472] In some aspects, the TILs exhibit increased expression of
CD27, CD28, PD1,
CD103, and TCF7 In some aspects, the TILs (e.g., the CD8+ TILs) cultured
according to the
methods disclosed herein exhibit an at least about 2-fold, at least about 3-
fold, at least about 4-
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fold, at least about 5-fold, at least about 6-fold, at least about 7-fold, at
least about 8-fold, at least
about 9-fold, at least about 10-fold, at least about 11-fold, at least about
12-fold, at least about 13-
fold, at least about 14-fold, or at least about 15-fold increase in the
expression of CD27, CD28,
PD1, CDI03, and TCF7. In some aspects, the TILs (e.g., the CD8 TILs) cultured
according to the
methods disclosed herein exhibit an at least about 10-fold increase in the
expression of CD27,
CD28, PD1, CD103, and TCF7. In some aspects, at least about 5%, at least about
10%, at least
about 15%, at least about 20%, at least about 25%, at least about 30%, at
least about 35%, at least
about 40%, at least about 45%, at least about 50%, at least about 55%, at
least about 60%, at least
about 65%, at least about 70%, or at least about 75% of the TILs are CD8+ CD27
+ CD28 + PD1'
CD103+ TCF7 + TILs. In some aspects, at least about 5%, at least about 10%, at
least about 15%,
at least about 20%, at least about 25%, at least about 30%, at least about
35%, at least about 40%,
at least about 45%, at least about 50% the CD8+ TILs are CD27 + CD28 PD1 +
CD103- TCF7.
[0473] In some aspects, the TILs exhibit increased expression of
CD39. In some aspects,
the TILs (e.g., the CD8+ TILs) cultured according to the methods disclosed
herein exhibit an at
least about 2-fold, at least about 3-fold, at least about 4-fold, at least
about 5-fold, at least about 6-
fold, at least about 7-fold, at least about 8-fold, at least about 9-fold, at
least about 10-fold, at least
about 11-fold, at least about 12-fold, at least about 13-fold, at least about
14-fold, or at least about
15-fold increase in the expression of CD39. In some aspects, the TILs (e.g.,
the CD8+ TILs)
cultured according to the methods disclosed herein exhibit an at least about
10-fold increase in the
expression of CD39. In some aspects, at least about 5%, at least about 10%, at
least about 15%, at
least about 20%, at least about 25%, at least about 30%, at least about 35%,
at least about 40%, at
least about 45%, at least about 50%, at least about 55%, at least about 60%,
at least about 65%, at
least about 70%, or at least about 75% of the immune cells are CD8+ CD39'
TILs. In some aspects,
at least about 5%, at least about 10%, at least about 15%, at least about 20%,
at least about 25%,
at least about 30%, at least about 35%, at least about 40%, at least about
45%, at least about 50%
the CD8+ Tits are CD39 .
[0474] In some aspects, the TILs exhibit increased expression of
CD39 and PD1. In some
aspects, the TILs (e.g., the CD8+ TILs) cultured according to the methods
disclosed herein exhibit
an at least about 2-fold, at least about 3-fold, at least about 4-fold, at
least about 5-fold, at least
about 6-fold, at least about 7-fold, at least about 8-fold, at least about 9-
fold, at least about 10-fold,
at least about 11-fold, at least about 12-fold, at least about 13-fold, at
least about 14-fold, or at least
about 15-fold increase in the expression of CD39 and PD1. In some aspects,
thee Tits (e.g., the
CD8 TILs) cultured according to the methods disclosed herein exhibit an at
least about 10-fold
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increase in the expression of CD39 and PD1. In some aspects, at least about
5%, at least about
10%, at least about 15%, at least about 20%, at least about 25%, at least
about 30%, at least about
35%, at least about 40%, at least about 45%, at least about 50%, at least
about 55%, at least about
60%, at least about 65%, at least about 70%, or at least about 75% of the
immune cells are CDS+
CD39+ PD1 + TILs. In some aspects, at least about 5%, at least about 10%, at
least about 15%, at
least about 20%, at least about 25%, at least about 30%, at least about 35%,
at least about 40%, at
least about 45%, at least about 50% the CD8 TILs are CD39+ PDT'.
[0475] In some aspects, the TILs exhibit increased expression of
PD1. In some aspects, the
TILs (e.g., the CD8+ TILs) cultured according to the methods disclosed herein
exhibit an at least
about 2-fold, at least about 3-fold, at least about 4-fold, at least about 5-
fold, at least about 6-fold,
at least about 7-fold, at least about 8-fold, at least about 9-fold, at least
about 10-fold, at least about
11-fold, at least about 12-fold, at least about 13-fold, at least about 14-
fold, or at least about 15-
fold increase in the expression of PD1. In some aspects, the TILs (e.g., the
CD8+ TILs) cultured
according to the methods disclosed herein exhibit an at least about 10-fold
increase in the
expression of PD1. In some aspects, at least about 5%, at least about 10%, at
least about 15%, at
least about 20%, at least about 25%, at least about 30%, at least about 35%,
at least about 40%, at
least about 45%, at least about 50%, at least about 55%, at least about 60%,
at least about 65%, at
least about 70%, or at least about 75% of the immune cells are CD8+/PD1+ TILs.
In some aspects,
at least about 5%, at least about 10%, at least about 15%, at least about 20%,
at least about 25%,
at least about 30%, at least about 35%, at least about 40%, at least about
45%, at least about 50%
the CD8+ TILs are PD1.
[0476] In some aspects, the TILs exhibit increased expression of
PD1 and CD27. In some
aspects, the TILs (e.g., the CD8+ TILs) cultured according to the methods
disclosed herein exhibit
an at least about 2-fold, at least about 3-fold, at least about 4-fold, at
least about 5-fold, at least
about 6-fold, at least about 7-fold, at least about 8-fold, at least about 9-
fold, at least about 10-fold,
at least about 11-fold, at least about 12-fold, at least about 13-fold, at
least about 14-fold, or at least
about 15-fold increase in the expression of PD1 and CD27. In some aspects, the
TILs (e.g., the
CDS+ TILs) cultured according to the methods disclosed herein exhibit an at
least about 10-fold
increase in the expression of PD1 and CD27. In some aspects, at least about
5%, at least about
10%, at least about 15%, at least about 20%, at least about 25%, at least
about 30%, at least about
35%, at least about 40%, at least about 45%, at least about 50%, at least
about 55%, at least about
60%, at least about 65%, at least about 70%, or at least about 75% of the Tits
are CD8+ PD1+
CD27. TILs. In some aspects, at least about 5%, at least about 10%, at least
about 15%, at least
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about 20%, at least about 25%, at least about 30%, at least about 35%, at
least about 40%, at least
about 45%, at least about 50% the CD8+ TILs are PD1+ CD27+.
[0477] In some aspects, the TILs exhibit increased expression of
CD103. In some aspects,
the TILs (e.g., the CD8 TILs) cultured according to the methods disclosed
herein exhibit an at
least about 2-fold, at least about 3-fold, at least about 4-fold, at least
about 5-fold, at least about 6-
fold, at least about 7-fold, at least about 8-fold, at least about 9-fold, at
least about 10-fold, at least
about 11-fold, at least about 12-fold, at least about 13-fold, at least about
14-fold, or at least about
15-fold increase in the expression of CD103. In some aspects, the TILs (e.g.,
the CD8+ Tits)
cultured according to the methods disclosed herein exhibit an at least about
10-fold increase in the
expression of CD103. In some aspects, at least about 5%, at least about 10%,
at least about 15%,
at least about 20%, at least about 25%, at least about 30%, at least about
35%, at least about 40%,
at least about 45%, at least about 50%, at least about 55%, at least about
60%, at least about 65%,
at least about 70%, or at least about 75% of the TILs are CD8+/CD103+ TILs. In
some aspects, at
least about 5%, at least about 10%, at least about 15%, at least about 20%, at
least about 25%, at
least about 30%, at least about 35%, at least about 40%, at least about 45%,
at least about 50% the
CD8-' TILs are CD103t
[0478] In some aspects, the TILs (e.g, CD8+ TILs) cultured
according to the methods
and/or in the medium disclosed herein have an increased number of less-
differentiated cells as
compared to comparable immune cells cultured according to conventional
methods. In some
aspects, the TILs (e.g., CD8+ TILs) cultured according to the methods
disclosed herein exhibit
increased expression of one or more marker typical of a stem-like phenotype.
In some aspects, TIL
(e.g., CDS+ TIL) populations cultured according to the methods and/or in a
metabolic
reprogramming medium disclosed herein have an increased number of effector-
like cells as
compared to comparable cells cultured according to conventional methods, e.g.,
in media
containing less than 5 mM Kt In some aspects, TIL (e.g., CDS+ TIL) populations
cultured
according to the methods and/or in a metabolic reprogramming medium disclosed
herein have both
an increased number of stem-like and effector-like cells as compared to
comparable cells cultured
according to conventional methods, e.g., in media containing less than 5 mM Kt
In some aspects,
TILs (e.g., CD8- TILs) cultured according to the methods disclosed herein
exhibit greater
proliferative potential compared to cells cultured according to conventional
methods. In some
aspects, the TILs (e.g., CDS+ TILs) cultured according to the methods
disclosed herein exhibit
increased in vivo viability upon transplantation in a subject. In some
aspects, the TILs (e.g., CDS+
TILs) cultured according to the methods disclosed herein exhibit increased
cell potency. In some
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aspects, the TILs (e.g., CD8 TILs) cultured according to the methods disclosed
herein exhibit
decreased cell exhaustion. In some aspects, the TILs (e.g., CD8+ TILs)
cultured according to the
methods disclosed herein exhibit increased in vivo persistence upon
transplantation in a subject. In
some aspects, the TILs (e.g., CD8' TILs) cultured according to the methods
disclosed herein
exhibit increased in vivo activity upon transplantation in a subject. In some
aspects, the TILs (e.g.,
CD8+ TILs) cultured according to the methods disclosed herein exhibit a more
durable in vivo
response upon transplantation in a subject. In some aspects, the subject is a
human.
[0479] In some aspects, at least about 5% of the TILs (e.g.,
CD8+ TILs) in the composition
have a stem-like phenotype. In some aspects, at least about 10% of the TILs
(e.g., CD8+ Tits) in
the composition have a stem-like phenotype. In some aspects, at least about
15% of the TILs (e.g.,
CD8+ TILs) in the composition have a stem-like phenotype. In some aspects, at
least about 20% of
the TILs (e.g., CD8+ TILs) in the composition have a stem-like phenotype. In
some aspects, at least
about 25% of the TILs (e.g., CD8+ TILs) in the composition have a stem-like
phenotype. In some
aspects, at least about 30% of the TILs (e.g., CD8+ TILs) in the composition
have a stem-like
phenotype. In some aspects, at least about 35% of the TILs (e.g., CD8+ TILs)
in the composition
have a stem-like phenotype. In some aspects, at least about 40% of the TILs
(e.g., CDS+ TILs) in
the composition have a stem-like phenotype. In some aspects, at least about
45% of the TILs (e.g.,
CD8+ TILs) in the composition have a stem-like phenotype. In some aspects, at
least about 50% of
the TILs (e.g., CD8+ TILs) in the composition have a stem-like phenotype. In
some aspects, at least
about 55% of the TILs (e.g., CD8 TILs) in the composition have a stem-like
phenotype. In some
aspects, at least about 60% of the TILs (e.g., CD8+ TILs) in the composition
have a stem-like
phenotype. In some aspects, at least about 65% of the TILs (e.g., CD8+ TILs)
in the composition
have a stem-like phenotype. In some aspects, at least about 70% of the TILs
(e.g, CDS+ TILs) in
the composition have a stem-like phenotype.
[0480] In some aspects, following culture of TILs (e.g., CDS+
TILs) according to the
methods disclosed herein, stem-like Tits (e.g., CD8+ TILs) constitute at least
about 10% to at least
about 70% of the total number of TTLs (e.g., CD8' TILs) in the culture. In
some aspects, following
culture of TILs (e.g., CD8+ TILs) according to the methods disclosed herein,
stem-like TILs (e.g.,
CD8+ TILs) constitute at least about 10%, at least about 20%, at least about
30%, at least about
40%, at least about 50%, at least about 60%, or at least about 70% of the
total number of TILs
(e.g., CDS+ TILs) in the culture In some aspects, following culture of Tits
(e.g., CDS+ TILs)
according to the methods disclosed herein, stem-like Tits (e.g., CD8 Tits)
constitute at least
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about 10%, at least about 20%, at least about 30%, at least about 40%, at
least about 50%, at least
about 60%, or at least about 70% of the total number of CD8+ TILs in the
culture.
[0481]
In some aspects, the number of TILs (e.g., CD8+ TILs) haying a stem-
like
phenotype in the composition is increased at least about L5-fold as compared
to the number of
TILs (e.g., CD8+ TILs) in the composition prior to the culture. In some
aspects, the number of TILs
(e.g., CD8+ TILs) haying a stem-like phenotype in the composition is increased
at least about 2.0-
fold as compared to the number of TILs (e.g., CD8+ TILs) in the composition
prior to the culture.
In some aspects, the number of Tits
CD8+ TILs) having a stem-like phenotype in the
composition is increased at least about 2,5-fold as compared to the number of
TILs (e.g., CD8+
TILs) in the composition prior to the culture. In some aspects, the number of
TILs (e.g., CD8+
TILs) haying a stem-like phenotype in the composition is increased at least
about 3.0-fold as
compared to the number of TILs (e.g., CD8+ TILs) in the composition prior to
the culture. In some
aspects, the number of TILs (e.g., CD8+ TILs) haying a stem-like phenotype in
the composition is
increased at least about 3.5-fold as compared to the number of TILs (e.g.,
CD8+ TILs) in the
composition prior to the culture. In some aspects, the number of TILs (e.g.,
CD8+ TILs) haying a
stem-like phenotype in the composition is increased at least about 4.0-fold as
compared to the
number of TILs (e.g, CD8+ TILs) in the composition prior to the culture. In
some aspects, the
number of cells haying a stem-like phenotype in the composition is increased
at least about 4.5-
fold as compared to the number of TILs (e.g., CD8+ TILs) in the composition
prior to the culture.
In some aspects, the number of TILs (e.g., CD8+ TILs) haying a stem-like
phenotype in the
composition is increased at least about 5.0-fold as compared to the number of
TILs (e.g., CD8+
TILs) in the composition prior to the culture. In some aspects, the number of
TILs (e.g., CD8+
TILs) haying a stem-like phenotype in the composition is increased at least
about 5.5-fold as
compared to the number of TILs (e.g., CD8+ TILs) in the composition prior to
the culture. In some
aspects, the number of TILs (e.g., CD8+ TILs) haying a stem-like phenotype in
the composition is
increased at least about 6.0-fold as compared to the number of TILs (e.g.,
CD8+ TILs) in the
composition prior to the culture. In some aspects, the number of TILs (e.g.,
CD8 TILs) haying a
stem-like phenotype in the composition is increased at least about 6.5-fold as
compared to the
number of TILs (e.g., CD8+ TILs) in the composition prior to the culture. In
some aspects, the
number of TILs (e.g., CD8+ TILs) haying a stem-like phenotype in the
composition is increased at
least about 7.0-fold as compared to the number of cells in the composition
prior to the culture. In
some aspects, the number of TILs (e.g., CD8+ TILs), having a stem-like
phenotype in the
composition is increased at least about 7.5-fold as compared to the number of
cells in the
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composition prior to the culture. In some aspects, the number of TILs (e.g.,
CD8 TILs) haying a
stem-like phenotype in the composition is increased at least about 8.0-fold as
compared to the
number of TILs (e.g., CD8+ TILs) in the composition prior to the culture. In
some aspects, the
number of TILs (e.g., CD8' TILs) having a stem-like phenotype in the
composition is increased at
least about 9.0-fold as compared to the number of TILs (e.g., CD8+ TILs) in
the composition prior
to the culture. In some aspects, the number of cells having a stem-like
phenotype in the composition
is increased at least about 10-fold as compared to the number of TILs (e.g.,
CD8+ TILs) in the
composition prior to the culture In some aspects, the number of cells having a
stem-like phenotype
in the composition is increased at least about 15-fold as compared to the
number of TILs (e.g.,
CD8+ TILs) in the composition prior to the culture. In some aspects, the
number of cells having a
stem-like phenotype in the composition is increased at least about 20-fold as
compared to the
number of TILs (e.g., CD8+ TILs) in the composition prior to the culture. In
some aspects, the
number of TILs haying a stem-like phenotype in the composition is increased at
least about 30-
fold as compared to the number of TILs in the composition prior to the
culture. In some aspects,
the number of Tits having a stem-like phenotype in the composition is
increased at least about 40-
fold as compared to the number of cells in the composition prior to the
culture. In some aspects,
the number of Tits haying a stem-like phenotype in the composition is
increased at least about 50-
fold as compared to the number of TILs in the composition prior to the
culture. In some aspects,
the number of Tits having a stem-like phenotype in the composition is
increased at least about 75-
fold as compared to the number of TILs in the composition prior to the
culture. In some aspects,
the number of TILs having a stem-like phenotype in the composition is
increased at least about
100-fold as compared to the number of TILs in the composition prior to the
culture. In some
aspects, the number of TILs haying a stem-like phenotype in the composition is
increased at least
about 500-fold as compared to the number of TILs in the composition prior to
the culture. In some
aspects, the number of TILs haying a stem-like phenotype in the composition is
increased at least
about 1000-fold as compared to the number of 1'1-Ls in the composition prior
to the culture.
[0482] In some aspects, following culture of TILs (e.g., CD8'
TILs) according to the
methods disclosed herein, at least about 10% to at least about 70% of the
total number of TILs
(e.g., CD8+ TILs) in the culture are CD397'TCF7+ T cells. In some aspects,
following culture of
TILs (e.g., CD8 TILs) according to the methods disclosed herein, at least
about 10%, at least about
15%, at least about 20%, at least about 25%, at least about 30%, at least
about 35%, or at least
about 40% of the total number of TILs (e.g., CDS+ TILs) in the culture are
CD39"/TCF7+ TILs
(e.g., CD8+ TILs).
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[0483] In some aspects, the cell composition comprises an
increased percentage of TILs
which express CD95. In some aspects, the cell composition comprises an
increased percentage of
TILs which do not express CD45RO. In some aspects, the cell composition
comprises an increased
percentage of TILs which express CD45RA. In some aspects, the cell composition
comprises an
increased percentage of TILs which express CCR7. In some aspects, the cell
composition
comprises an increased percentage of TILs which express CD62L. In some
aspects, the cell
composition comprises an increased percentage of TILs which express TCF7. In
some aspects, the
cell composition comprises an increased percentage of TILs which express CD3.
In some aspects,
the cell composition comprises an increased percentage of TILs which express
CD27 In some
aspects, the cell composition comprises an increased percentage of TILs which
express CD45RA.
In some aspects, the cell composition comprises an increased percentage of
TILs which express
CD95 and CD45RA. In some aspects, the cell composition comprises an increased
percentage of
TILs which express CD45RA and CCR7. In some aspects, the cell composition
comprises an
increased percentage of TILs which express CD95, CD45RA, and CCR7. In some
aspects, the cell
composition comprises an increased percentage of TILs which express CD45RA,
CCR7, and
CD62L. In some aspects, the cell composition comprises an increased percentage
of TILs which
express CD95, CD45RA, CCR7, and CD62L. In some aspects, the cell composition
comprises an
increased percentage of TILs which express CD45RA, CCR7, CD62L, and TCF7. In
some aspects,
the cell composition comprises an increased percentage of TILs which express
CD95, CD45RA,
CCR7, CD62L, and TCF7. In some aspects, the cell composition comprises an
increased
percentage of TILs which express CD45RA, CCR7, CD62L, TCF7, and CD27. In some
aspects,
the cell composition comprises an increased percentage of TILs which express
CD95, CD45RA,
CCR7, CD62L, TCF7, and CD27. In some aspects, the cell composition comprises
an increased
percentage of TILs which express CD45RA, CCR7, CD62L, TCF7, and CD27, and
which are
CD45R010. In some aspects, the cell composition comprises an increased
percentage of TILs
which express CD95, CD45RA, CCR7, CD62L, TCF7, and CD27, and which are
CD45R010. In
some aspects, the cell composition comprises an increased percentage of TILs
which express
CD45RA, CCR7, CD62L, TCF7, and CD27, and which do not express CD45RO.In some
aspects,
the cell composition comprises an increased percentage of TILs which express
CD95, CD45RA,
CCR7, CD62L, TCF7, and CD27, and which do not express CD45RO.
[0484] In some aspects, the cell composition comprises an
increase in the percent of TILs
which do not express CD39 and CD69. In some aspects, the cell composition
comprises an increase
in the percent of TILs which express CD8, and which do not express CD39 and
CD69. In some
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aspects, following culture of TILs according to the methods disclosed herein,
at least about 10%
to at least about 40% of the total number of TILs in the culture are
CD39"/CD69" TILs. In some
aspects, following culture of TILs according to the methods disclosed herein,
at least about 10%,
at least about 15%, at least about 20%, at least about 25%, at least about
30%, at least about 35%,
or at least about 40% of the total number of TILs in the culture are
CD39"/CD69" TILs.
[0485] In some aspects, the TILs (e.g., CD8+ TILs) cultured
according to the methods
and/or in the medium disclosed herein express one or more stem-like markers
and one or more
effector-like markers. In some aspects, the TILs (e.g., CD8+ Tits) cultured
according to the
methods and/or in the medium disclosed herein express at least two stem-like
markers and one or
more effector-like markers. In some aspects, the TILs (e.g., CD8+ TILs)
cultured according to the
methods and/or in the medium disclosed herein express at least three stem-like
markers and one or
more effector-like markers. In some aspects, the TILs (e.g., CD8+ TILs)
cultured according to the
methods and/or in the medium disclosed herein express at least four stem-like
markers and one or
more effector-like markers. In some aspects, the TILs (e.g., CD8+ TILs)
cultured according to the
methods and/or in the medium disclosed herein express one or more stem-like
markers and at least
two effector-like markers. In some aspects, at least about 40%, at least about
50%, at least about
60%, at least about 70%, at least about 80%, at least about 85%, at least
about 90%, at least about
95%, at least about 99%, or about 100% of the TILs express one or more stem-
like markers and
one or more effector-like markers. In some aspects, at least about 40% of the
TILs express one or
more stem-like markers and one or more effector-like markers. In some aspects,
at least about 50%
of the TILs express one or more stem-like markers and one or more effector-
like markers. In some
aspects, at least about 60% of the TILs express one or more stem-like markers
and one or more
effector-like markers. In some aspects, at least about 70% of the TILs express
one or more stem-
like markers and one or more effector-like markers. In some aspects, at least
about 75% of the TILs
express one or more stem-like markers and one or more effector-like markers.
In some aspects, at
least about 80% of the TILs express one or more stem-like markers and one or
more effector-like
markers. In some aspects, at least about 85% of the TlLs express one or more
stem-like markers
and one or more effector-like markers. In some aspects, at least about 90% of
the T1Ls express one
or more stem-like markers and one or more effector-like markers. In some
aspects, at least about
95% of the TILs express one or more stem-like markers and one or more effector-
like markers. In
some aspects, at least about 96% of the TILs express one or more stem-like
markers and one or
more effector-like markers. In some aspects, at least about 97% of the Tits
express one or more
stem-like markers and one or more effector-like markers. In some aspects, at
least about 98% of
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the TILs express one or more stem-like markers and one or more effector-like
markers. In some
aspects, at least about 99% of the TILs express one or more stem-like markers
and one or more
effector-like markers.
[0486] In some aspects, the stem-like markers are selected from
CD45RA+, CD62L+,
CCR7+, CD27+, CD28+, BACH2+, LEF1+, TCF7+, and any combination thereof In some
aspects
the stem-like markers comprise CD45RA I, CD62L I, CCR7 , and TCF7 I, or any
combination
thereof. In some aspects, the TIL expresses CD45R010. In some aspects, the
stem-like markers
comprise one or more genes listed herein as part of a gene-signature (see
supra; see, e.g., Gattinoni,
L., et al., Nat Med 17(10): 1290-1297 (2011) or Galletti et al. Nat Immunol
21, 1552-1562 (2020)).
In some aspects, the effector-like markers are selected from pSTAT5+, STAT5+,
pSTAT3+,
STAT3+, and any combination thereof. In some aspects, the effector-like marker
comprises a
STAT target selected from the group consisting of AKT1, AKT2, AKT3, BCL2L1,
CBL, CBLB,
CBLC, CCND1, CCND2, CCND3, CISH, CLCF1, CNTF, CNTFR, CREBBP, CRLF2, CSF2,
CSF2RA, CSF2RB, CSF3, CSF3R, CSH1, CTF1, EP300, EPO, EPOR, GH1, GH2, GHR,
GRB2,
IFNA1, IFNA10, IFNA13, IFNA14, IFNA16, IFNA17, IFNA2, 1FNA21, IFNA4, IFNA5,
IFNA6,
IFNA7, IFNA8, IFNAR1, IFNAR2, IFNB1, IFNE, IF'NG, IFNGR1, IFNGR2, IFNK, IFNL1,
IFNL2, IFNL3, IFNLR1, IFNW1, IL10, ILI ORA, IL 1 ORB, IL11, IL 11RA, IL12A,
IL12B,
IL12RB1, IL12RB2, IL13, IL13RA1, IL13RA2, IL15, IL15RA, IL19, IL2, IL20,
IL20RA,
IL20RB, IL21, IL21R, IL22, IL22RA1, 1L22RA2, IL23A, IL23R, IL24, IL26, IL2RA,
IL2RB,
IL2RG, IL3, IL3RA, IL4, IL4R, IL5, 1L5RA, 1L6, IL6R, IL6ST, IL7, IL7R, IL9,
IL9R, IRF9,
JAK1, JAK2, JAK3, LEP, LEPR, LIF, LIFR, MPL, MYC, OSM, OSMR, PIAS1, PIAS2,
PIAS3,
PIAS4, PIK3CA, PIK3CB, PIK3CD, PIK3CG, PIK3R1, PIK3R2, PIK3R3, PIK3R5, PIM1,
PRL,
PRLR, PTPN11, PTPN6, SOCS1, SOCS2, SOCS3, SOCS4, SOCS5, SOCS7, SOS1, SOS2,
SPRED1, SPRED2, SPRY1, SPRY2, SPRY3, SPRY4, STAM, STAM2, STAT1, STAT2, STAT3,
STAT4, STAT5A, STAT5B, STAT6, TPO, TSLP, TYK2, and any combination thereof In
some
aspects, the TILs (e.g., CD8+ TILs) cultured according to the methods and/or
in the medium
disclosed herein are CD45RA+, STAT5+, and STAT3+. In some aspects, the TILs
(e.g., CD8+
TILs) cultured according to the methods and/or in the medium disclosed herein
are CD62L+,
STAT5+, and STAT3+. In some aspects, the TILs (e.g., CDS+ TILs) cultured
according to the
methods and/or in the medium disclosed herein are TCF7+, STAT5+, and STAT3+.
In some
aspects, the TILs (e.g., CD8+ TILs) cultured according to the methods and/or
in the medium
disclosed herein are CD45RA+, CD62L+, CCR7+, CD27+, CD28+, BACH2+, LEF1+,
TCF7+,
STAT5+, and STAT3+. In some aspects the TILs (e.g., CD8 TILs) cultured
according to the
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methods and/or in the medium disclosed herein are CD45RA+, CD62L+, CCR7+,
CD27+, CD28+,
BACH2+, LEF1+, TCF7+, pSTAT5+, STAT5+, pSTAT3+, and STAT3+. In some aspects,
the
TILs (e.g., CD8+ TILs) cultured according to the methods and/or in the medium
disclosed herein
are CD45RA+, CD62L+, CCR7+, CD27+, CD28+, BACH2+, LEF1+, 1CF7+, pSTAT5+,
STAT5+, pSTAT3+, and STAT3+. In some aspects, the Tits (e.g., CD8+ TILs)
cultured according
to the methods and/or in the medium disclosed herein are CD45RA I, CD45R010w,
CD62L I,
CCR7+, CD27+, CD28+, BACH2+, LEF1+, TCF7+, pSTAT5+, STAT5+, pSTAT3+, and
STAT3+.
[0487] In some aspects, an TIL comprises one or more markers
selected from CD45RA+,
CD62L+, CCR7+, CD27+, CD28+, BACH2+, LEF1+, TCF7+, and any combination thereof
and
one or more markers selected from pSTAT5+, STAT5+, pSTAT3+, STAT3+, and any
combination thereof. In some aspects, a TIL comprises one or more markers
selected from
CD45RA+, CD62L+, CCR7+, CD27+, CD28+, BACH2+, LEF1+, TCF7+, and any
combination
thereof and one or more effector-like markers. In some aspects, a TIL
comprises one or more stem-
like markers and one or more markers selected from pSTAT5+, STAT5+, pSTAT3+,
STAT3+,
and any combination thereof. In some aspects, the effector-like marker
comprises a STAT target
selected from the group consisting of AKT1, AKT2, AKT3, BCL2L1, CBL, CBLB,
CBLC,
CCND1, CCND2, CCND3, CISH, CLCF1, CNTF, CNTFR, CREBBP, CRLF2, CSF2, CSF2RA,
CSF2RB, CSF3, CSF3R, CSH1, CTF1, EP300, EPO, EPOR, GH1, GH2, GHR, GRB2, IFNA1,
IFNAIO, IFNA13, IFNA14, IFNA16, IFNA17, IFNA2, IFNA21, IFNA4, IFNA5, IFNA6,
IFNA7,
IFNA8, IFNAR1, IFNAR2, IFNBI, IFNE, IFNG, IFNGR1, IFNGR2, IFNK, IFNL1, IFNL2,
IFNL3, IFNLR1, IFNW1, IL10, ILlORA, ILlORB, IL11, IL11RA, IL12A, IL12B,
IL12RB1,
IL12RB 2, IL13, IL13RA1, IL 13RA2, IL15, IL15RA, IL 19, IL2, IL20, IL20RA,
IL20RB, IL21,
IL21R, IL22, IL22RA1, IL22RA2, IL23A, IL23R, IL24, IL26, IL2RA, IL2RB, IL2RG,
IL3,
IL3RA, IL4, IL4R, IL5, IL5RA, IL6, IL6R, IL6ST, IL7, IL7R, IL9, IL9R, IRF9,
JAK1, JAK2,
JAK3, LEP, LEPR, LIF, LIFR, MPL, MYC, OSM, OSMR, PIAS1, PIAS2, PIAS3, PIAS4,
PIK3CA, PIK3CB, PIK3CD, PIK3CG, P11K3R1, PIK3R2, PIK3R3, PIK3R5, PIM1, PRL,
PRLR,
PTPN11, PTPN6, SOCS1, SOCS2, SOCS3, SOCS4, SOCS5, SOCS7, SOS1, SOS2, SPRED1,
SPRED2, SPRY1, SPRY2, SPRY3, SPRY4, STAM, STA1V12, STAT1, STAT2, STAT3, STAT4,
STAT5A, STAT5B, STAT6, TPO, TSLP, TYK2, and any combination thereof.
[0488] In some aspects, the TILs that expresses one or more stem-
like markers and one or
more effector-like marker is a T stem/effector (TsE) cell In some aspects, the
TSE cell retains a less
differentiated state (e.g., expreses one or more stem-like markers, is capable
of proliferation, is
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capable of differentiation, or any combination thereof) and the cell has
effector function (e.g.,
expresses one or more effector-like markers, is capable of targeting and/or
killing tumor cells,
exhibits polyfunctionality, or a combination thereof). In some aspects, a TSE
cell disclosed herein
expresses CD45RA+, CD62L+, CCR7+, CD27+, CD28+, BACH2+, LEF I+, TCF7+,
pSTAT5+,
STAT5+, pSTAT3+, and STAT3+. In some aspects, a TSE cell disclosed herein
expresses
CD45RA I, CD62L I, CCR7 , TCF7 I, pSTAT5 I, STAT5 I, pSTAT3 I, and STAT3 I. In
some
aspects, the TSE cell is CD45R010
.
[0489] Some aspects of the present disclosure are directed to an
expanded population of
TILs comprising one or more TSE cell. In some aspects, at least about 40%, at
least about 50%, at
least about 60%, at least about 70%, at least about 80%, at least about 85%,
at least about 90%, at
least about 95%, at least about 99%, or about 100% of the expanded population
of TILs are TSE
cells. In some aspects, at least about 40% of the expanded population of TILs
are TSE cells. In some
aspects, at least about 50% of the expanded population of TILs are TSE cells.
In some aspects, at
least about 60% of the expanded population of TILs are TSE cells. In some
aspects, at least about
70% of the expanded population of TILs are TSE cells. In some aspects, at
least about 75% of the
expanded population of TILs are TSE cells. In some aspects, at least about 80%
of the expanded
population of TILs are TSE cells. In some aspects, at least about 85% of the
expanded population
of TILs are TSE cells. In some aspects, at least about 90% of the expanded
population of TILs are
TSE cells. In some aspects, at least about 95% of the expanded population of
TILs are TSE cells. In
some aspects, at least about 98% of the expanded population of TILs are TSE
cells. In some aspects,
at least about 99% of the expanded population of TILs are TSE cells. In some
aspects, about 100%
of the expanded population of TILs in the population are TSE cells.
[0490] Some aspects of the present disclosure are directed to a
TIL, which expresses one
or more stem-like markers and one or more effector-like marker. In some
aspects, the TIL
expresses at least two stem-like markers and one or more effector-like
markers. In some aspects,
the TIL expresses at least three stem-like markers and one or more effector-
like markers. In some
aspects, the TIL expresses at least four stem-like markers and one or more
effector-like markers.
In some aspects, the TIL expresses one or more stem-like markers and at least
two effector-like
markers. In some aspects, the stem-like markers are selected from CD45RA+,
CD62L+, CCR7+,
CD27+, CD28+, BACH2+, LEF1+, TCF7+, and any combination thereof. In some
aspects, the
stem-like markers are selected from CD45RA+, CD62L+, CCR7+, TCF7+, and any
combination
thereof, In some aspects, the stem-like markers comprise one or more genes
listed herein as part
of a gene-signature (see supra; see, e.g., Gattinoni, L., et al., Nat Med
17(10): 1290-1297 (2011)
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or Galletti et al. Nat Immunol 21, 1552-1562 (2020)). In some aspects, the
effector-like markers
are selected from pSTAT5+, STAT5+, pSTAT3+, STAT3+, and any combination
thereof In some
aspects, the TIL expresses CD45RA+, STAT5+, and STAT3+. In some aspects, the
TIL expresses
CD62L+, STAT5+, and STAT3+. In some aspects, the TIL expresses TCF7+, STAT5+,
and
STAT3+. In some aspects, the TIL expresses CD45RA+, CD62L+, CCR7+, CD27+,
CD28+,
BACII2 I, LEF1 I, TCF7 I, STAT5 I , and STAT3 I . In some aspects, the TIL
expresses CD45RA I,
CD62L+, CCR7+, CD27+, CD28+, BACH2+, LEF1+, TCF7+, pSTAT5+, STAT5+, pSTAT3+,
and STAT3+. In some aspects, the TIL expresses CD45RA+, CD62L+, CCR7+, CD27+,
CD28+,
BACH2+, LEF1+, TCF7+, CD45R010, pSTAT5+, STAT5+, pSTAT3+, and STAT3+.
[0491] Some aspects of the present disclosure are directed to a
cell composition comprising
a population of TILs, wherein the population of Tits comprises (i) a first sub-
population of TILs
expressing one or more stem-like markers (e.g., stem-like TILs) and (ii) a
second sub-population
of TILs expressing one or more effector-like marker (e.g., effector-like
TILs), wherein the
population of TILs comprises a higher percentage (i.e., the number of stem-
like TILs/the total
number of TILs) of the first sub-population of TILs expressing one or more
stem-like markers, as
compared to a population of TILs cultured in a control media. In some aspects,
the TILs cultured
according to the methods dislosed herein result in these cell compositions. In
some aspects, TILs
cultured according to the methods disclosed herein have increased expression,
e.g., a higher
percentage of TILs that express, GZMB, MHC-II, LAG3, TIGIT, and/or NKG7, and
decreased
expression, e.g., a lower percentage of TILs that express, IL-32. Cells
highest for NKG7 have been
shown to be better killers (Malarkannan et al. 2020 Nat. Immuno.), whereas
cells higher in IL-32
have been shown to have activation-induced cell death (Goda et al., 2006 Int.
Immunol). In some
aspects the TILs with higher expression of GZMB, MHC-II, LAG3, TIGIT, and/or
NKG7 are
CD8+ TILs expressing effector-like markers. In some aspects the TILs with
lower expression of
IL-32 are CD8+ TILs expressing effector-like markers.
[0492] Some aspects of the present disclosure are directed to a
cell composition comprising
TILs expressing one or more stem-like markers and one or more effector-like
marker. Some aspects
of the present disclosure are directed to a population of cells comprising the
TIL, e.g., the TIL
expressing one or more stem-like markers and one or more effector-like marker.
In some aspects,
at least about 40%, at least about 50%, at least about 60%, at least about
70%, at least about 80%,
at least about 85%, at least about 90%, at least about 95%, at least about
99%, or about 100% of
the cell composition comprise Tits expressing one or more stem-like markers
and one or more
effector-like marker.
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[0493] In some aspects, the TIL that expresses one or more stem-
like markers and one or
more effector-like markers is a T stem/effector (TsE) cell. In some aspects,
the TSE cell retains a
less differentiated state (e.g., expreses one or more stem-like markers, is
capable of proliferation,
is capable of differentiation, or any combination thereof) and the cell has
effector function (e.g.,
expresses one or more effector-like markers, is capable of targeting and/or
killing tumor cells, or a
combination thereof). In some aspects, a TsE, cell disclosed herein expresses
CD45RA I, CD62L I,
CCR7+, CD27+, CD28+, BACH2+, LEF1+, TCF7+, pSTAT5+, STAT5+, pSTAT3+, and
STAT3+. In some aspects, a TSE cell disclosed herein expreses CD45RA+, CD62T+,
CCR7+,
CD27+, CD28+, BACH2+, LEF1+, TCF7+, STAT5+, and STAT3+. In some aspects, a TSE
cell
disclosed herein expreses CD45RA+, CD62L+, CCR7+, CD27+, CD28+, BACH2+, LEF1+,
TCF7+, pSTAT5+, STAT5+, pSTAT3+, and STAT3+. In some aspects, a TSE cell
disclosed herein
expreses CD45RA+, CD62L+, CCR7+, CD27+, CD28+, BACH2+, LEF1+, TCF7+, CD45R010
,
pSTAT5+, STAT5+, pSTAT3+, and STAT3+. Some aspects of the present disclosure
are directed
to a population of expanded TILs comprising one or more TSE cell. In some
aspects, at least about
40%, at least about 50%, at least about 60%, at least about 70%, at least
about 80%, at least about
85%, at least about 90%, at least about 95%, at least about 99%, or about 100%
of the expanded
TILs are TSE cells. In some aspects, at least about 40% of the expanded TILs
are TSE cells. In some
aspects, at least about 50% of the expanded Ills are TSE cells. In some
aspects, at least about 60%
of the expanded TILs are TSE cells. In some aspects, at least about 70% of the
expanded TILs are
TSE cells. In some aspects, at least about 75% of the expanded TILs are TSE
cells. In some aspects,
at least about 80% of the expanded TILs are TSE cells. In some aspects, at
least about 85% of the
expanded TILs are TSE cells. In some aspects, at least about 90% of the
expanded TILs are TSE
cells. In some aspects, at least about 95% of the expanded TILs are TSE cells.
In some aspects, at
least about 98% the expanded TILs are TSE cells. In some aspects, at least
about 99% of the
expanded TILs are TSE cells. In some aspects, about 100% the expanded TILs are
TSE cells.
[0494] As further described herein (see, e.g., Example 14), in
some aspects, the
characteristics of a cell (e.g., TILs) can be assessed using tran seri ptome
analysis by comparing the
upregulation and/or downregulation of different sets of genes associated with
stem-like cells (see,
e.g., Table 3, Panel 1-4), exhaustion (see, e.g., Table 3, Panel 5-10), and
metabolic fitness (see,
e.g., Table 3, Panel 11-19).
[0495] In some aspects, the terminally exhausted T cells are
characterized using a TTE-
associated gene set described in Oliveira et al., Nature 596: 119-125 (2021).
In some aspects, the
gene signature for TTE cells comprises one or more or all of the genes
selected from: KRT86,
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RDH10, ACP5, CXCR6, HMOX1, LAYN, CLIC3, HAVCR2, AC243829.4, PRF1, SLC2A8,
CHST12, GALNT2, ENTPD1, LAG3, GZMB, PDCD1, CARD16, CTLA4, SLA2, CD27, RALA,
VCAM1, SYNGR2, NKG7, LSP1, CCL5, RARRES3, CD7, CTSW, MTSS1, PTMS, BATF,
KIR2DL4, AKAP5, CD38, RAB27A, GZMH, IGFLR1, ATP8B4, CD63, HOPX, TNFRSF 18,
ADGRG1, PLPP1, CSFI, TNFSF10, SNAP47, LINC01871, MY01E, ZBED2, AH11, ABI3,
FASLG, TYMP, ZBTB38, CTSB, PLSCR1, AFAP1L2, ITGAE, TNS3, DUSP16, CASP1, CARS,
DUSP5, IFIT1, SLC1A4, GOLIM4, RSAD2, DNPH1, NBL1, ACOT9, ABEID6, OAS1,
SLC27A2, ZBP1, CD200R1, OAS3, CMPK2, 'TNFSF4, POLR1E, CADM1, HELZ2, SYTL2,
AGPAT2, UBE2F, GIMAP6, ZBTB32, RIN3, PLEKFIF 1, CHPF, PACSIN2, ABCB1, SPATS2L,
USP18, TMEM9, KLRC1, MPST.
[0496] In some aspects, the TIL is an engineered TIL. As used
herein, an "engineered" TIL
refers to a TIL that has been manipulated in a way, e.g., according to the
methods discosed herein,
that confers on the TIL one or more physical and/or functional properties that
are not characteristic
of a naturally occurring TIL. For example, in some aspects, an engineered TIL
can be generated
by modifying a TIL to express one or more proteins heterologous to the cell
(e.g., chimeric antigen
receptor or T cell receptor) so that the engineered TIL is not naturally
occurring. In some aspects,
an engineered TIL can be generated by culutirng a TIL in a particular way,
e.g., culturing in
hyperkalemic medium, wherein the resulting engineered TIL has one or more
physical and/or
functional properties that are not shown in naturally occurring cells.
[0497] In some aspects, the cell composition after the initial
culture comprises at least
about 2 x 106, at least about 3 x 106, at least about 4 x 106, at least about
5 x 106, at least about 6 x
106, at least about 7 x 106, at least about 8 x 106, at least about 9 x 106,
or at least about 10 x 106
cells (e.g., TILs). In some aspects, the cell composition after the initial
culture compriss about 2 x
106 to about 10 x 106, e,g., about 2 x 106, about 3 x 106, about 4 x 106,
about 5 x 106, about 6 x 106,
about 7 x 106, about 8 x 106, about 9 x 106, or about 10 x 106, cells (e.g.,
TILs). In some aspects,
the composition after the initial culture comprises about 2 x 106 cells (e.g.,
TILs) to about 3 x 106
cells (e.g., TILs). In some aspects, the composition after the initial culture
comprises about 3 x 106
cells (e.g., TILs) to about 4 x 106 cells (e.g., TILs). In some aspects, the
composition after the initial
culture comprises about 4 x 106 cells (e.g., TILs) to about 5 x 106 cells
(e.g., TILs). In some aspects,
the composition after the initial culture comprises about 5 x 106 cells (e.g.,
TILs) to about 6 x 106
cells (e.g., TILs). In some aspects, the composition after the initial culture
comprises about 6 x 106
cells (e.g., Tits) to about 7 x 106 cells (e.g., TILs). In some aspects, the
composition after the initial
culture comprises about 7 x 106 cells (e.g., TILs) to about 8 x 106 cells
(e.g., TILs). In some aspects,
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the composition after the initial culture comprises about 8 x 106 cells (e.g.,
TILs) to about 9 x 106
cells (e.g., TILs). In some aspects, the composition after the initial culture
comprises about 9 x 106
cells (e.g., TILs) to about 10 x 106 cells (e.g., TILs).
[0498] In some aspects, the cell composition after the second
TIL expansion comprises at
least about 5 x 107, at least about 3 x 107, at least about 4 x 107, at least
about 5 x 107, at least about
6 x 107, at least about 7 x 107, at least about 8 x 107, at least about 9 x
107, at least about 10 x 107,
at least about 11 x 107, at least about 12 x 107, at least about 13 x 107, at
least about 14 x 107, at
least about 15 x 107, at least about 16 x 107, at least about 17 x 107, at
least about 18 x 107, at least
about 19 x 107, or at least about 20 x 107 cells (e.g., TILs), In some
aspects, the cell composition
after the second expansion comprises about 5 x 107 to about 20 x 107, e,g.,
about 5 x 107, about 6
x 107, about 7 x 10, about 8 x 107, about 9 x 107, about 10 x 107, about 11 x
107, about 12 x 107,
about 13 x 107, about 14 x 107, about 15 x 107, about 16 x 107, about 17 x
107, about 18 x 107,
about 19 x 107, or about 20 x 107 cells (e.g., TILs). In some aspects, the
composition after the
second expansion comprises about 5 x 107 to about 6 x 107 cells (e.g., TILs),
about 6 x 107 to about
7 x 107 cells (e.g., TILs), about 7 x 107 to about 8 x 107 cells (e.g-.,
TILs), about 8 x 107 to about 9
x 107 cells (e.g., TILs), about 9 x 107 to about 10 x 107 cells (e.g., TILs),
about 10 x 107 to about
11 x 107 cells (e.g., TILs), about 11 x 107 to about 12 x 107 cells (e.g,
TILs), about 12 x 107 to
about 13 x 107 cells (e.g., TILs), about 13 x 107 to about 14 x 107 cells
(e.g., TILs), about 14 x 107
to about 15 x 107 cells (e.g., TILs), about 15 x 107 to about 16 x 107 cells
(e.g., TILs), about 16 x
107 to about 17 x 107 cells (e.g., TILs), about 17 x 107 to about 18 x 107
cells (e.g., TILs), about
18 x 107 to about 19 x 107 cells (e.g., TILs), or about 19 x 107 to about 20 x
107 cells (e.g., TILs).
In some aspects, the composition after the second expansion comprises about 5
x 107 to about 6 x
107 cells (e.g., TILs). In some aspects, the composition after the second
expansion comprises about
6 x 107 to about 7 x 107 cells (e.g., TILs). In some aspects, the composition
after the second
expansion comprises about 7 x 107 to about 8 x 107 cells (e.g., TILs). In some
aspects, the
composition after the second expansion comprises about 8 x 107 to about 9 x
107 cells (e.g., Tits).
In some aspects, the composition after the second expansion comprises about 9
x 107 to about 10
x 107 cells (e.g., TILs). In some aspects, the composition after the second
expansion comprises
about 10 x 107 to about 11 x 107 cells (e.g., TILs). In some aspects, the
composition after the second
expansion comprises about 11 x 107 to about 12 x 107 cells (e.g., TILs) In
some aspects, the
composition after the second expansion comprises about 12 x 107 to about 13 x
107 cells (e.g.,
TILs). In some aspects, the composition after the second expansion comprises
about 13 x 107 to
about 14 x 107 cells (e.g., TILs). In some aspects, the composition after the
second expansion
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comprises about 14 x 107 to about 15 x 107 cells (e.g., TILs). In some
aspects, the composition
after the second expansion comprises about 15 x 107 to about 16 x 107 cells
(e.g., TILs). In some
aspects, the composition after the second expansion comprises about 16 x 107
to about 17 x 107
cells (e.g., TILs). In some aspects, the composition after the second
expansion comprises about 17
x 107 to about 18 x 107 cells (e.g., TILs). In some aspects, the composition
after the second
expansion comprises about 18 x 107 to about 19 x 107 cells (e.g., TILs). In
some aspects, the
composition after the second expansion comprises about 19 x 107 to about 20 x
107 cells (e.g.,
TILs).
[0499] In some aspects, the cell composition after the final TIL
expansion comprises at
least about 40 x 109, at least about 50 x 109, at least about 60 x 109, at
least about 70 x 109, at least
about 80 x 109, at least about 90 x 109, or at least about 100 x 109 cells
(e.g., TILs). In some aspects,
the cell composition after the final expansion comprises about 40 x 109 to
about 100 x 109, e,g.,
about 40 x 109, about 50 x 109, about 60 x 109, about 70 x 109, about 80 x
109, about 90 x 109, or
about 100 x 109 cells (e.g., TILs). In some aspects, the composition after the
final expansion
comprises about 40 x 109 to about 50 x 109 cells (e.g., TILs), about 50 x 109
to about 60 x 109 cells
(e.g., TILs), about 60 x 109 to about 70 x 109 cells (e.g., TILs), about 70 x
109 to about 80 x 109
cells (e.g, TILs), about 80 x 109 to about 90 x 109 cells (e.g , TILs), or
about 90 x 109 to about 100
x 109 cells (e.g., TILs). In some aspects, the composition after the final
expansion comprises about
40 x 109 to about 50 x 109 cells (e.g., TILs). In some aspects, the
composition after the final
expansion comprises about 50 x 109 to about 60 x 109 cells (e.g., TILs). In
some aspects, the
composition after the final expansion comprises about 60 x 109 to about 70 x
109 cells (e.g., TILs).
In some aspects, the composition after the final expansion comprises about 70
x 109 to about 80 x
109 cells (e.g., TILs). In some aspects, the composition after the final
expansion comprises about
80 x 109 to about 90 x 109 cells (e.g., TILs). In some aspects, the
composition after the final
expansion comprises about 90 x 109 to about 100 x 109 cells (e.g., TILs).
[0500] In some aspects, the cell composition suitable for
administration to a subject
comprises at least about 2 x 109, at least about 3 x 109, at least about 4 x
109, at least about 5 x 109,
at least about 6 x 109, at least about 7 x 109, at least about 8 x 109, at
least about 9 x 109, or at least
about 1 x 1010, or at least about 10 x 1010, or at least about 15 x 1010, or
at least about 20 x 1010, or
at least about 25 x101 , or at least about 30 x 1010 CD8- TILs. In some
aspects, the cell composition
suitable for administration to a subject comprises at least about 2 x 109 CDS+
TILs. In some aspects,
the cell composition suitable for administration to a subject comprises at
least about 5 x 109 CDS+
TILs. In some aspects, the cell composition suitable for administration to a
subject comprises at
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least about 9 x 109 CD8 TILs. In some aspects, the cell composition suitable
for administration to
a subject comprises at least about 1 x 1010 CD8+ TILs. In some aspects, the
cell composition
suitable for administration to a subject comprises at least about 10 x 1010
CD8+ TILs. In some
aspects, the cell composition suitable for administration to a subject
comprises at least about 20 x
1010 CDS+ TILs. In some aspects, the cell composition suitable for
administration to a subject
comprises at least about 30 x 1010 CD8+ TILs.
[0501] In some aspects, the methods disclosed herein yield a
composition comprising TILs
that are at least about 80%, at least about 85%, at least about 90%, at least
about 94%, at least about
95%, at least about 96%, at least about 97%, at least about 98%, or at least
about 99% viable.
IV. Methods of Treatment
[0502] Some aspects of the present disclosure are directed to a
population of TILs cultured
according to any of the methods disclosed herein. In some aspects, the TILs
are tumor-infiltrating
T cells. In some aspects, the TILs comprise both CD4+ T cells and CD8+ T
cells. In some aspects,
the TILs comprise CDS+ T cells. In some aspects, the TILs are enriched for
tumor reactive (e.g.,
tumor specific) TILs. In some aspects, the TILs are enriched for stem-like
TILs.
[0503] In some aspects, the composition comprising the
population of TILs is administered
to a subject in need thereof In some aspects the TILs prepared using the
methods disclose herein
are administered to a subject to treat a cancer, e.g., a tumor. In some
aspects, the method of treating
comprises administering to the subject an effective amount of a TIL
composition of the disclosure,
e.g., a composition comprising a population of TILs prepared according to the
methods disclosed
herein, e.g., a population of TILs enriched for CD8+ TILs, tumor-specific
TILs, and/or stem-like
TILs. Non-limiting examples of tumors that can be treated are further
described elsewhere in the
present disclosure. In some aspects, the tumor is refractory to a checkpoint
inhibitor. Non-limiting
examples of checkpoint inhibitors include a PD-1 antagonist (e.g., anti-PD-1
antibody or anti-PD-
Li antibody), a CTLA-4 antagonist (e.g., anti-CTLA-4 antibody), a TIIVI3
antagonist (e.g., anti-
TIM3 antibody), a GITR antagonist (e.g., anti-GITR antibody), a KIR antagonist
(e.g., anti-KIR
antibody), a LAG3 antagonist (e.g., anti-LAG3 antibody), or any combination
thereof In some
aspects, the tumor is relapsed. In some aspects, the tumor is metastatic. In
some aspects, the tumor
that can be treated with the present disclosure is refractoiy to a checkpoint
inhibitor and relapsed.
In some aspects, the tumor is refractory to a checkpoint inhibitor and
metastatic. In some aspects,
the tumor is refractory to a checkpoint inhibitor, relapsed, and metastatic.
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[0504] The present disclosure also provides a method of
stimulating a T cell-mediated
immune response to a target cell population or tissue in a subject, comprising
administering an
effective amount of a TIL composition of the disclosure, e.g., a population of
TILs prepared
according to the methods disclosed herein, e.g., a population of TILs enriched
for CDS+ TILs.
[0505] In some aspects, the population of TILs administered in
the cell composition of the
disclosure comprises autologous TILs.
[0506] In some aspects, the method comprises administering at
least about 1 x 104, at least
about 5 x 104, at least about 1 x 105, at least about 5 x 105, at least about
1 x 106, at least about 2 x
106, at least about 3 x 106, at least about 4 x 106, at least about 5 x 106,
at least about 6 x 106, at
least about 7 x 106, at least about 8 x 106, at least about 9 x 106, at least
about 1 x 107, at least about
x 107, at least about 1 x 108 TILs to the subject. In some aspects, the method
comprises
administering at least about 1 x 104, at least about 5 x 104, at least about 1
x 105, at least about 5 x
105, at least about 1 x 106, at least about 2 x 106, at least about 3 x 106,
at least about 4 x 106, at
least about 5 x 106, at least about 6 x 106, at least about 7 x 106, at least
about 8 x 106, at least about
9 x 106, at least about 1 x 107, at least about 5 x 107, at least about 1 x
108, at least about 1 x 109,
at least about 2 x 109, at least about 3 x 109, at least about 4 x 109, at
least about 5 x 109, at least
about 6 x 109, at least about 7 x 109, at least about 8 x 109, at least about
9 x 109, at least about 1 x
1010, at least about 10 x 1010, at least about 15 x 1010, at least about 20 x
1010, at least about 25
x101 , or at least about 30 x 1010 cells to the subject. In some aspects, the
cells are TILs. In some
aspects, the TILs are CD8+ TILs.
[0507] In some aspects, the method comprises administering at
least about 5 x 109, 10 x
109, at least about 20 x 109, at least about 30 x 109, at least about 40 x
109, at least about 50 x 109,
at least about 60 x 109, at least about 70 x 109, at least about 80 x 109, at
least about 90 x 109, or at
least about 100 x 109 cells (e.g., TILs). In some aspects, the method
comprises administering about
x 109 to about 100 x 109, e,g., about 10 x 109, about 20 x 109, about 30 x
109, about 40 x 109,
about 50 x 109, about 60 x 109, about 70 x 109, about 80 x 109, about 90 x
109, or about 100 x 109,
cells (e.g., TILs). In some aspects, the method comprises administering about
10 x 109 to about 20
x 109 cells (e.g., TILs), about 20 x 109 to about 30 x 109 cells (e.g., TILs),
about 30 x 109 to about
40 x 109 cells (e.g., TILs), about 40 x 109 to about 50 x 109 cells (e.g.,
TILs), about 50 x 109 to
about 60 x 109 cells (e.g., TILs), about 60 x 109 to about 70 x 109 cells
(e.g., TILs), about 70 x 109
to about 80 x 109 cells (e.g., TILs), about 80 x 109 to about 90 x 109 cells
(e.g., TILs), or about 90
x 109 to about 100 x 109 cells (e.g., Tits). In some aspects, the method
comprises administering
about 10 x 109 to about 20 x 109 cells (e.g., TILs). In some aspects, the
method comprises
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administering about 20 x 109 to about 30 x 109 cells (e.g., TILs). In some
aspects, the method
comprises administering about 30 x 109 to about 40 x 109 cells (e.g., TILs).
In some aspects, the
method comprises administering about 40 x 109to about 50 x 109 cells (e.g.,
TILs). In some aspects,
the method comprises administering about 50 x 109 to about 60 x 109 cells
(e.g., TILs). In some
aspects, the method comprises administering about 60 x 109 to about 70 x 109
cells (e.g., TILs). hi
some aspects, the method comprises administering about 70 x 109 to about 80 x
109 cells (e.g.,
TILs). In some aspects, the method comprises administering about 80 x 109 to
about 90 x 109 cells
(e.g., Tits). In some aspects, the method comprises administering about 90 x
109 to about 100 x
109 cells (e.g., TILs). In some aspects, the TILs are CD8+ TILs. In some
aspects, the method
comprises administering about 5 x 109 to about 8 x 109 cells (e.g , TILs). In
some aspects, the
method comprises administering about 10 x 109to about 30 x 109 cells (e.g.,
TILs). In some aspects,
the method comprises administering about 10 x 109 to about 40 x 109 cells
(e.g., TILs). In some
aspects, the method comprises administering about 40 x 109 to about 80 x 109
cells (e.g., TILs) In
some aspects, the method comprises administering about 50 x 109 to about 80 x
109 cells (e.g.,
TILs). In some aspects, the method comprises administering about 90 x 109 to
about 110 x 109
cells (e.g., TILs).
[0508] In some aspects, the TILs are administered at a ratio of
TILs to tumor cells of at
least about 2:1, at least about 2.5:1, at least about 3:1, at least about
3.5:1, or at least about 4:1. In
some aspects, the TILs are administered at a ratio of TILs to tumor cells of
at least about 2:1. In
some aspects, the TILs are administered at a ratio of TILs to tumor cells of
at least about 2.5:1. In
some aspects, the TILs are administered at a ratio of TILs to tumor cells of
at least about 3:1. In
some aspects, the TILs are administered at a ratio of TILs to tumor cells of
at least about 3.5:1. In
some aspects, the TILs are administered at a ratio of TILs to tumor cells of
at least about 4:1.
[0509] In some aspects, administering the cell composition of
the disclosure (e.g.,
comprising a population of TILs prepared according to the methods disclosed
herein (e.g., enriched
for CDS+ TILs, tumor-specific TILs, and/or stem-like TILs)) reduces a tumor
volume in the subject
compared to a reference tumor volume. In some aspects, the reference tumor
volume is the tumor
volume in the subject prior to the administration of the engineered cell. In
further aspects, the
reference tumor volume is the tumor volume in a corresponding subject that did
not receive the
administration. In some aspects, the tumor volume in the subject is reduced by
at least about 5%,
at least about 10%, at least about 15%, at least about 30%, at least about
40%, at least about 50%,
at least about 60%, at least about 70%, at least about 80%, at least about
90%, or at least about
100% after the administration compared to the reference tumor volume.
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[0510]
In some aspects, treating a tumor comprises reducing a tumor weight
in the subject.
In some aspects, administering the cell composition of the disclosure (e.g.,
comprising a population
of TILs prepared according to the methods disclosed herein (e.g., enriched for
CD8+ TILs, tumor-
specific TILs, and/or stem-like TILs)) can reduce the tumor weight in a
subject when administered
to the subject. In some aspects, the tumor weight is reduced by at least about
5%, at least about
10%, at least about 15%, at least about 30%, at least about 40%, at least
about 50%, at least about
60%, at least about 70%, at least about 80%, at least about 90%, or at least
about 100% after the
administration compared to a reference tumor weight. In some aspects, the
reference tumor weight
is the tumor weight in the subject prior to the administration of the cell
composition of the
disclosure. In further aspects, the reference tumor weight is the tumor weight
in a corresponding
subject that did not receive the administration_
[0511]
In some aspects, administering the cell composition of the disclosure
(e.g.,
comprising a population of TILs prepared according to the methods disclosed
herein (e.g., enriched
for CD8+ TILs, tumor-specific TILs, and/or stem-like TILs)) to a subject,
e.g., suffering from a
tumor, can increase the number and/or percentage of TILs
CD8+ TILs) in a tumor and/or a
tumor microenvironment (TME) of the subject. In some aspects, the number
and/or percentage of
TILs (e.g., CD8+ TILs) in a tumor and/or TME is increased by at least about
5%, at least about
10%, at least about 15%, at least about 20%, at least about 25%, at least
about 30%, at least about
35%, at least about 40%, at least about 45%, at least about 50%, at least
about 55%, at least about
60%, at least about 65%, at least about 70%, at least about 75%, at least
about 80%, at least about
85%, at least about 90%, at least about 95%, at least about 100%, at least
about 110%, at least
about 120%, at least about 130%, at least about 140%, at least about 150%, at
least about 160%,
at least about 170%, at least about 180%, at least about 190%, at least about
200%, at least about
210%, at least 220%, at least about 230%, at least about 240%, at least about
250%, at least about
260%, at least about 270%, at least about 280%, at least about 290%, or at
least about 300% or
more compared to a reference (e.g., corresponding value in a subject that did
not receive the cell
composition of the present disclosure or the same subject prior to the
administration of the cell
composition of the present disclosure).
[0512]
In some aspects, administering the cell composition of the disclosure
(e.g.,
comprising a population of TILs prepared according to the methods disclosed
herein (e.g., enriched
for CDS+ TILs, tumor-specific TILs, and/or stem-like TILs)) to a subject,
e.g., suffering from a
tumor, can increase the duration of an immune response in a subject relative
to the duration of an
immune response in a subject administered a similar cell therapy comprising
cells prepared
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according to conventional methods, e.g., cultured in a medium not comprising a
potassium ion
concentration of at least about 40 mM to at least about 90 mM, e.g., at least
50 mM. In some
aspects, the duration of the immune response is increased by at least about
10%, at least about
20%, at least about 30%, at least about 40%, at least about 50%, at least
about 75%, at least about
100%, at least about 150%, at least about 200%, at least about 300%, at least
about 400%, at least
about 500%, or at least about 1000% or more compared to a reference (e.g., a
subject administered
a similar cell therapy comprising cells prepared according to conventional
methods, e.g., cultured
in a medium not comprising a potassium ion concentration of at least 50 mM).
In some aspects,
the duration of the immune response is increased by at least about 2-fold, at
least about 3-fold, at
least about 4-fold, at least about 5-fold, at least about 6-fold, at least
about 7-fold, at least about 8-
fold, at least about 9-fold, or at least about 10-fold or more compared to a
reference (e.g., a subject
administered a similar cell therapy comprising cells prepared according to
conventional methods,
e.g., cultured in a medium not comprising a potassium ion concentration of at
least about 40 mM
to at least about 90 mM, e.g., at least 50 mM).
[0513] In addition to the above, administering the cell
composition of the disclosure (e.g.,
comprising a population of TILs prepared according to the methods disclosed
herein (e.g., enriched
for CD8+ TILs, tumor-specific TILs, and/or stem-like TILs)) can have other
effects which are
conducive for the treatment of a tumor.
[0514] As described herein, the cell composition of the
disclosure (e.g., comprising a
population of TILs prepared according to the methods disclosed herein (e.g.,
enriched for CD8+
TILs, stem-like TILs, tumor-specific TILs, and/or naïve TILs)) can be used to
treat variety of
cancer types, e.g., a tumor derived from a cancer comprising a breast cancer,
head and neck cancer,
uterine cancer, brain cancer, skin cancer, renal cancer, lung cancer,
colorectal cancer, prostate
cancer, liver cancer, bladder cancer, kidney cancer, pancreatic cancer,
thyroid cancer, esophageal
cancer, eye cancer, stomach (gastric) cancer, gastrointestinal cancer, ovarian
cancer, carcinoma,
sarcoma, leukemia, lymphoma, myeloma, or a combination thereof. In some
aspects, the cancer
comprises a solid tumor. In some aspects, the cancer comprises a solid tumor
derived from a
melanoma, a colon cancer, a lung cancer, a cervical cancer, a gastrointestinal
cancer, a breast
cancer, a prostate cancer, a liver cancer, bone cancer, a pancreatic cancer, a
small cell carcinoma
of the head and neck, lung squamous cell carcinoma, lung adenocarcinoma,
pancreatic
adenocarcinoma, head and neck squamous cell carcinoma, testicular germ cell
tumors, stomach
adenocarcinoma, skin cutaneous melanoma, mesothelioma, kidney renal clear cell
carcinoma,
cervical squamous cell carcinoma and endocervical adenocarcinoma, esophageal
carcinoma,
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bladder urothelial carcinoma, breast invasive carcinoma, kidney renal
papillary cell carcinoma,
colon adenocarcinoma, or any combination thereof. In some aspects, the cancer
comprises a
melanoma. In some aspects, the cancer comprises colorectal cancer. In some
aspects, the cancer
comprises a colon cancer. In some aspects, the cancer comprises pancreatic
cancer. In some
aspects, the cancer comprises head and neck cancer. In some aspects, the
cancer comprises cervical
cancer. In some aspects, the cancer comprises ovarian cancer. In some aspects,
the cancer
comprises a lung cancer. In some aspects, the cancer comprises a
gastrointestinal cancer. In some
aspects, the cancer comprises a breast cancer. In some aspects, the cancer
comprises a prostate
cancer. In some aspects, the cancer comprises a liver cancer. In some aspects,
the cancer comprises
bone cancer. In some aspects, the cancer comprises a small cell carcinoma of
the head and neck.
In some aspects, the cancer comprises lung squamous cell carcinoma. In some
aspects, the cancer
comprises lung adenocarcinoma. In some aspects, the cancer comprises
pancreatic
adenocarcinoma. In some aspects, the cancer comprises head and neck squamous
cell carcinoma.
In some aspects, the cancer comprises a testicular germ cell tumor. In some
aspects, the cancer
comprises stomach adenocarcinoma. In some aspects, the cancer comprises skin
cutaneous
melanoma. In some aspects, the cancer comprises mesothelioma. In some aspects,
the cancer
comprises kidney renal clear cell carcinoma. In some aspects, the cancer
comprises cervical
squamous cell carcinoma. In some aspects, the cancer comprises endocervical
adenocarcinoma. In
some aspects, the cancer comprises esophageal carcinoma. In some aspects, the
cancer comprises
bladder urothelial carcinoma. In some aspects, the cancer comprises breast
invasive carcinoma. In
some aspects, the cancer comprises kidney renal papillary cell carcinoma. In
some aspects, the
cancer comprises colon adenocarcinoma. In some aspects, the cancer comprises a
uterine cancer.
In some aspects, the cancer comprises a brain. In some aspects, the cancer
comprises a thyroid
cancer. In some aspects, the cancer comprises an esophageal cancer. In some
aspects, the cancer
comprises an eye cancer. In some aspects, the cancer comprises a stomach
(gastric) cancer. In some
aspects, the cancer comprises a gastrointestinal cancer. In some aspects, the
cancer comprises a
sarcoma. In some aspects, the cancer comprises a leukemia In some aspects, the
cancer comprises
a lymphoma. In some aspects, the cancer comprises a myeloma.
[0515] As such, some aspects of the present disclosre are
directed to methods of treating a
melanoma in a subject in need thereof, comprising administering to the subject
a cell composition
disclosed herein. Some aspects of the present disclosure are directed to
methods of treating a
colorectal cancer in a subject in need thereof, comprising administering to
the subject a cell
composition disclosed herein. Some aspects of the present disclosure are
directed to methods of
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treating a colon cancer in a subject in need thereof, comprising administering
to the subject a cell
composition disclosed herein. Some aspects of the present disclosure are
directed to methods of
treating pancreatic cancer in a subject in need thereof, comprising
administering to the subject a
cell composition disclosed herein. Some aspects of the present disclosure are
directed to methods
of treating head and neck cancer in a subject in need thereof, comprising
administering to the
subject a cell composition disclosed herein. Some aspects of the present
disclosure are directed to
methods of treating cervical cancer in a subject in need thereof, comprising
administering to the
subject a cell composition disclosed herein. Some aspects of the present
disclosure are directed to
methods of treating ovarian cancer in a subject in need thereof, comprising
administering to the
subject a cell composition disclosed herein. Some aspects of the present
disclosure are directed to
methods of treating a lung cancer in a subject in need thereof, comprising
administering to the
subject a cell composition disclosed herein. Some aspects of the present
disclosure are directed to
methods of treating a gastrointestinal cancer in a subject in need thereof,
comprising administering
to the subject a cell composition disclosed herein. Some aspects of the
present disclosure are
directed to methods of treating a breast cancer in a subject in need thereof,
comprising
administering to the subject a cell composition disclosed herein. Some aspects
of the present
disclosure are directed to methods of treating a prostate cancer in a subject
in need thereof,
comprising administering to the subject a cell composition disclosed herein.
Some aspects of the
present disclosure are directed to methods of treating a liver cancer in a
subject in need thereof,
comprising administering to the subject a cell composition disclosed herein.
Some aspects of the
present disclosure are directed to methods of treating bone cancer in a
subject in need thereof,
comprising administering to the subject a cell composition disclosed herein.
Some aspects of the
present disclosure are directed to methods of treating a small cell carcinoma
of the head and neck
in a subject in need thereof, comprising administering to the subject a cell
composition disclosed
herein. Some aspects of the present disclosure are directed to methods of
treating lung squamous
cell carcinoma in a subject in need thereof, comprising administering to the
subject a cell
composition disclosed herein Some aspects of the present disclosure are
directed to methods of
treating lung adenocarcinoma in a subject in need thereof, comprising
administering to the subject
a cell composition disclosed herein. Some aspects of the present disclosure
are directed to methods
of treating pancreatic adenocarcinoma in a subject in need thereof, comprising
administering to the
subject a cell composition disclosed herein. Some aspects of the present
disclosure are directed to
methods of treating head and neck squamous cell carcinoma in a subject in need
thereof,
comprising administering to the subject a cell composition disclosed herein.
Some aspects of the
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present disclosure are directed to methods of treating a testicular germ cell
tumor in a subject in
need thereof, comprising administering to the subject a cell composition
disclosed herein. Some
aspects of the present disclosure are directed to methods of treating stomach
adenocarcinoma in a
subject in need thereof, comprising administering to the subject a cell
composition disclosed
herein. Some aspects of the present disclosure are directed to methods of
treating skin cutaneous
melanoma in a subject in need thereof, comprising administering to the subject
a cell composition
disclosed herein. Some aspects of the present disclosure are directed to
methods of treating
mesothelioma in a subject in need thereof, comprising administering to the
subject a cell
composition disclosed herein Some aspects of the present disclosure are
directed to methods of
treating kidney renal clear cell carcinoma in a subject in need thereof,
comprising administering to
the subject a cell composition disclosed herein. Some aspects of the present
disclosure are directed
to methods of treating cervical squamous cell carcinoma in a subject in need
thereof, comprising
administering to the subject a cell composition disclosed herein. Some aspects
of the present
disclosure are directed to methods of treating endocervical adenocarcinoma in
a subject in need
thereof, comprising administering to the subject a cell composition disclosed
herein. Some aspects
of the present disclosure are directed to methods of treating esophageal
carcinoma in a subject in
need thereof, comprising administering to the subject a cell composition
disclosed herein. Some
aspects of the present disclosure are directed to methods of treating bladder
urothelial carcinoma
in a subject in need thereof, comprising administering to the subject a cell
composition disclosed
herein. Some aspects of the present disclosure are directed to methods of
treating breast invasive
carcinoma in a subject in need thereof, comprising administering to the
subject a cell composition
disclosed herein. Some aspects of the present disclosure are directed to
methods of treating kidney
renal papillary cell carcinoma in a subject in need thereof, comprising
administering to the subject
a cell composition disclosed herein. Some aspects of the present disclosure
are directed to methods
of treating colon adenocarcinoma in a subject in need thereof, comprising
administering to the
subject a cell composition disclosed herein. Some aspects of the present
disclosure are directed to
methods of treating a uterine cancer in a subject in need thereof, comprising
administering to the
subject a cell composition disclosed herein. Some aspects of the present
disclosure are directed to
methods of treating a brain tumor in a subject in need thereof, comprising
administering to the
subject a cell composition disclosed herein. Some aspects of the present
disclosure are directed to
methods of treating an esophageal cancer in a subject in need thereof,
comprising administering to
the subject a cell composition disclosed herein. Some aspects of the present
disclosure are directed
to methods of treating a thyroid cancer in a subject in need thereof,
comprising administering to
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the subject a cell composition disclosed herein. Some aspects of the present
disclosure are directed
to methods of treating an eye cancer in a subject in need thereof, comprising
administering to the
subject a cell composition disclosed herein. Some aspects of the present
disclosure are directed to
methods of treating a stomach (gastric) cancer in a subject in need thereof,
comprising
administering to the subject a cell composition disclosed herein. Some aspects
of the present
disclosure are directed to methods of treating a gastrointestinal cancer in a
subject in need thereof,
comprising administering to the subject a cell composition disclosed herein.
Some aspects of the
present disclosure are directed to methods of treating a sarcoma in a subject
in need thereof,
comprising administering to the subject a cell composition disclosed herein.
Some aspects of the
present disclosure are directed to methods of treating a leukemia in a subject
in need thereof,
comprising administering to the subject a cell composition disclosed herein.
Some aspects of the
present disclosure are directed to methods of treating a lymphoma in a subject
in need thereof,
comprising administering to the subject a cell composition disclosed herein.
Some aspects of the
present disclosure are directed to methods of treating a myeloma in a subject
in need thereof,
comprising administering to the subject a cell composition disclosed herein.
[0516] In some aspects, the cell composition of the disclosure
(e.g., comprising a
population of TILs prepared according to the methods disclosed herein (e.g.,
enriched for CD8+
TILs, stem-like TILs, tumor-specific TILs, and/or naïve TILs)) can be used in
combination with
other therapeutic agents (e.g., anti-cancer agents and/or immunomodulating
agents). Accordingly,
in some aspects, a method of treating a tumor disclosed herein comprises
administering the cell
composition of the disclosure in combination with one or more additional
therapeutic agents.
[0517] In some aspects, the cell composition of the disclosure
(e.g., comprising a
population of TILs prepared according to the methods disclosed herein (e.g.,
enriched for CD8+
TILs, stem-like TILs, tumor-specific TILs, and/or naïve TILs)) can be used in
combination with
one or more anti-cancer agents, such that multiple elements of the immune
pathway can be
targeted. In some aspects, an anti-cancer agent comprises an immune checkpoint
inhibitor (i.e.,
blocks signaling through the particular immune checkpoint pathway).
[0518] Non-limiting examples of immune checkpoint inhibitors
that can be used in the
present methods comprise a CTLA-4 antagonist (e.g., anti-CTLA-4 antibody), PD1
antagonist
(e.g., anti -PD1 antibody, anti-PD-Li antibody), TIM-3 antagonist (e.g., anti-
TIM-3 antibody), or
combinations thereof. Non-limiting examples of such antagonists are provided
elsewhere in the
present disclosure. In some aspects, the checkpoint inhibitor is a PD1
antagonist. In some aspects,
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the checkpoint inhibitor is an anti-PD1 antibody. A comprehensive and non-
limiting list of
combination treatment is disclosed in detail elsewhere in this application.
[0519] In some aspects, the cell composition of the disclosure
(e.g., comprising a
population of TILs prepared according to the methods disclosed herein (e.g.,
enriched for CDS+
TILs, stem-like TILs, tumor-specific TILs, and/or naive TILs)) is administered
to the subject prior
to or after the administration of the additional therapeutic agent. In other
aspects, the cell
composition of the disclosure (e.g., comprising a population of TILs prepared
according to the
methods disclosed herein (e.g., enriched for CDS+ TILs, stem-like TILs, tumor-
specific TILs,
and/or naive TILs)) is administered to the subject concurrently with the
additional therapeutic
agent. In some aspects, the cell composition of the disclosure (e.g.,
comprising a population of
TILs prepared according to the methods disclosed herein (e.g., enriched for
CD8+ TILs, stem-like
TILs, tumor-specific TILs, and/or naïve TILs)) and the additional therapeutic
agent can be
administered concurrently as a single composition in a pharmaceutically
acceptable carrier. In
other aspects, the cell composition of the disclosure (e.g., comprising a
population of TILs prepared
according to the methods disclosed herein (e.g., enriched for CDS+ TILs, stem-
like TILs, tumor-
specific TILs, and/or naive TILs)) and the additional therapeutic agent are
administered
concurrently as separate compositions.
[0520] In some aspects, the subject is a nonhuman animal such as
a rat or a mouse. In some
aspects, the subject is a human.
[0521] In some aspects, a cell composition disclosed herein
(e.g., comprising a population
of TILs prepared according to the methods disclosed herein (e.g., enriched for
CD8+ TILs, stem-
like TILs, tumor-specific TILs, and/or naive TILs)) can be used in combination
with other
therapeutic agents (e.g., anti-cancer agents and/or immunomodulating agents).
Accordingly, in
some aspects, a method of treating a tumor disclosed herein comprises
administering a cell
composition of the present disclosure (e.g., comprising a population of TILs
prepared according to
the methods disclosed herein (e.g., enriched for CDS+ TILs, stem-like Ins,
tumor-specific TILs,
and/or naive TILs)) in combination with one or more additional therapeutic
agents to a subject.
Such agents can include, for example, chemotherapeutic drug, targeted anti-
cancer therapy,
oncolytic drug, cytotoxic agent, immune-based therapy, cytokine, surgical
procedure, radiation
procedure, activator of a costimulatory molecule, immune checkpoint inhibitor,
a vaccine, a
cellular i mmunotherapy, or any combination thereof.
[0522] In some aspects, a cell composition disclosed herein
(e.g., comprising a population
of TILs prepared according to the methods disclosed herein (e.g., enriched for
CD8+ TILs, stem-
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like TILs, tumor-specific TILs, and/or naive TILs)) can be used in combination
with a standard of
care treatment (e.g., surgery, radiation, and chemotherapy). Methods described
herein can also be
used as a maintenance therapy, e.g., a therapy that is intended to prevent the
occurrence or
recurrence of tumors.
[0523] In some aspects, a cell composition of the present
disclosure (e.g., comprising a
population of TILs prepared according to the methods disclosed herein (e.g.,
enriched for CD8+
TILs, stem-like TILs, tumor-specific TILs, and/or naïve TILs)) can be used in
combination with
one or more anti-cancer agents, such that multiple elements of the immune
pathway can be
targeted. Non-limiting of such combinations include: a therapy that enhances
tumor antigen
presentation (e.g., dendritic cell vaccine, GM-CSF secreting cellular
vaccines, CpG
oligonucleotides, imiquimod); a therapy that inhibits negative immune
regulation e.g., by
inhibiting CTLA-4 and/or PD1/PD-L1/PD-L2 pathway and/or depleting or blocking
Tregs or other
immune suppressing cells (e.g., myeloid-derived suppressor cells); a therapy
that stimulates
positive immune regulation, e.g., with agonists that stimulate the CD-137, OX-
40, and/or CD40 or
GITR pathway and/or stimulate T cell effector function; a therapy that
increases systemically the
frequency of anti-tumor T cells; a therapy that depletes or inhibits Tregs,
such as Tregs in the
tumor, e.g., using an antagonist of CD25 (e.g., daclizumab) or by ex vivo anti-
CD25 bead depletion;
a therapy that impacts the function of suppressor myeloid cells in the tumor;
a therapy that enhances
immunogenicity of tumor cells (e.g., anthracyclines); an additional adoptive T
cell or NK cell
transfer including genetically engineered cells, e.g., cells engineered to
express a chimeric antigen
receptor (CAR-T therapy); a therapy that inhibits a metabolic enzyme such as
indoleamine
dioxigenase (IDO), dioxigenase, arginase, or nitric oxide synthetase; a
therapy that
reverses/prevents T cell anergy or exhaustion; a therapy that triggers an
innate immune activation
and/or inflammation at a tumor site; administration of immune stimulatory
cytokines; blocking of
immuno repressive cytokines; or any combination thereof
[0524] In some aspects, an anti-cancer agent comprises an immune
checkpoint inhibitor
(i.e., blocks signaling through the particular immune checkpoint pathway). Non-
limiting examples
of immune checkpoint inhibitors that can be used in the present methods
comprise a CTLA-4
antagonist (e.g., anti-CTLA-4 antibody), PD1 antagonist (e.g., anti-PD1
antibody, anti-PD-Li
antibody), TIM-3 antagonist (e.g., anti-TIM-3 antibody), or combinations
thereof. Non-limiting
examples of such immune checkpoint inhibitors include the following: anti-PD1
antibody (e.g.,
nivolumab (OPDIV0'), pembrolizumab (KEYTRUIDA ; 1V1K-3475), pidilizumab (CT-
011),
PDR001, MEDI0680 (AMP-514), TSR-042, REGN2810, JS001, AMP-224 (GSK-2661380),
PF-
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06801591, BGB-A317, BI 754091, SHR-1210, and combinations thereof); anti-PD-Li
antibody
(e.g., atezolizumab (TECENTRIQc); RG7446; MPDL3280A; R05541267), durvalumab
(MEDI4736, IMFINZI*), BMS-936559, avelumab (BAVENCIO
LY3300054, CX-072
(Proclaim-CX-072), FAZ053, KN035, MDX-1105, and combinations thereof); and
anti-CTLA-4
antibody (e.g., ipilimumab (YERVOY*), tremelimumab (ticilimumab; CP-675,206),
AGEN-1884,
ATOR-1015, and combinations thereof). Additional description of immune
checkpoint inhibitors
that are useful for the present disclosure are provided below.
[0525]
In some aspects, the cell composition of the present disclosure
(e.g., comprising a
population of TILs prepared according to the methods disclosed herein (e.g.,
enriched for CD8+
TILs, stem-like TILs, tumor-specific TILs, and/or naive TILs)) is administered
to a subject in
combination with a PD1 antagonist. In some aspects, the cell composition of
the present disclosure
(e.g., comprising a population of TILs prepared according to the methods
disclosed herein (e.g.,
enriched for CD8+ TILs, stem-like TILs, tumor-specific TILs, and/or naive
TILs)) is administered
to a subject in combination with an anti-PD1 antibody. In some aspects, the
cell composition of
the present disclosure (e.g., comprising a population of TILs prepared
according to the methods
disclosed herein (e.g., enriched for CD8 TILs, stem-like TILs, tumor-specific
TILs, and/or naive
TILs)) is administered to a subject in combination with nivolumab. In some
aspects, the cell
composition of the present disclosure (e.g., comprising a population of TILs
prepared according to
the methods disclosed herein (e.g., enriched for CDS+ TILs, stem-like TILs,
tumor-specific TILs,
and/or naive TILs)) is administered to a subject in combination with
pembrolizumab.
[0526]
In some aspects, the cell composition of the present disclosure
(e.g., comprising a
population of TILs prepared according to the methods disclosed herein (e.g.,
enriched for CD8+
TILs, stem-like TILs, tumor-specific TILs, and/or naive TILs)) is administered
to a subject in
combination with a PD-L1 antagonist. In some aspects, the cell composition of
the present
disclosure (e.g., comprising a population of TILs prepared according to the
methods disclosed
herein (e.g., enriched for CDS+ TILs, stem-like TILs, tumor-specific TILs,
and/or naive TILs)) is
administered to a subject in combination with an anti-PD-L1 antibody. In some
aspects, the cell
composition of the present disclosure (e.g., comprising a population of TILs
prepared according to
the methods disclosed herein (e.g., enriched for CDS+ TILs, tumor-specific
TILs, and/or naive
TILs)) is administered to a subject in combination with atezolizumab. In some
aspects, the cell
composition of the present disclosure (e.g., comprising a population of TILs
prepared according to
the methods disclosed herein (e.g., enriched for CDS+ TILs, tumor-specific
TILs, and/or naive
TILs)) is administered to a subject in combination with durvalumab. In some
aspects, the cell
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composition of the present disclosure (e.g., comprising a population of TILs
prepared according to
the methods disclosed herein (e.g., enriched for CDS+ TILs, tumor-specific
TILs, and/or naïve
TILs)) is administered to a subject in combination with avelumab.
[0527] In some aspects, the cell composition of the present
disclosure (e.g., comprising a
population of TILs prepared according to the methods disclosed herein (e.g.,
enriched for CD8+
TILs, stem-like TILs, tumor-specific TILs, and/or naïve TILs)) is administered
to a subject in
combination with a CTLA-4 antagonist. In some aspects, the cell composition of
the present
disclosure (e.g., comprising a population of TILs prepared according to the
methods disclosed
herein (e.g., enriched for CD8+ TILs, stem-like TILs, tumor-specific TILs,
and/or naïve TILs)) is
administered to a subject in combination with an anti- CTLA-4 antibody. In
some aspects, the cell
composition of the present disclosure (e.g., comprising a population of TILs
prepared according to
the methods disclosed herein (e.g., enriched for CDS+ TILs, stem-like TILs,
tumor-specific TILs,
and/or naïve TILs)) is administered to a subject in combination with
ipilimumab. In some aspects,
the cell composition of the present disclosure (e.g., comprising a population
of TILs prepared
according to the methods disclosed herein (e.g., enriched for CDS+ TILs, stem-
like TILs, tumor-
specific TILs, and/or naïve TILs)) is administered to a subject in combination
with tremelimumab.
[0528] In some aspects, an anti-cancer agent comprises an immune
checkpoint activator
(i.e., promotes signaling through the particular immune checkpoint pathway).
In some aspects,
immune checkpoint activator comprises 0X40 agonist (e.g., anti-0X40 antibody),
LAG-3 agonist
(e.g. anti-LAG-3 antibody), 4-1BB (CD137) agonist (e.g., anti-CD137 antibody),
GITR agonist
(e.g., anti-GITR antibody), TIM3 agonist (e.g., anti-TIM3 antibody), or
combinations thereof In
some aspects, the additional therapeutic agent comprises a cytokine. In some
aspects, the cytokine
comprises IL-2, IL-21, IL-7, IL-15, or any combination thereof.
[0529] In some aspects, a cell composition disclosed herein
(e.g., comprising a population
of TILs prepared according to the methods disclosed herein (e.g., enriched for
CD8+ TILs, stem-
like TILs, tumor-specific TILs, and/or naive TILs)) is administered to the
subject prior to or after
the administration of the additional therapeutic agent. In other aspects, cell
composition disclosed
herein (e.g., comprising a population of TILs prepared according to the
methods disclosed herein
(e.g., enriched for CD8+ TILs, stem-like TILs, tumor-specific TILs, and/or
naïve TILs)) is
administered to the subject concurrently with the additional therapeutic
agent. In some aspects, the
cell composition disclosed herein (e.g., comprising a population of TILs
prepared according to the
methods disclosed herein (e.g., enriched for CDS+ TILs, stem-like TILs, tumor-
specific TILs,
and/or naïve TILs)) and the additional therapeutic agent can be administered
concurrently as a
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single composition in a pharmaceutically acceptable carrier. In other aspects,
the cell composition
disclosed herein (e.g., comprising a population of TILs prepared according to
the methods
disclosed herein (e.g., enriched for CD8+ TILs, stem-like TILs, tumor-specific
TILs, and/or naive
TILs)) and the additional therapeutic agent are administered concurrently as
separate compositions.
In some aspects, the additional therapeutic agent and the cell composition
disclosed herein (e.g.,
comprising a population of TILs prepared according to the methods disclosed
herein (e.g., enriched
for CD8 TILs, stem-like TILs, tumor-specific TILs, and/or naive TILs)) are
administered
sequentially.
[0530] In some aspects, a cell composition disclosed herein
(e.g., comprising a population
of TILs prepared according to the methods disclosed herein (e.g., enriched for
CD8+ TILs, stem-
like TILs, tumor-specific TILs, and/or naive TILs)) is administered to the
subject in combination
with a checkpoint inhibitor (e.g., an anti-PD1 antibody). In some aspects, the
cell composition is
administered before the checkpoint inhibitor (e.g., an anti-PD1 antibody). In
some aspects, the cell
composition is administered after the checkpoint inhibitor (e.g., an anti-PD1
antibody).
[0531] In some aspects, the subject is administered a
lymphodepleting therapy prior to
receiving the cell composition. Any lymphodepleting therapy can be used in the
method disclosed
herein. In some aspects, the lymphodepleting therapy comprises a chemotherapy.
In some aspects,
the lymphodepleting therapy comprises cyclophosphamide. In some aspects, the
lymphodepleting
therapy comprises fludarabine. In some aspects, the lymphodepleting therapy
comprises
cyclophosphamide and fludarabine. In some aspects, the lymphodepleting therapy
is administered
at least about 3 days, at least about 4 days, at least about 5 days, at least
about 7 days, at least about
8 days, at least about 9 days, at least about 10 days, at least about 11 days,
at least about 12 days,
at least about 13 days, or at least about 14 days prior to the cell
composition.
[0532] As described herein, a cell composition disclosed herein
(e.g., comprising a
population of TILs prepared according to the methods disclosed herein (e.g.,
enriched for CD8+
TILs, stem-like Tits, tumor-specific TILs, and/or naïve TILs)) can be used to
treat various types
of tumors. As it is generally known in the art, certain tumors are less
responsive to currently
available anti-cancer treatments (e.g., such as those described above). For
instance, some tumors
are refractory to a checkpoint inhibitor. As used herein, the term
"refractory" refers to a disease or
condition that does not respond to treatment. Where the term is used to
describe a tumor (e.g.,
"refractory tumor"), the term refers to tumors that do not respond to
treatment and includes
circumstances where the tumor is resistant at the beginning of treatment or
the tumor becomes
resistant during treatment.
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[0533] Accordingly, some aspects of the present disclosure are
related to a method of
treating a tumor in a subject in need thereof, comprising administering to the
subject any of the
cell compositions described herein, wherein the tumor is refractory to one or
more checkpoint
inhibitors. In some aspects, the checkpoint inhibitor comprises a PD-1
antagonist, a CTLA-4
antagonist, a TIM3 antagonist, a GITR antagonist, a KIR antagonist, a LAG3
antagonist, or any
combination thereof In some aspects, the checkpoint inhibitor comprises an
anti-PD1 antibody, an
anti-CTLA-4 antibody, an anti-TIM3 antibody, an anti-GITR antibody, an anti-
KIR antibody, an
anti-LAG3 antibody, or any combination thereof. Accordingly, in some aspects,
a tumor that can
be treated with the present disclosure is refractory to a PD-1 antagonist
(e.g., anti-PD-1 antibody).
In some aspects, the tumor is refractory to a CTLA-4 antagonist (e.g., anti-
CTLA-4 antibody). In
some aspects, the tumor is refractory to a PD-1 antagonist (e.g., anti-PD-1
antibody) and a CTLA-
4 antagonist (e.g., anti-CTLA-4 antibody). In some aspects, the tumor is
refractory to a TIM3
antagonist (e.g., anti-TIM3 antibody). In some aspects, the tumor is
refractory to a GITR antagonist
(e.g., anti-GITR antibody). In some aspects, the tumor is refractory to a KIR
antagonist (e.g., anti-
KIR antibody). In some aspects, the tumor is refractory to a LAG3 antagonist
(e.g., anti-LAG3
antibody). In some aspects, the tumor is refractory to a PD-1 antagonist, a B-
raf enzyme inhibitor,
and a MEK protein inhibitor. In some aspects, the tumor is refractory to one
or more PD-1
antagonists, radiation, a B-raf enzyme inhibitor, and/or a MEK protein
inhibitor. In some aspects,
the tumor is refractory to one or more PD-1 antagonists, radiation and/or an
oncolytic virus.
[0534] In some aspects, provided herein is a method of treating
a tumor in a subject in need
thereof, comprising administering to the subject any of the cell compositions
described herein,
wherein the tumor is refractory to a PD-1 antagonist.
[0535] In some aspects, the PD-1 antagonist is an anti-PD-1
antibody. In some aspects, the
PD-1 antagonist includes any known anti-PD1 antibodies in the art. Antibodies
(e.g., human
antibodies) that bind specifically to PD-1 with high affinity have been
disclosed in U.S. Patent
Nos. 8,008,449 and 8,779,105, each of which is hereby incorporated by
reference. Other anti-PD-
1 mAbs have been described in, for example, U.S. Patent Nos. 6,808,710,
7,488,802, 8,168,757
and 8,354,509, and PCT Publication No. WO 2012/145493, each of which is hereby
incorporated
by reference. Additional examples of anti-PD-1 antibodies include
lambrolizumab (MK-3475)
described in WO 2008/156712, and AMP-514 described in WO 2012/145493, the
teachings of
which are hereby incorporated by reference. Further known anti-PD-1 antibodies
and other PD-1
inhibitors include those described in WO 2009/014708, WO 03/099196, WO
2009/114335 and
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WO 2011/161699, the teachings of which are hereby incorporated by reference.
In some aspects,
anti-PD-1 antibodies include monoclonal antibodies 5C4 (referred to herein as
Nivolumab or
BMS-936558), 17D8, 2D3, 4H1, 4A11, 7D3, and 5F4, described in WO 2006/121168,
the
teachings of which are hereby incorporated by reference, can be used. In some
aspects, the anti-
PD-1 antibody is pidilizumab (CT-011). Antibodies or antigen binding fragments
thereof that
compete with any of these antibodies or inhibitors are also relevant for the
present disclosure.
[0536]
In some aspects, the anti-PD-1 antibody cross-competes with
nivolumab. In some
aspects, the anti-PD-1 antibody binds to the same epitope as nivolumab. In
some aspects, the anti-
PD-1 antibody has the same CDRs as nivolumab. In some aspects, the anti-PD-1
antibody is
nivolumab. Accordingly, some aspects of the present disclosure is related to a
method of treating
a tumor which is refractory to nivolumab in a subject in need thereof, wherein
the method
comprises administering to the subject any of the cell compositions described
herein to the subject.
[0537]
In some aspects, the anti-PD-1 antibody cross-competes with
pembrolizumab. In
some aspects, the anti-PD-1 antibody binds to the same epitope as
pembrolizumab. In some
aspects, the anti-PD-1 antibody has the same CDRs as pembrolizumab. In some,
the anti-PD-1
antibody is pembrolizumab. Pembrolizumab (also known as "Keytruda ",
lambrolizumab, and
MK-3475) is a humanized monoclonal IgG4 antibody directed against human cell
surface receptor
PD-1 (programmed death-1 or programmed cell death-1). Pembrolizumab is
described, for
example, in U.S. Patent Nos. 8,354,509 and 8,900,587; see also
worldwideweb.cancer.gov/drugdictionary?cdrid=695789 (last accessed: May 25,
2017), each of
which is hereby incorporated by reference. Accordingly, some aspects of the
present disclosure is
related to a method of treating a tumor which is refractory to pembrolizumab
in a subject in need
thereof, wherein the method comprises administering to the subject any of the
cell compositions
described herein to the subject.
[0538]
In some aspects, the anti-PD-1 antibody cross-competes with MEDI0608.
In some
aspects, the anti-PD-1 antibody binds to the same epitope as MEDI0608. In some
aspects, the anti-
PD-1 antibody has the same CDRs as MEDI0608 In soem aspects, the anti-PD-1
antibody is
MEDI0608 (formerly AMP-514), which is a monoclonal antibody. MEDI0608 is
described, for
example, in U.S. Patent No. 8,609,089
or in
worldwideweb.cancer.gov/drugdictionary?cdrid=756047 (last accessed May 25,
2017), each of
which is hereby incorporated by reference. Accordingly, some aspects of the
present disclosure is
related to a method of treating a tumor which is refractory to MEDI0608 in a
subject in need
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thereof, wherein the method comprises administering to the subject any of the
cell compositions
described herein to the subject.
[0539] In some aspects, the anti-PD-1 antibody cross-competes
with BGB-A317. In some
aspects, the anti-PD-1 antibody binds the same epitope as BGB-A317. In some
aspects, the anti-
PD-1 antibody has the same CDRs as BGB-A317. In some aspects, the anti-PD-1
antibody is BGB-
A317, which is a humanized monoclonal antibody. BGB-A317 is described in U.S.
Publ. No.
2015/0079109, which is hereby incorporated by reference. Accordingly, some
aspects of the
present disclosure is related to a method of treating a tumor which is
refractory to BGB-A317 in a
subject in need thereof, wherein the method comprises administering to the
subject any of the cell
compositions described herein to the subject.
[0540] In some aspects, the PD-1 antagonist is an anti-PD-Li
antibody_ In some aspects,
the PD-1 antagonist includes any known anti-PD-Li antibodies in the art. In
some aspects, human
anti-PD-Li antibodies disclosed in U.S. Pat. No. 7,943,743, the contents of
which are hereby
incorporated by reference, are within the scope of the present disclosure.
Such anti-PD-Li
antibodies include 3G10, 12A4 (also referred to as BMS-936559), 10A5, 5F8,
10H10, 1B12, 7H1,
11E6, 12B7, and 13G4. Other art recognized anti-PD-Li antibodies include those
described in, for
example, U.S. Pat. Nos. 7,635,757 and 8,217,149, U.S. Publication No.
2009/0317368, and PCT
Publication Nos. WO 2011/066389 and WO 2012/145493, the teachings of which
also are hereby
incorporated by reference. Additional examples of an anti-PD-Li antibody
include atezolizumab
(TECENTRIQ , RG7446), or durvalumab (IM1FINZI , MEDI4736). Antibodies or
antigen
binding fragments thereof that compete with any of these art-recognized
antibodies or inhibitors
for binding to PD-Li are also relevant for the present disclosure.
[0541] In some aspects, the anti-PD-Li antibody cross-competes
with BMS-936559
(formerly 12A4 or MDX-1105) (see, e.g., U.S. Patent No. 7,943,743; WO
2013/173223, both of
which are hereby incorporated by reference). In some aspects, the anti-PD-L1
antibody binds to
the same epitope as BMS-936559. In some aspects, the anti-PD-Li antibody has
the same CDRs
as BMS-936559. In some aspects, the anti-PD-Li antibody is BMS-936559.
Accordingly, some
aspects of the present disclosure is related to a method of treating a tumor
which is refractory to
BMS-936559 in a subject in need thereof, wherein the method comprises
administering to the
subject any of the cell compositions described herein to the subject.
[0542] In some aspects, the anti-PD-Li antibody cross-competes
with MPDL3280A (also
known as RG7446 and atezolizumab) (see, e.g., Herbst el al. 2013 J Clin Oncol
31(suppl):3000;
U.S. Patent No. 8,217,149, both of which are hereby incorporated by
reference), MEDI4736
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(Khleif, 2013, In: Proceedings from the European Cancer Congress 2013;
September 27-October
1, 2013; Amsterdam, The Netherlands. Abstract 802, which is hereby
incorporated by reference).
In some aspects, the anti-PD-L1 antibody binds to the same epitope as
MPDL3280A. In some
aspects, the anti-PD-Li antibody has the same CDRs as MPDL3280A. In some
aspects, the anti-
PD-Li antibody is MPDL3280A. Accordingly, some aspects of the present
disclosure is related to
a method of treating a tumor which is refractory to MPDL3280A in a subject in
need thereof,
wherein the method comprises administering to the subject any of the cell
compositions described
herein to the subject.
[0543] In some aspects, the anti-PD-Li antibody cross-competes
with MSB0010718C
(also called Avelumab; see US 2014/0341917, which is hereby incorporated by
reference). In some
aspects, the anti-PD-Li antibody binds to the same epitope as MSB0010718C. In
some aspects,
the anti-PD-Li antibody has the same CDRs as MSB0010718C. In some aspects, the
anti-PD-Li
antibody is MSB0010718C. Accordingly, some aspects of the present disclosure
is related to a
method of treating a tumor which is refractory to MSB0010718C in a subject in
need thereof,
wherein the method comprises administering to the subject any of the cell
compositions described
herein to the subject.
[0544] In some aspects, provided herein is a method of treating
a tumor in a subject in need
thereof, comprising administering to the subject any of the cell compositions
described herein,
wherein the tumor is refractory to a CTLA-4 antagonist.
[0545] In some aspects, the CTLA-4 antagonist is an anti-CTLA-4
antibody. In some
aspects, the CTLA-4 antagonist includes any known anti-CTLA-4 antibodies in
the art. For
instance, human antibodies that bind specifically to CTLA-4 with high affinity
have been disclosed
in U.S. Patent Nos. 6,984,720 and 7,605,238, each of which is hereby
incorporated by reference.
Additional examples of anti-CTLA-4 antibodies are described in, for example,
U.S. Patent Nos.
5,977,318, 6,051,227, 6,682,736, and 7,034,121, each of which is hereby
incorporated by
reference. Other non-limiting examples of anti-CTLA-4 antibodies that are
within the scope of the
present disclosure include: MK-1308 (Merck) and AGEN-1884 (Agenus Inc.; see WO
2016/196237). Anti-CTLA-4 antibodies relevant for the present disclosure also
include isolated
antibodies that bind specifically to human CTLA-4 and cross-compete for
binding and/or binding
to the same epitope region as one or more of the anti-CTLA-4 antibodies
disclosed herein (e.g.,
ipilimumab, tremelimumab, MK-1308, or AGEN-1884).
[0546] In some aspects, the anti-CTLA-4 antibody cross-competes
with ipilimumab. In
some aspects, the anti-CTLA-4 antibody binds to the same epitope as
ipilimumab. In some aspects,
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the anti-CTLA-4 antibody has the same CDRs as ipilimumab. In some aspects, the
anti-CTLA-4
antibody is ipilimumab. Ipilimumab (also known as 10D1 and marketed as YERVOY
) is a fully
human, IgG1 monoclonal antibody that blocks the binding of CTLA-4 to its B7
ligands.
ipilimumab is further described in U.S. Patent No. 6,984,720, which is hereby
incorporated by
reference. Accordingly, some aspects of the present disclosure is related to a
method of treating a
tumor which is refractory to ipilimumab in a subject in need thereof, wherein
the method comprises
administering to the subject any of the cell compositions described herein to
the subject.
[0547] As is apparent from at least the above disclosure, in
some aspects, provided herein
is a method of treating a tumor that is refractory to a TfM3 antagonist (e.g.,
anti-T1M3 antibody)
in a subject in need thereof, comprising administering to the subject any of
the cell compositions
described herein. In some aspects, provided herein is a method of treating a
tumor that is refractory
to a GITR antagonist (e.g., anti-GITR antibody) in a subject in need thereof,
comprising
administering to the subject any of the cell compositions described herein. In
some aspects,
provided herein is a method of treating a tumor that is refractory to a KIR
antagonist in a subject
in need thereof, comprising administering to the subject any of the cell
compositions described
herein. In some aspects, provided herein is a method of treating a tumor that
is refractory to a
LAG3 antagonist in a subject in need thereof, comprising administering to the
subject any of the
cell compositions described herein.
[0548] For any of the treatment methods described above, in some
aspects, the tumor is
relapsed. In some aspects, the tumor is metastatic. In some aspects, the tumor
is both relapsed and
metastatic.
[0549] As described and demonstrated herein, in some aspects,
the culturing processes
described herein (e.g., expanding and culturing cells in MRM) can improve one
or more properties
of TILs of a tumor that is refractory to one or more immune checkpoint
inhibitors. For instance, in
some aspects, culturing such TILs using the MRM processes described herein can
increase the
sternness of the TILs (i.e., the TILs are more stem-like as compared to the
TILs prior to the
culturing and/or as compared to corresponding TILs that were cultured using a
non-MRM process).
In some aspects, after culturing with the MRM process, the sternness of the
TILs from the
refractory tumor is increased by at least about 10%, at least about 20%, at
least about 30%, at least
about 40%, at least about 50%, at least about 60%, at least about 70%, at
least about 80%, at least
about 90%, or at least about 100% as compared to reference TILs (e.g., the
TILs prior to the
culturing with the MR1\4 process and/or corresponding TILs that were cultured
using a non-MRM
process). In some aspects, after culturing with the MRM process, the stemness
of the TILs from
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the refractory tumor is increased by at least about 2-fold, at least about 3-
fold, at least about 4-fold,
at least about 5-fold, at least about 6-fold, at least about 7-fold, at least
about 8-fold, at least about
9-fold, at least about 10-fold, at least about 15-fold, at least about 20-
fold, at least about 25-fold,
at least about 30-fold, at least about 40-fold, or at least about 50-fold as
compared to the reference
TILs. Accordingly, as is apparent from the present disclosure, in some
aspects, the MRM process
described herein can be used to increase the sternness of TILs from tumors
that were previously
treated with one or more immune checkpoint inhibitors.
[0550] Exemplary methods of measuring the sternness of TILs are
provided elsewhere in
the present disclosure. For instance, in some aspects, sternness can be
measured by determining
the expression of one or more stem-like markers, e.g., CD45RA+, CD62L+, CCR7+,
CD27+,
CD28+, BACH2+, LEF1+, TCF7+, CD39-, CD69-, or a combination thereof. In some
aspects, the
sternness of the TILs can be measured by measuring the expression of CD39 and
CD69 on the
TILs. As further described elsewhere in the present disclosure, stem-like
cells are CD39- and
CD69-. Accordingly, in some aspects, after culturing TILs from a tumor that is
refractory to one
or more immune checkpoint inhibitors with the MRI\4 process, at least about
15%, at least about
20%, at least about 25%, at least about 30%, at least about 35%, at least
about 40%, at least about
45%, at least about 50%, at least about 55%, at least about 60%, at least
about 65%, or at least
about 70% of the expanded TILs are CD39- and CD69-.
EXAMPLES
Example 1. Methods
[0551] Media preparation: T cell conditioned media (TCM) was
supplemented with
immune Cell Serum Replacement (Thermo Fisher), 2 mM L-glutamine (Gibco), 2 mM
Glutamax
(Gibco), MEM Non-Essential Amino Acids Solution (Gibco), Sodium pyruvate
(Gibco), IL-2, 200
IU/mL; IL-7 ,120 IU/ml, IL-15, 20 IU/ml.
[0552] For hypotonic conditioning medium, TCM media with varying
concentrations of
sodium, potassium, glucose and calcium were adjusted by adding NaCl, glucose,
and calcium free
RPMI. After adding defined NaC1 free RPMI to TCM, the final concentrations
were in the range
of: NaCl (40-80 mM), KC1 (40-80 mM), Calcium (0.5-2.8mM), Glucose (10-24mM)
and
osmolality (-250-260 mOsmol). See Table. 1.
Table 1. Hypotonic conditioning medium with varying concentrations of
potassium, sodium,
glucose, and calcium
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Media K NaC1 Glucose Ca Osmolality
Tonicity*
(mM) (mM) (mM) (mM) (mOsmol) (mOsmol)
Basal Media 4 118.47 -24mM -2.8mM 245
245
Hyper K 80 mM 55.6mM 15mM 1.2mM -262.26
271.2
Hyper K 75 59.3 15.4 1.3 -260
268.6
Hyper K 70 63.9 15.9 1.4 -259.7
267.8
Hyper K 65 67.6 16.3 1.5 -257.5
265.2
Hyper K 60 72.2 16.8 1.6 -257.2
264.4
Hyper K 55 76 17.2 1.7 -255.2
262
Hyper K 50 80.5 17.7 1.8 -254.7
261
RPMI Gibco + 5.34 103 11.1 0.4
216.7
IC SR
RPMI 1640 + 50 55.34 103
316.7
mM K+
*Tonicity is calculated based on the following formula: 2 X (concentration of
K +
concentration of NaCl)
[0553] We also tested the effect of tonicity on T cells by
maintaining constant tonicity
conditions (250 mOsmol - hypotonic, 280m Osmol - isotonic, 320 mOsmol -
hypertonic) with
varying potassium concentrations. Final concentrations in hypotonic
conditions, NaC1 (35-75mM),
KC1 (50-90 mM), final concentrations in isotonic conditions NaCl (50-90 mM),
KC1 (50-90 mM),
final concentrations in hypertonic conditions NaCl (70-110 mM), KC1 (50-90
mM). See Table. 2.
Table 2. Hypotonic, isotonic, hypertonic solutions with varying concentrations
of potassium and
NaCl
Tonicity* K NaCl
mOsmol
(mM) (mM)
50 75
60 65
Hypotonic 250 70 55
80 45
90 35
50 90
60 80
Isotonic 280 70 70
80 60
90 50
50 110
Hypertonic 320 60 100
70 90
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so 80
90 70
* Tonicity is calculated according to the formula: Tonicity = ([K] + [MICA]) x
2
wherein "[K]" is the potassium concentration and "[NaCl]' is the sodium
chloride concentation of the media.
[0554] Cell culture and Transduction: Healthy donor
cryopreserved human CD4 and
CD8 cells were activated with TransAct (Miltenyi) in T cell conditioned media-
TCM, basal media,
or hypotonic conditioning medium. After 24 hours of activation in the TCM,
basal media, or
hypotonic conditioning medium, T cells were transduced with lentiviral
particles to introduce
chimeric antigen receptor (anti-CD19 CAR) in Grex plates (Wilson Wolf). The
following day after
transduction, T cells were supplemented with fresh media to dilute the
TransAct and end T-cell
activation. Depending on the cell growth and density, T cells were fed with
warm 2X cytokine
media by aspirating half of the media in the Grex plate. On day 7, cells were
harvested, counted
and analyzed for the expression of sternness markers by flow cytometry.
[0555] Intracellular Cytokine assays: On day 7, T cells were
washed and placed in
control media and subjected to a 5 hour re-stimulation with phorbol myrystate
acetate (PMA) and
ionomycin in the presence of brefeldin A to measure intracellular cytokines,
IL-2, IFNy, and
TNFa. T cells were stained with surface antibody staining in FACS buffer
containing fixable live/
dead solution. Cells were stained with respective antibodies for intracellular
cytokines following
fixation and permeabilization. Quantification of intracellular cytokine
expression was assessed
using flow cytometry.
[0556] Sternness phenotype CAR expression measurement via flow
cytometry: On day
7, live T cells from the respective treatments were assessed via flow
cytometry. Cells were first
washed with cell staining buffer and stained with anti-CCR7 for 15 minutes at
37 C. Following
this, a 2x master mix of the antibodies against several other antigens (as
detailed below) was added
to cells and incubated for 20 minutes at 4 C. Cells were washed with cell
staining buffer and
permeabilized with the foxp3 staining kit (ebioscience) as per manufacturers'
protocol. After
fixing, the cells were stained for TCF7 for twenty minutes at 4 C following
which, cells were
analyzed by flow cytometry on aurora (cytek). The following are the list of
antibodies used for
assessing the sternness markers: CD8 (BD-4563795), CD4 (BD-4 612936), CD27 (BD-
4612829),
CD3 (Thermo-4 612893), CD28 (Biolegend- 4302936), CD62L, CAR-EGFR (Thermo-
4352911),
CD45R0 (BD4564290), CD39 (Biolegend- 4328236), TCF7 (Cell signaling -#14456),
CCR7
(BD-#562381), CD127 (Bio legend- 4351324), CD45RA (BD-4560673).
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Example 2. Methods of Preparing Media and Culturing TILs
[0557] Control Media: Commercially available T cell media (e.g.,
CT STm
OPTIMIZERTm, INIMUNOCULTTm or TEXMACSTm).
[0558] Metabolic reprogramming media ("MRM"): The inorganic salt
ion
concentrations of T cell media were adjusted using NaCl free T cell media. The
final concentrations
of MRM were the following: NaC1 (40-80 mM), KC1 (40-90 mM), Calcium (0.5-2.8
mM), Glucose
(10-24 mM) and osmolality (-250-340 mOsmol).
[0559] TIL media preparation used for initial culture and
secondary and final TIL
expansions: Either Control media or MRM was supplemented with 2.5% serum
supplement
(CTSTm Immune Cell SR, Thermo Fisher), 2 mM L-glutamine (Gibco), 2 mM L-
glutamax (Gibco),
MEM Non-Essential Amino Acids Solution (Gibco), Pen-strep (Gibco), 2014/m1
FUNGE\FTM
(InvivoGen), Sodium pyruvate (Gibco), and 1mM of O-Acetyl-L-carnitine
hydrochloride (Sigma).
[0560] Initial TIL Culture: FIG 1 is a schematic depicting
generally certain aspects of the
methods of culturing TILs described herein. Multiple tumors surgically
resected from various
tumor types (colon, lung, hepatocellular carcinoma, renal, pancreas, breast,
melanoma, and
prostate) with an average size of 1-10mm3 were seeded in 24-well plates in 2m1
of either control
media or MRM as described above, both supplemented with IL-2 (300ng/mL) and IL-
21
(30ng/m1). Tumor fragments were cultured in a heat jacketed incubator at 37 C
incubator with 5%
CO2 until colony formation was visible Fresh media (control or MRM)
supplemented with IL-2
(300ng/mL) and IL-21 (30ng/m1) were replenished every 3 days depending on the
growth of the
cells. This method resulted in a yield of about 2x106-10x106 cells per
fragment at the end of the
initial culture. A subset of cells for analysis were passed through a 40p.m
strainer and pheonotyped
with multi color flow cytometry using various biomarkers including CD62L,
CD27, CD28,
CD45RO, CD39, TI1V13, CD127, PD1, CD103, CD45RA, and TCF7.
[0561] Secondary TIL Expansion: When cell yield from the initial
culture reached about
2x106-10x106 cells per cultured fragment (usually at about day 14 to day 19),
the TILs cultured in
either control media or MRM, both supplemented with IL-2 (73.6ng/m1), IL-
21(10ng/m1), and IL-
15 (0.4ng/m1), were stimulated by adding 1:100 T cell TRANSACT' (Miltenyi
Biotec), 5 jig/ml
recombinant human CD27 ligand (R&D systems), and 1 n.g/m1 recombinant human 4-
1BB
ligand/TNFSF9 (R&D systems). Cells were maintained in culture until about
5x107 to 20x107 cells
were obtained (about 7 to 11 days post-stimulation). At the end of the
secondary expansion period,
TILs were analyzed with multicolor flow cytometry using various biomarkers
including, CD62L,
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CD27, CD28, CD45RO, CD39, TIM3, CD127, PD1, CD103, CD45RA and TCF7. Only live
and
CD3+ cells were analyzed.
[0562] Final TIL Expansion: When the cultures reached a yield of
about 5x107 to 20x107
cells, the TILs cultured in either control media or MRM were transferred to
fresh control media
supplemented with IL-2 (73.6ng/m1), IL-21(10ng/m1), and 1L-15 (0.4ng/m1). TILs
were stimulated
for a second time with 1:100 TRANSACTTm (Mil tenyi Biotec), 5ps/m1 recombinant
human CD27
ligand (R&D systems), and 1 ,g/m1 recombinant human 4-1BB ligand/TNF SF9 (R&D
systems).
The cells were cultured in static GREX or stirred tank until a yield of about
10x109-100x109 cells
per fragment was achieved (about 14 days) and analyzed with multi color flow
cytometry for
various bi markers, including CD62L, CD27, CD28, CD45RO, CD39, TIM3, CD 127,
PD I ,
CD103, CD45RA, and TCF7. To check for polyfunctionality of the cells, the
resultant TILs were
stimulated with PMA/ionomycin (1:500) for 4 hours and intracellular staining
was performed using
the following markers: CD4, CDR, CD27, IL2, IFNy, TNFa and TCF7. Only live and
CD3+ cells
were analyzed.
Example 3. MRM results in expansion of CD8+, tumor-reactive, less
differentiated TILs
[0563] TILs were grown as described in Example 1 (FIG. 1). After
the initial T1L culture
(i.e., 14 days), multiparameter flow cytometry was performed to quantify the
percentages of CD4+
and CD8+ TILs present in the cell culture. Cells cultured in MRM had
significantly enriched CD8+
TILs by ¨20-80% as compared to the cells cultured in control media (FIGs. 2A-
2C and data not
shown). Although CD4 TlLs are capable of eradicating solid tumors, superior
cytolytic activity
towards tumors is primarily mediated by CD8+ TILs. Tumor cells predominantly
express MT1C
class I associated tumor antigens, which are recognized by CD8+ TILs. Thus,
having a greater
proportion of CD8+ TILs in the TIL therapy infusion product is therapeutically
beneficial. Use of
MRM to culture TILs unexpectedly enriched CD8+ TILs as compared to TILs
cultured in control
media (FIG. 2C).
[0564] Tits obtained at the end of the initial culture in MRM
(about day 14) also
demonstrated consistent expression of several cell surface markers of tumor-
reactive TTLs (e.g.,
CD39, CD103, CD226, and/or PD1). Initial culturing in MRM produced TILs with
enhanced
expression of CD39 and PD1 (greater than 20%) as compared to TILs cultured in
control media
(FIGs. 2A-2B). Previously used methods show that PD1 expression is completely
lost in the PD1
subsets during initial TIE culture, indicating undesirable loss of tumor-
reactive TILs (see, e.g.,
Poschke et al, Oncoimmunology 5(12):e1240859 (2016); and Gros et al, JCI
124(5):2246-59
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(2014)). We obseverved that maintenace of PD1 expression after T cell
stimulation was dependent
on donor (data not shown). Similar results were obtained for expression of
other markers of tumor
reactivity including CD39 and CD103 (see FIGs. 2A-2B and 5A-5B, and data not
shown).
However, clonal repertoire observed at day 14 is maintained throughout entire
TIL process (data
not shown).
[0565] We also observed co-expression of PD1 (indicative of a
tumor-reactive metabolic
state) and CD27, in both CD4 and CD8- TILs cultured in MRM (FIGs. 3A-3E). CD27
expression,
which is constitutively expressed on naïve and memory committed T-cells, is
indicative of a stem-
like phenotype in T cells. Previous reports indicate that CD27 expression is
reduced in CD8+ T
cells during cell expansion following T cell stimulation (see, e.g., Tran et
al., J Immunotherapy
31(8):742-51 (2008); and Rosenberg et al., Clinical Cancer Research
17(13):4550-557 (2011),
Huang et al, J. Immunology 176(12).7726-35 (2006)). In contrast, TILs cultured
in MRM,
disclosed herein, have preserved CD27 expression throughout the culturing
process, allowing for
selective expansion of stem-like tumor-reactive clones. As expected, not all
CD27 + cells at day 14
co-expressed PD1, indicating that not all stem-like cells displayed a tumor-
reactive metabolic state.
[0566] Anti-tumor function and survival of TILs are dependent on
the consolidated signals
received by the TCR, cytokine, and costimulatory receptors. Inadequate
exposure of any of these
signals will result in anergy and atrophy of the Tits. Previously used methods
of culturing and
expanding TILs result in loss of CD27 expression in the TILs. However, TILs
that maintain CD27
expression in an infusion product, e.g., in minimally expanded TILs, have been
shown to be
associated with tumor regression following adoptive T cell therapy (see, e.g.,
Tran et al., J.
Immunotherapy 31(8)742-51 (2008); and Rosenberg et al., Clinical Cancer
Research
17(13):4550-557 (2011)). In addition, elevated expression of costimulatory
receptors, e.g, CD27
and CD28, is associated with in vivo therapeutic efficacy (Tran et al., J.
Immunotherapy 31(8):742-
51(2008), Geltink et al., Cell 171, 385-397 (2017)). Expression of these
costimulatory receptors
has also been associated with stemness and longer telomere lengths that
correlated with young TIL
cultures. In contrast to previously used methods, e.g., control media, the use
of MRM described
herein enriched TILs with CD27 and CD28 expression to about 20% to about 80%
of the total
number of TILs across several tumor types (FIG. 4 and data not shown).
Enrichment of CD27 and
CD28 was not unique to the CD8 T cell subset but was also observed in the
CD4' subset (data not
shown).
[0567] Expression of CD27 and CD62L is correlated with less
differentiated T cells and is
associated with efficient trafficking of the T cells to tumor tissues and
lymph nodes. Expression of
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CD27, CD28, and CD62L is also linked to longer telomere length, which is
indirectly linked to the
age of the TIL and in vivo therapeutic efficacy (see, e.g., Tran et al., J.
Inentunotherapy 31(8):742-
511 (2008); and Rosenberg et al., Clinical Cancer Research 17(13):4550-557
(2011)). However,
TILs cultured in MR1\4, as disclosed herein, maintained both CD27 and CD62L
expression
throughout the process, similar to that of minimally cultured TILs. We
observed a ¨50% increase
in CD27 and CD62L expression in TILs cultured in MRI\4 as compared to those
cultured in control
media (data not shown).
[0568] Tumor-reactive clones that mediate tumor regression post
immune checkpoint
blockade are believed to be derived from the CD8+ T cells that expressed PD1
and transcription
factor TCF-7 (Im et al., Nature 537:417-21 (2016); and Feldman et at., Cell
175(4):998-1013
(2018)). TILs cultured in MRM disclosed herein displayed enrichment of tumor-
reactive TIL
biomarkers (PD1 and CD103) with a concurrent 4-fold to 50-fold higher level of
TCF7 expression
(FIGs. 5A-5C, 6D, 6H, 10). Expression of TCF7 is indicative of more stem-like
cells. Despite the
expression of PD1, these cells retained proliferative capacity and maintained
less differentiated
cells upon further stimulation (FIGs. 6A-6H).
Example 4: MR1V1 preserves tumor reactivity of TILs
[0569] Tumors are heterogenous in nature and often contain
common mutations in genes
such as KRAS, P53, and BRAF (public neoantigens). The methods using MRM as
disclosed herein
enriched for TILs that recognize such neoantigens.
[0570] Tumor resections were obtained from a patient with
pancreatic adenocarcinoma, a
cancer that predominantly has tumor cells with KRASG12V, KRAsG12C, and
KRASG12D mutations.
HPLC-purified 9mer, 1 Omer, and 25mer peptides of the above-mentioned KRAS hot
spot
mutations were purified and used to pulse immature dendritic cells (DCs)
generated from patient-
matched peripheral blood monocytes. These peptide pool-pulsed DCs were co-
cultured with TILs
obtained from the same pancreatic adenocarcinoma patient and cultured
according to the methods
described in the Examples above. Culture of the TILs in MRM resulted in ¨30%
more CDS+ T
cells with significant co-expression of CD27, CD28, PD1, and TCF-7 (FIGs. 7A-
7H). These results
indicate that MRI\4 disclosed herein preserves tumor-reactive TCR clones that
recognize public
antigens.
[0571] As expected, TILs pulsed with wild type KRAS peptides did
not result in specific
expansion of KRAS-specific TILs. However, we consistently observed increased
expression of
CD27, CD28, PD1, and TCF7 expression by TILs cultured in MR1\4. These results
indicate that
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culturing cells in MRM preserves the growth and proliferation of TILs that
recognize rare public
neoantigens (FIGs. 8A-8H).
[0572] Preferential enrichment of CDS+ TILs and markers
associated with tumor reactivity
(CD39 and PD I) were maintained during this expansion process (FIGs. 9A-9B).
These results
show that MRM further enhances PD1 expression in tumor-reactive TILs even
after initial culture.
In addition, TILs cultured in MRM according to the methods disclosed herein
exhibited a greater
than 50-fold increase in TCF7 expression as measured by qPCR (FIG. 10). These
data are
consistent with the role of TCF7, which is a master transcriptional regulator
required for self-
renewal and proliferative burst of the Tits post re-stimulation.
[0573] Expression of CD103 in TILs is reported to be correlated
with TIL infiltration in
tumors with high mutation antigen burden. In addition to CD39 and PD1
expression, our data
demonstrated that expansion of TILs in MRM increased the number of PD1 CD39
CD103+ cells
(FIGs. 11A-11L) while also increasing expression of sternness-associated
genes. Therefore, culture
of TILs in MRM may be sufficient to increase the number of cells with both
tumor-reactivity and
higher proportion of stem-like cells in the resulting expanded cell products.
[0574] The data presented herein demonstrate that expansion of
TILs in MRM generates
cell populations with not only increased sternness, but also increased
expression of several cell
surface markers associated with tumor reactivity. The methods described herein
using MRM
produce TILs that have characteristics and properties of an improved
therapeutic product.
Example 5. Methods of Treatment
[0575] Tits that are used for infusion are derived from a
pateint's tumor excised from
primary or metastatic tuomors or lymph nodes. TIL cultures are initiated by
plating the small tumor
fragments (-1-10 mm3) in 24 well plates containing MRM as described above in
Example 1. These
fragments are grown until about 2 x 106 to about 10 x 106 cells / tumor
fragment are obtained
(typically about 2-3 weeks). The resuting cells from all the fragments are
pooled and plated at a
density of 2 x 106/well for T cell stimulation, e.g., by adding TRANSACTTm,
and optionally CD27
agonist, 41BB agonist, and/or OX-40 agonist. Cells are maintained in culture
until about 5 x 10'
to about 20 x 107 cells are obtained. These cells are further stimulated,
e.g., using TRANSACTTAI
and optionally CD27 agonist, 41BB agonist, and/or OX-40 agonist to achieve
about 1000-2000
fold increase in the number of cells (-A -150 x 109-Tits) for the infusion
product.
[0576] Prior to administration of the TIL infusion product,
patients are administered a
lymphodepletion treatment, e.g., comprising cyclophosphamide and fludarabine.
In addition to TIL
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infusion, patients may also receive an anti-PD1 checkpoint inhibitor (e.g.,
pembrolizumab or
nivolumab) after infusion of TILs cutltured as disclosed herein.
Example 6. Analysis of Clonal Diversity
[0577] To assess the clonal diversity of TILs cultured in MRM,
according to the methods
disclosed above, tumor fragments and TILs obtained from the tumor fragments
were cultured in
either control media or metabolic reprogramming media (MRM). Total genomic DNA
was isolated
from tumor and TIL samples using DNeasy Blood and Tissue Kit (QIAGEN) and
sequenced using
Immuno-seq for TCRf3 and CDR3 regions (Adaptive Biotechnologies, Seattle, WA).
[0578] The diversity metrics of the samples were assessed by
Simpsons clonality
where pi is the proportional abundance of clone i in a given sample. Simpsons
clonality and
productive rearrangements were examined in tumor fragments ("tumor"), TILs
cultured in control
media ("control"), and TILs cultured in MRM ("MRM") (FIG. 12). TILs cultured
in MRM (post-
expansion) displayed Simpsons clonality of approximately 0.04, which indicates
high clonal
diversity, similar to what is seen in the tumor fragments (FIG. 12).
Maintenance of TCR13 diversity
represent preservation of TEL clonotypes that infilatrated the tumors and are
tumor reactive. In
contrast, TILs cultured in control media significantly lose clonal diversity
and displayed Simpsons
clonality of approximately 0.4 (i.e., a significantly less diverse clonality,
indicating that many
tumor reactive clones are lost in the culturing process using control media)
(FIG. 12).
[0579] Differential abundance (DA) plots were generated using
the data presented in FIG.
12 (ImmunoSeq, Adaptive Biotechnolgies). Such DA analysis calculates TCR
overlap (see, e.g.,
Emerson et al, J. Path. (2013), which is incorporated by reference herein in
its entirety), Morisita's
Index, and the Jaccard Index for any pair of samples. These plots show that
the differential
abundance of clones are significantly different between tumor vs TILs expanded
in control media
(FIG. 13A) as compared to tumor vs TILs expanded in MRM (FIG. 13B).
[0580] The Tit repertoire present in tumor fragments were
represented approximately 4-
fold more in Tits cultured in MRM process compared to TILs cultured in control
media. When
expanded, TILs are typically outgrown by infrequent clonotypes with
preferential proliferative
potential (these clones are shown expanded along the y-axis in FIGs. 13A-13B).
In contrast, TILs
cultured in MR1\4 showed significantly better preservation and expansion of a
wider repertoire of
clonotypes that are present in the tumor. The preservation and expansion of
clonal diversity of the
TIL population is critical for optimization for effective therapy.
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[0581] To further analyze TIL clonal diversity, the top 50 most
dominant prevalent TCRs
in initial tumor digests were compared to TILs expanded in control media
versus MRM. Culture
in MRM preserves both dominant (i.e. prevalent) and rare T1L clonotypes. The
density of lines in
FIG. 13D show that the majority of T cell clones expanded in MRI\4 recognize
tumor antigens
where they do not in T cell clones cultured in control media (FIG. 13C). The
majority of dominant
tumor clones are recognized by T cell clones expanded in MRM, as indicated by
the connections
above the dotted line of FIG. 13D, as compared to the T cell clones cultured
in control media (FIG.
13C). Of the top 50 dominant tumor TCRs, 2% are preserved in TILs cultured in
control media
(FIG. 13C) as compared to 57% in Tits cultured in MRM (FIG. 13D). These data
demonstrate
that MRM expanded TILs retain a more faithful representation of initial tumor
TCR clonotypes
than TILs expanded in control media.
Example 7. MRM preserves tumor reactivity of TILs
[0582] The ability of TILs generated using the methods described
herein to preserve KRAS
mutant reactivity was evaluated. TILs expanded in control media or MRM were
mixed with
autologous dendritic cells (DCs) that had been pulsed with mutant KRAS
peptide. After 10 days
total genomic DNA was isolated from the TILs using DNeasy Blood and Tissue Kit
(Qiagen) and
the TCR-Vb CDR3 motif was sequenced and analyzed using Immuno-seq for TCRI3
and CDR3
regions (Adaptive Biotechnologies, Seattle, WA). FIG. 14 shows that TILs
cultured in MRM
preserve KRAS mutant reactivity, whereas TILs cultured in control media are
not able to do so.
Specifically, TILs cultured in MRM shared similar CDR3 amino acid motifs as
seen in Tits from
the tumor fragment. These were not detected in TILs cultured in control media.
Thus, the data
show that culture in MRM preserves TIL clonotypes that are represented in the
original tumor
fragment.
Example 8. TILs generated using MRM demonstrate increased tumor recognition
and
tumor killing
[0583] TILs obtained from a melanoma tumor were obtained and
expanded with either
control media or MRM as described above. An autologous tumor cell line was
derived from the
same melanoma sample as the TILs. Briefly, the melanoma cell line was
generated by
enzymatically digesting a fragment of melanoma (using collagenase and DNAse),
plating the
resulting single-cell suspension, followed by serial passaging of the
outgrowing cells. The ability
of the TILs derived from the melanoma tumor to generate inflammatory cytokines
in response to
the melanoma derived cell line was assessed. TILs generated using either
control media or MRM
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were co-cultured with the autologous melanoma cell line for 24 hours. 100,000
tumor cells from
the autologous melanoma cell line were plated in a 48 well plate in 300 ul of
complete RPMI media
(w/ 10% FBS) and allowed to incubate overnight. The next day, the supernatant
was removed and
TILs were added (some TILs were plated in the absence of tumor cells as a
control). After 24 hrs,
media supernatant was collected from the wells and secreted interferon gamma
and TNF-alpha was
measured using MSD. In addition, TILs generated with MRM were incubated in
control media
overnight, and IL-2 secretion was assessed by MSD. These data show that TILs
expanded using
MR_M exhibit significantly increased ability to respond to autologous tumor
cells in terms of
inflammatory cytokine production (FIGs. 15A-15B). Moreover, the Tits generated
using MRM
secrete significantly more IL-2 at steady state with no stimulation indicating
that these cells are
more fit.
[0584] The ability of the TILs derived from the melanoma tumor
to kill the melanoma
derived cell line was also assessed. The number of live tumor cells remaining
after co-culture was
assessed by detaching the tumor cells with trypsin and counting live cells by
flow cytometry. Based
on this live tumor cell count, percent tumor killing by the TILs (normalized
to wells of tumor cells
that did not experience TIL co-culture) was calculated. TILs generated using
MRM show a
significantly better ability to kill the tumor cells (FIG. 15C).
[0585] The above experiment was repeated as described above,
however some wells were
treated with the W6/32 antibody to block HLA Class I interactions. FIG. 15D
demonstrates that
the recognition of autologous tumor cells by TILs generated using MRM is
dependent on HLA
Class I interactions since blocking Class I eliminates the increase in
interferon gamma production.
Example 9. TIL clearance of tumor cells ex vivo
[0586] Patient autologous melanoma tumor cells were plated at
10,000 cells/well in the
xCELLigence impedance assay well plate. The following day, matched TILs that
were expanded
in control media or MRM were added to the plate at various effector T cell (E)
to tumor cell (T)
ratios, including 1:1, 2.1, and 4.1. TIL clearance of tumor cells was
monitored over time. FIG. 16
shows that TILs cultured in MRM displayed superior tumor cell lysis in an ex
vivo assay as
compared to TILs cultured in control media across a range of relative doses,
with a 4:1 ratio of
effector T cells cultured in MR_I\4 to tumor cells resulting in complete tumor
eradication.
Example 10. Analysis of stemness of tumor cells ex vivo
[0587] Non-small cell lung cancer (NSCLC) TILs produced
following standard TIL
expansion methods were compared to TIL produced in MRM, according to the
methods disclosed
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herein. While the control process generated TILs highly enriched for CD8+ T
cells, these cells
were not enriched for a stemlike CD8+CD39-CD69- T cell population and
expressed low levels of
central memory markers and co-stimulatory receptor, CD27 (FIGs. 17A-17D). The
control
expansion process generated T cell products with enriched stemlike CD8+CD39-
CD69- T cells
and higher expression of co-stimulatory receptor CD27, but resulted in reduced
expansion of CD8+
T cells. TILs cultured in MRM yield highly enriched populations of CD8 I T
cells with enhanced
abundance of CD8+CD39-CD69- stemlike T cells, central memory markers
(CD45RO+CD62L+),
and markedly higher expression of co-stimulatory receptor CD27 (FIGs. 17E-
17H). Collectively
these markers demonstrate attributes of stem-like cells that are correlated
with clinical responses.
[0588] TIL products with enhanced stemlike properties, including
retention of key co-
stimulatory receptors CD27, elongated telomeres, memory cell phenotypes and
the presence of
populations of cells enriched with CD391CD69- CD8+ T cells, are associated
with improved
clinical responses. Additional differentiation state-linked characteristics of
T cells, including
ability to secrete polyfunctional cytokines, and expression of co-stimulatory
receptor CD28 have
also been correlated with anti-tumor potency. TILs expanded in MRM exhibit
favorable
phenotypic attributes, including an increased abundance of CD8+CD39-CD69- T
cells when
compared with control TIL expansion process in melanoma, NSCLC, and colorectal
cancers (FIGs.
18A-18C).
Example 11. TIL culture in MR1V1
[0589] The data presented herein show that culturing TILs in a
culture medium comprising
increased potassium generates a TIL population that has increased expansion of
CD8+ TILs and
increased sternness, relative to TILs cultured in medium having lower levels
of potassium (e.g.,
less than about 40 mM potassium ion, e.g., 5 mM potassium ion). Further, data
suggest that
exceedingly high levels of potassium (e.g., greater than about 80 mM, greater
than about 90 mM,
or greater than about 100 mM potassium ion) in the culture medium can lead to
decreased TIL
yield, likely due to decreased TIL growth and expansion, data not shown. To
further characterize
the effects of MRM on TIL culture, (1) TILs will be cultured in control medium
(an isotonic RPMI
formulation comprising 55 mM potassium ion) during an initial expansion stage
followed by
culture in MR1V1 medium comprising between 50-70 mM (e.g., 50 mM, 55 mM, 60
mM, 65 mM,
or 70 mM) potassium ion and between 50-70 mM (e.g., 50 mM, 55 mM, 60 mM, 65
mM, or 70
mM) NaC1 during a final expansion stage, (2) TILs will be cultured in MRM
medium comprising
between 50-70 mM (e.g., 50 mM, 55 mM, 60 mM, 65 mM, or 70 mM) potassium ion
and between
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50-70 mM (e.g., 50 mM, 55 mM, 60 mM, 65 mM, or 70 mM) NaC1 during an initial
expansion
stage followed by culture in control medium during a final expansion stage;
and (3) TILs will be
cultured in MRM medium comprising between 50-70 mM (e.g., 50 mM, 55 mM, 60 mM,
65 mM,
or 70 mM) potassium ion and between 50-70 mM NaCl during an initial expansion
stage followed
by culture in MRM medium comprising between 50-70 mM (e.g., 50 mM, 55 mM, 60
mM, 65
mM, or 70 mM) potassium ion and between 50-70 mM (e.g., 50 mM, 55 mM, 60 mM,
65 mM, or
70 mM) NaCl during a final expansion stage. In all experiments, initial
culture conditions will
further comprise 6000 IU/mL IL-2, and final culture conditions will further
comprise 3000 IU/mL
IL-2. Tits cultured according to this method will be characterized for
expression of sternness and
effector markers, cytotoxicity, and yield.
Example 12 Analysis of Clonal Diversity
[0590] To assess the clonal diversity of TILs cultured in MRM,
according to the methods
disclosed above, tumor fragments and TILs obtained from the tumor fragments
(e.g., dissociated
tumor fragments) were cultured/expanded in either control media or metabolic
reprogramming
media (MRM). Total genomic DNA was isolated from tumor and TIL samples using
DNeasy
Blood and Tissue Kit (QIAGEN) and sequenced using Immuno-seq for TCRI3 and
CDR3 regions
(Adaptive Biotechnologies, Seattle, WA).
[0591] The diversity metrics of the non-small cell lung cancer
(NSCLC) and melanoma
samples were assessed by Simpsons clonality AiIpi2 where pi is the
proportional abundance of
clone i in a given sample. Simpsons clonality and productive rearrangements
were examined in
tumor fragments ("tumor"), Tits cultured in control media ("control"), and
TILs cultured in MRM
("MRM") for NSCLC and melanoma (FIG. 19A and FIG. 19B, respectively) TILs
cultured in
MRM (post-expansion) displayed Simpsons clonality of approximately 0.28 for
NSCLC and
approximately 0.38 for melanoma, which indicates high clonal diversity (FIG.
19A and FIG. 19B).
Maintenance of TCRI3 diversity represent preservation of TIL clonotypes that
infilatrated the
tumors and are tumor reactive. In contrast, TILs cultured in control media
significantly lose clonal
diversity and displayed Simpsons clonality of approximately 0.65 for NSCLC and
approximately
0.8 for melanoma (i.e., a significantly less diverse clonality, indicating
that many tumor reactive
clones are lost in the culturing process using control media) (FIG. 19A and
FIG. 19B).
Example 13: Large-Scale Expansion of TIL
[0592] The present example provides a sample protocol for the
large scale production of
less differentiated TILs. Tumor fragments are coarsely chopped in MRM media
(as described
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herein). Chopped tumor fragments are dissociated using the gMACS machine in
MRM media.
Dissociated cells are filtered and then either cryopreserved or taken directly
to pre-REP.
[0593] All cells from the dissociated tumor fragment are used at
Day 0 of the
manufacturing process. At day 0, all cells isolated from the patient's tumor
are seeded at a density
of 0.5 x 106/mL at 2 mL per well. The cells are then cultured in MRM
supplemented with IL-2 at
6000 IU/mL and IL-21 at 30 ng/mL. At pre-REP Day 4 the cells are split (1:1
vol:vol) if pII is <
7.1, and the culture medium is supplemented with 10 1 of TRANSACTTm and 14/mL
of 4-1BBL
per 2 mL of MRM cell culture media. At pre-REP Day 7, 8, or 9, split cells
(1:1) if pH is < 7.1. At
pre-REP Day 10, cells are counted. If about 30 million cells are present, the
cells are advanced to
secondary expansion. If less than 30 million cells are present, the cells are
split (1:1) if pH is < 7.1,
and pre-REP is continued for one more day. Pre-REP should not exceed 14 days.
[0594] Secondary expansion comprises two steps: static-REP and
dynamic-REP. Briefly,
TILs were isolated from tumor samples by mechanical and/or chemical
dissocation of the tumor
followed by pre-REP expansion, as described above. On day 4 of pre-REP, 4-1BB
ligand and
TRANSACTTh are added to the culture medium. Cells are harvested on day 10. If
less than 30
million cells are obtained at day 10, cells are allowed to culture for one or
two more days in the
pre-REP step.
[0595] Following pre-REP, TILs arc transferred to PL750 culture
bags for static-REP. TILs
are cultured in the presence of irradiated PBMCs (1:200) and OKT-3. The cell
culture bag(s) are
gently mixed and placed in a static incubator at 37 C and 5% C07 for a target
of 5 days. Upon
completion of the static-REP, cells are forward processed into the dynamic-
REP.
[0596] The dynamic-REP unit operation includes the inoculation
of a bioreactor and
expansion using cell culture media (MRM) The dynamic-REP unit operation is
performed to
expand the number of TILs to achieve dose requirements. A rocking motion
bioreactor
(BIOSTAT R1\4 TX, Sartorius) is used for expansion.
[0597] Based on the volume seeded in the static-REP, cell
culture media is added into the
bioreactor, and the bioreactor controls are set to predefined values.
[0598] After bioreactor setup, static-REP cells are inoculated
into the bioreactor to achieve
a working volume between 2 ¨ 3L. The culture is sampled and tested as part of
the in-process
testing. Upon conclusion of inoculation, the expansion recipe is initiated on
the Multifunctional
Control Software (MFCS) program that controls perfusion and volume addition in
the bioreactor.
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[0599] The perfusion program begins with perfusion of 25% of the
working volume every
24 hours for the initial 48 hours of expansion. Sampling and testing will
occur daily throughout
the duration of dynamic-REP. After 2 days, volume increases, and increases to
perfusion volume
may begin based on the viable cell density achieved in the bioreactor. These
volume increases
occur by entering the new pre-determined setpoints into the MFCS program, and
the perfusion
volume will be increased to maintain a perfusion rate of 25% of the working
volume.
[0600] Once the reactor has achieved the max working volume of
5L, the perfusion rate is
increased to 50% of the working volume for the duration of the expansion.
[0601] After 9 days of expansion, the total viable cell count
will be used to determine if an
extension of additional 3 or 4 days of expansion will be applied, to help
achieve the number cells
required to meet the intended dose. After the dynamic-REP expansion is
completed, cells will
proceed to harvest.
[0602] The harvest unit operation objective is to end the cell
expansion phase of the process
and prepare the cells for formulation and fill. Once harvest criteria are met
based on the process
control strategy, the MFCS protocol will be terminated to conclude the
programmed perfusion
schedule. Using the Sartorius RIVITX Harvest Device, the reactor bag can be
inserted to gravity
draining the entire cell culture into a transfer bag. Once transferred, the
weight and cell count are
captured to determine the harvested culture TVC. The cells are spiked with HSA
(2% w/v) prior
to forward processing to formulation.
[0603] The drug product formulation unit operation is comprised
of washing, buffer
exchanging, and resuspending the expanded T1L product. The Sefia S-2000 and
respective sterile
single use kit is used to allow for automated concentration, wash, and buffer
exchange of the
expanded cell culture. Once the equipment and kit are setup the cells are
volume reduced to reduce
the up to 5.5 L culture down to less than 200 mL and spent cell culture media
is transferred to
waste. The cells are then washed with Plasmalyte containing 2% (w/v) HSA
before the cells are
concentrated to 1x109 cell s/mL and diluted with a 6%-7%DMS0 cryopreservation
buffer (mixture
of CryoStor CS10 and Cryostor 5) The final drug product volume is calculated
to achieve a
formulated drug product cell density of 200x106 cell s/mL, with additional
manual dilution, using
6%-7% DMSO cryopreservation buffer, executed in cases where the target
formulated volume is
greater than 300 mL maximum output volume of the Sefia. Once the Sefia and
additional dilution
as necessary is completed the formulated drug product is weighed, sampled for
release assays
according to the sample plan, and transferred into CryoMACS 250 bags. The
CryoMACS 250 bags
are filled to a target fill volume of 50 mL, and a final bag fill volume of
between 30 mL to 70 mL.
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Once each bag is filled the excess air is removed from the bag and sealed
using a sealer designed
for EVA tubing. The sealed bags are placed individually in cassettes and
frozen via controlled rate
cryopreservation to ramp cells to -100 C. Once the control rate freezer
profile has completed the
frozen drug product cassettes and vials are transferred via temperature-
controlled storage device
and stored in LN2 until the therapeutic drug product can be administered to
the patient.
[0604] We assessed whether MR1\4 TIL products generated at
research (small) scale and
large scale are comparable. TIL from three metastatic melanoma tumors
underwent the 1VIR1\4
production at both small and large scale. The TIL products were analyzed for
CD4+:CD8+ T cell
composition, sternness characteristics (%CD39-CD69- and %CD27+) and exhaustion
(%PD1+) in
CD8+ T cells and sternness characteristics (%CD39-CD69-) in CD4+ T cells, with
no significant
differences observed (data not shown). Additionally, bulk RNAseq analysis
showed consistent
gene expression of stemness and exhaustion markers between small and large
scale Epi-R samples
(data not shown). These results indicate that TIL expanded using small and
large scale MRM
procedures have comparable phenotypes.
Example 14: Transcriptional Profiling of DO gMACS and TIL Products by CITE-Seq
[0605] Cryopreserved DO gMACS (i.e., single cell suspentions)
and TIL products (both
small and large scale) were processed post thaw for Cellular Indexing of
Transcriptomes and
Epitopes by Sequencing (CITE-Seq) analysis CITE-Seq allows simultaneous
measurement of
single cell RNA and cell surface protein.
[0606] Cryopreserved DO gMACS and TIL products were thawed and
processed for CITE-
seq on separate days. Cell viability and counts were assessed using the
Cellometer K2 Cell Counter
(Nexcelom). For sorting, cells were stained with Live/Dead eFluor780 reagent
for 10 min at RT,
blocked with 10% Human TruStain FcX blocking reagent for 10 min at 4 C,
followed by staining
with a mix of fluorochrome-conjugated antibodies against CD45, CD4 and CD8,
DNA-conjugated
Total-SeqC antibodies against multiple cell surface proteins, and unique
hashtag antibodies
containing DNA barcodes for 30 min at 4 C. LiveCD45+ T cells from DO gMACS,
and equal
proportion of LiveCD45+ CD4+ and LiveCD45+ CD8+ T cells from TIL products were
FACS
sorted into PBS containing 2% BSA using the BD FACSAriaTM Fusion Cell Sorter
(BD
Biosciences). Sorted cells from all uniquely barcoded samples with hashtag
antibodies were pooled
together and then counted using the Cellometer K2 Cell Counter (Nexcelom).
Pooled cells were
loaded into each channel of the Chromium Next GEM Chip K using a Chromium Next
GEM Single
Cell 5' v2 Reagent Kit (10x Genomics). After single cell capture and lysis,
cDNA was synthesized,
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and along with DNA conjugated to antibodies bound to cell surface proteins,
amplified over 13
cycles according to the manufacturer's protocol (10x Genomics). The amplified
cDNA from
polyadenylated mRNA and the amplified DNA from cell surface protein antibody
barcodes were
separated by size selection for generating independent barcoded 5' Gene
Expression (GEX)
libraries and Antibody derived tag (ADT) libraries, respectively. The GEX and
ADT libraries
prepared from multiple channels of the Chromium Next GEM Chip K were
quantified, pooled, and
sequenced together using the NovaSeq 6000 System (Illumina). An average
sequencing depth of
at least 30,000 reads per cell for GEX libraries and at least 5,000 reads per
cell for ADT libraries
was obtained.
[0607] The CD8+ TIL product obtained according to the methods
disclosed herein in MRM
was found to be enriched for stem-like phenotype, by both bulk and single-cell
RNA-seq analysis.
TILs generated from culture in MR1VI had generally higher expression of genes
associated with a
stem-like phenotype, which were generally expressed at lower levels by TILs
generated using AIM
V or a control media (FIG. 20A). CITEseq + scTCRseq was also conducted on each
of the
products. Using the Rosenberg DN stem score and CD39 and CD69 protein
expression we
identified the stemlike populations in each product. The TMRM product has
significantly more
stemlike cells than both control and AIM V, as calculated by a 1 sided t-test
(FIG. 20B). The
proportion of stemlike cells was gerenerally higher in the CD8+ TIL product
obtained using MRM
as compared to control or AIM V (FIG. 20C; each data set representing an
individual donor). A
similar effect was also observed in the CD4+ TIL products (FIGs. 20G-201).
[0608] Gene sets used in the analysis described in this example
are shown in Table 3.
Table 3: Gene Sets.
Panel Name Genes
1 Caushi.Stem- SELL, CCR7, S1PR1, KLF3, TCF7, GPR183,
SCSD,
like.memory FAAH2, LTB, SESN3, MAL, TSHZ2, LEF1,
AP3M2,
SLC2A3, ICAM2, PLAC8, SCML1, IL7R, ABLIM1,
RASGRP2, TRABD2A, SATB1, ALG13, ARIDSA, BACH2,
PABPC1, GPCPD1, NELL2, TAF4B, FCMR, ARRDC2,
Clorf162, FAM177A1, ANKRD12, TXK, SORL1, AQP3,
ADTRP, FXYD7, CD28, P2RY8, CRYBC1, TNFSF8, 13EX2,
PGAP1, PTGER4, MAML2, BEX3, PCSK1N, INPP4B,
AC119396.1, CXCRS, LINC00402, CCR4, IL6R, ZBTB10,
ITGA6, ARMH1, RILPL2, FOXP1, TESPA1, YPEL5, LPAR6,
CMSS1, RIPOR2, ZNF331, EMP3, GIMAP7, WDR74, RIC3,
CYSLTR1, ITGB1, CDS, SAMHD1, SERINCS
2 Caushi.CD8.Stem- CCR7, SELL, IL7R, LEF1, KLF3, S1PR1,
BACH2,
like.memory GPR183, PABPC1, SESN3, LTB, NFKB1,
SCML1, P2RY8,
TRABD2A, ARMH1, PCSK1N, BEX2, FAAH2, PGAP1, LM07,
CMSS1, YES1, ACTN1, NELL2, ZBTB10, SCSD, ICAM2,
GPCPD1, FAM177A1, TCF7, SATB1, YBX3, CXCR3,
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JMJD1C, AP3M2, RGCC, FAM107B, PRNP, SLC2A3, ELL2,
TESPA1, TNFAIP8, TAF4B, TXK, PLAC8, RASA3, FOXP1,
ABLIM1, SDCBP, TLE4, ANKRD12, ALG13, EMP3, FCMR,
SAMHD1, PTGER2, LITAF, SORL1, TNFSF8, MAL, NSG1,
APBA2, RIPOR2, ITGA6, NOSIP, SERINC5, FXYD7, ATM,
CD28
3 DN stem S100A10, ZFP36L2, ANXA1, RPL35A,
RPL34, TPT1,
AQP3, RPL30, SORL1, S100A11, NOSIP, RPL14, NACA,
ODF2L, PLP2, FXYDS, RPL10, TIMP1, PIK3IP1,
LEPROTL1, LDLRAP1, MYC, LEF1, CDSS, EEF1A1, RPL39,
RGS10, SMDT1, TCF7, RPS28, FAM102A, RPL5, GPR155
4 Jansen Stem.like ENPP5, PLAC8, RAPGEF5, CCR6, IL18R1,
SSBP2,
GALNT3, MPP7, KLF3, RASGRF2, RPS6KA2, SPART, RORA,
IL7R, MYC, PLCB1, DENNDSA, CY5561, GCSAM, CD226,
KLRB1, IL18RAP, TGFBI, TPRG1, RIMKLB, ITGA5,
LRRC2, FAM241A, ANXA1, TCF7, GPR183, SPEF2,
TGFBR3, JAML, DPP4, TNFSF14, SESN1, TMEM71,
SLAMF1, MYBL1, STAT4, EGR1, SORL1, LY9, ADRB2,
CD83, SCML4, ABCB1, PRNP, GLIPR1, KCNA3, MPZL3
Zhang TEX 3Tumors HAVCR2, PDCD1, ENTPD1, TNFRSF9, SIRPG, CTLA4,
CXCL13, TOX, IGFLR1, MY07A, ITM2A, VCAM1, TIGIT,
SN7P47, LAG3, PARK?, NDFIP2, CD27, ACP5, BST2,
CCL3, CD27-AS1, CD38, CD63, CTSD, CXCR6, DUSP4,
FKBP1A, GZMB, HLA-DRA, IFNG, ITGAE, MIR155HG,
PHLDA1
6 Zhang TEX onlyColon CXCL13, HAVCR2, CCL3, VCAM1, CD82,
PDCD1, RBPJ,
GZMB, CXCR6, SIRPG, ITGAE, MIR155, CD27, ACP5,
MIR155HG, CD27-AS1, CD63, IFNG, TIGIT, TNFSF4,
FASLG, FKBP1A, HLA-DRA, TNFRSF9, PHLDA1, HLA-DQA1,
NDFIP2, ANXAS, APOBEC3C, APOBEC3G, IFI6, ITM2A,
BST2, CTLA4, TOX, HLA-DRB5, MIR497HG, HLA-DRB6,
ENTPD1, MY07A, AFAP1L2, LAG3, BATF, GZMH, NAB1,
FKBP1A-SDCBP2, PKM, HLA-DRB1, CD38, TNFRSF18, HLA-
DQB1, SIT1, DUSP4, TNIP3, CCL4L1, IGFLR1, CTSD,
CCL4, CCR1, ISG15, SN7P47, PARK7, CTSW, COX5A,
IFI27L2, FUT8, GSTO1
7 SadeF CD8 3_EXHAUST CCL3, EPSTI1, CD38, FASLG, IFI44L,
GIMAP6, TRAFD1,
LGALS9, CXCR6, RAB37, CCR5, ZBP1, SAMD9L, SIRPG,
MX1, HAVCR2, ACPS, DDX60, PDCD1, SH2D3C, GPR174,
RPS6KA1, GBPS, GBP1, PTPN6, SlOOPBP, IFI35, OAS3,
SNAP47, GIMAP4, PARP9, IFNG, SIT1, PYCARD, RGS3,
XAF1, OAS2, C5orf56, GIMAP5, ABT3, SNX20, VAMPS,
IRF2, UBASH3A, PARP10, GIMAP7, GBP4, PVRIG, CYTH4,
DTX3L, RHOC, SASH3, CCL4L2, IFI6, BCAS4, IKZF3,
GIMAP2, ADORA2A, ARPC5L, GYG1, SLFN5, CHST12,
APOBEC3D, WARS, UBE2L6, TMEM140, CSK, F2R, CTSS,
SLAMF7, CXCR3, CD27, PPP1R18, TOX, CTSC, SLAMF6,
STAT1, FUT8, IDH2, PCED1B, BST2, PSMB10, STAT2,
RNASET2, RBCK1, SEL1L3, C14orf159, HLA-DRA, GZMA,
CD63, DENND2D, HLA-DQB1, PRF1, CD84, TIGIT,
CCL4L1, PLSCR1, LAG3, DAXX, PHF11, IGFLR1,
ATP6V1E1, ELA-DQA1, CMTM3, DNAJC4, LASP1, HLA-DMA,
NCKAP1L, OASL, TMEM179B, CCL4, USB1, UBE2F, CHMP5,
C19orf66, PPM1M, ST8SIA4, YARS, TBC1D10C, DRAP1,
POLD4, TRAPPC1, PKN1, JAKMIP1, LCP2, CASP4, APOL2,
CASP1, ABCA2, HLA-DRB5, IFI27L2, SYNRG, ARHGAP30,
IRF7, RARRES3, HMOX2, GZMH, ISG15, CHFR, TRPV2,
ZNHIT1, HLA-DPA1, UBA7, ADAM8, GOLIM4, SERPINB1,
ATF6B, SHISA5, ITGB7, TMBIM4, TRAF3IP3, GPR171,
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TRAF5, ARHGEF3, PSMB8, IL2RB, APOBEC3G, CALC00O2,
DTHD1, LY6E, PPCS, CAPN1, GBP2, PYHIN1, FKBP1A,
NUDT22, CTSD, TRIM14, SLC25A45, KLRD1, UCP2,
UNC13D, PSMB9, GSDMD, IRF9, MPG, MY01F, SLFN12L,
FERMT3, MUS81, APOL6, C17orf62, FCRL3, ICAM3,
SP140
8 EM.associated.GSE233 MTCH2, RAB6C, KIAA0195, SETD2, C2orf24,
NRD1,
21 GNA13, COPA, SELT, TNIP1, CBFA2T2,
LRP10, PRKCI,
ARE, ANKS1A, PNPLA6, WDFY1, MAPK1, GPR153, SHKBP1,
MAP1LC3B2, PIP4K2A, HCN3, GTPBP1, TLN1, C4orf34,
KIF3B, TCIRG1, PPP3CA, ATG4D, TYMP, TRAF6,
C17orf76, WIPF1, FAM108A1, MYL6, SPCS2, GGT3P,
GALK1, CLIP4, ARL4C, YWHAQ, LPCAT4, ATG2A, IDS,
TBC1D5, DMPK, ST6GALNAC6, REEPS, ABHD6, KIAA0247,
EMB, TSEN54, SPIRE2, PIWIL4, ZSCAN22, ICAM1, CHD9,
LPIN2, SETD8, ZC3H12A, ULBP3, IL15RA, HLA-DQB2,
LCP1, CHP, RUNX3, TMEM43, MEF2D, ABL1, TMEM39A,
PCBP4, PLCD1, CHST12, RASGRP1, C1orf58, Cllorf63,
C6orf129, FHOD1, DKFZp434F142, PIK3CG, ITPR3,
C4orf50, CNNM3, IFI16, AK1, CDK2AP1, REL, BCL2L1,
MVD, TTC39C, PLEKNA2, FKBP11, EML4, FUCA2, MF9D10,
TBCD, CAPN2, IQGAP1, CHST11, PIK3R1, MYOSA,
KIR2DL3, DLG3, MXD4, RALGDS, S1PR5, WSB2, CCR3,
TIPARP, SP140, CD151, SOX13, KRTAP5-2, NF1, PEA15,
PARP8, RNF166, UEVLD, LIMK1, CACNB1, TMX4, SLC6A6,
LBA1, SV2A, LLGL2, IRF1, PPP2R5C, CD99, RAPGEF1,
PPP4R1, OSBPL7, FOXP4, SLA2, TBC1D2B, ST7, JAZFl,
GGA2, PI4K2A, CD68, LPGAT1, STX11, ZAK, FAM160B1,
RORA, C8orf80, APOBEC3F, TGFBI, DNAJC1, GPR114,
LRP8, CD69, CMIP, NAT13, TGFB1, FLJ00049, ANTXR2,
NR4A3, IL12RB1, NTNG2, MLLT4, GPRIN3, ADCY9,
CD300A, SCD5, ABI3, PTPN22, LGALS1, SYTL3, BMPR1A,
TBK1, PMAIP1, RASGEF1A, GCNT1, GABARAPL1, STOM,
CALHM2, ABCA2, PPP1R16B, SYNE2, PAM, Cl2orf75,
CLCF1, MXRA7, APOBEC3C, CLSTN3, ACOT9, HIP1, LAG3,
TNFATP, DCRID1, KLF6, CACNR, RNF19A, R7\R27A,
FADS3, DLG5, APOBEC3D, TNFRSF1B, ACTN4, TBKBP1,
ATXN1, ARAP2, ARHGEF12, FAM53B, MAN1A1, FAM38A,
PLXNC1, GRLF1, SRGN, HLA-DRB5, B4GALT5, WIPI1,
PTPRJ, SLFN11, DUSP2, ANXAS, AHNAK, NE01, CLIC1,
EIF2C4, MAP3K5, IL2RB, PLEKHG1, MY06, GTDC1,
EDARADD, GALM, TARP, ADAM8, MSC, HNRPLL, SYT11,
ATP2B4, NHSL2, MATK, ARHGAP18, SLFN12L, SPATS2L,
RAB27B, PIK3R3, TP53INP1, MBOAT1, GYG1, FAM46C,
ZC3HAV1L, ANXA2P2, CTNNA1, NPC1, C3AR1, CRIM1,
SH2D2A, ERNI, YPEL1, TBX21, SLC1A4, FASLG,
PHACTR2, GALNT3, ADRB2, PIK3AP1, TLR3, PLEKHA5,
DUSP10, GNA01, PTGDR, FRMD4B, ANXA2, EOMES, CADM1,
MAF, TPRG1, NBEAL2, PPP2R2B, PELO, SLC4A4, KLRF1,
FOSL2, ROS2, TGFBR3, PRF1, MY01F, GAB3, Cl7orf66,
MICAL2, CYTT-13, TOX, HLA-DRA, SYNE1, PYNIN1, F2R,
PLD1, THBS1, CD58, FAS, NET02, CXCR6, ST6GALNAC2,
DUSP4, AUTS2, Clorf21, KLRG1, TNIP3, GZMA, PRR5L,
PRDM1, ST8SIA6, PLXND1, PTPRM, GFPT2, MYBL1,
SLAMF7, FLJ16686, GNLY, 7E132, CST7, IL18RAP, CCLS,
KLRD1, KLRB1
9 Oliveira.TTE KRT86, RDH10, ACP5, CXCR6, HMOX1,
LAYN, CLIC3,
HAVCR2, AC243829.4, PRF1, SLC2A8, CHST12, GALNT2,
ENTPD1, LAGS, GZMB, PDCD1, CARD16, CTLA4, SLA2,
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CO27, RALA, VCAM1, SYNGR2, NKG7, LSP1, CCL5,
RARRES3, CD7, CTSW, MTSS1, PTMS, BATF, KIR2DL4,
AKAP5, C038, RAB27A, GZMH, IGFLR1, ATP8B4, C063,
HOPX, TNFRSF18, ADGRG1, PLPP1, CSF1, TNFSF10,
SNAP47, LINC01871, MY01E, ZBED2, AHIl, ABI3,
FASLG, TYMP, ZBTB38, CTSB, PLSCR1, AFAP1L2, ITGAE,
TNS3, DUSP16, CASP1, CARS, DUSP5, IFIT1, SLC1A4,
GOLIM4, RSAD2, DNPH1, NBL1, ACOT9, ABHD6, OAS1,
SLC27A2, ZBP1, CD200R1, OAS3, CMPK2, TNFSF4,
POLR1E, CADM1, HELZ2, SYTL2, AGPAT2, UBE2F,
GIMAP6, ZBTB32, RIN3, PLEKHF1, CHPF, PACSIN2,
ABCB1, SPATS2L, USP18, TMEM9, KLRC1, MPST
Oliveira.Tumor- KRT86, RDH10, HMOX1, AFAP1L2, ACP5, MY01E, LAYN,
spec.TTE TNS3, TNFSF4, AKAPS, HAVCR2, ENTPD1,
SLC2A8,
AC243829.4, ZBED2, CAV1, GOLIM4, VCAM1, PON2,
MTSS1, C038, MS4A6A, TOX2, CSF1, GALNT2, FXYD2,
PLPP1, LMCD1, MYL6B, LAG3, IGFLR1, CCDC50, CO27,
KIAA1324, CDKN2A, CD70, ABHD6, CTLA4, PDCD1, GEM,
TOX, CXCR6, HOPX, CLIC3, INPP5F, SNAP47, TSHZ2,
HLA-DMA, SIT1, PYCARD, ADGRG1, PRF1, PTMS, HIPK2,
CHST12, LSP1, FAM3C, SLC1A4, NUDT1, DNP1I1, ONLY,
SLA2, CD7, ISG15, ISG20, OASL, ANXA1, GZMB, MX1,
RPS26, PBXIP1, LEPROTL1, RSAD2, SELPLG, ARHGAP9,
ATP1B3, CCL5, RARRES3, S100A11, ANXA2, IFIT1,
IFIT3, CREM, CRIP1, TAGLN2, SRGN, SLC7AS, RALA,
APOBEC3G, PLSCR1, LITAF, FTH1, TGFB1, TNFSF10,
GGA2, IFITM3
11 REACTOME CHOLESTEROL CYP51A1, MSM01, IDI1, FDFT1, SOLE,
SC5D, MVK,
BIOSYNTHESIS HMGCS1, HMGCR, DHCR24, ACAT2, HSD17B7,
REP, NSDHL,
1012, TM7SF2, GGPS1, LSS, FDPS, PMVK, MVD, DHCR7,
ARV1, PLPP6
12 HALLMARK OXIDATIVE P NDUFS3, UOCRB, NDUFS2, SDHA, UQCRC1,
NDUFA9,
HOSPHORYLATION NDUFS4, NDUFS1, NDUFA2, NDUFS8, SDHB,
NNT, ATP5P0,
ATP5MC3, NDUFS7, ATP5F1A, NDUFV1, COX5B, UQCRH,
NDUFA1, ATP5F1C, ATP5F1B, COX7B, SDHD, CYCS,
NDUFA6, NDUFAB1, COX8A, ACO2, ATP5MC1, CYCl,
NDUFB6, ATP5F1E, COX5A, UOCRC2, COX6A1, ATP5F1D,
COX6C, ATP5PF, NDUFB3, IDH3B, OGDH, NDUFB8, SURF1,
COX6B1, NDUFB5, NDUFA4, NDUFB1, COX4I1, COX7C,
UOCRFS1, SDHC, ATP6V1F, COX7A2, SUCLG1, NDUFS6,
NDUFA7, FH, NDUFV2, OXA1L, NDUFC1, UOCR11, NDUFA5,
CS, ATP6V1G1, ATP5PB, HCCS, HADHB, ATP5PD, PDHAl,
NDUFA8, OLD, OPA1, ATP5ME, PDHB, ATP5MF, NDUFB7,
IDH2, MTX2, VDAC3, MDH1, ATPSMC2, IMMT, MDH2,
SLC25A3, ATP6V1D, VDAC2, ACADM, COX7A2L, TIMM17A,
ATP6V1E1, NDUFA3, SLC25A6, IDH3G, ACADVL, ETFA,
TIMM9, IDH3A, TIMM8B, ATP6AP1, TIMM13, UQCRQ,
ABCB7, VDAC1, ATPSMG, PHB2, DECR1, SUCLA2, GOT2,
DLAT, ATP6V1H, NDUFB2, FDX1, HADHA, ATP6V1C1,
MAOB, NDUFB4, UOCR10, ETFDH, GPX4, PDHX, MFN2,
AIFM1, ACAA2, ETFE, COX11, ECHS1, PMPCA, ATP6V0B,
SLC25A5, DLST, COX15, CYB5A, ALAS1, SLC25A4,
CPT1A, SLC25A20, MTRR, COX17, CYB5R3, TOMM22,
ACAT1, MRPS11, ATP6VOC, PDK4, TIMM10, LDHA, ECI1,
MRPL11, FXN, MRPS12, COX10, RHOT1, ACAA1, ACADSB,
LDHB, MRPS30, ATP1B1, BDH2, 5LC25Al2, TIMM50,
MRPL34, ISCA1, MRPL35, IDH1, HSPA9, MRPL15,
MRPS15, TOMM70, TCIRG1, ISCU, POLR2F, NO02,
NDUFC2, MRPS22, POR, ATP6V0E1, PHYH, MPC1, GPI,
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AFG3L2, HSD17B10, CASP7, PRDX3, MGST3, HTRA2,
BCKDHA, LRPPRC, RETSAT, ECH1, RHOT2, BAX, MTRF1,
GLUD1, SUPV3L1, GRPEL1, PDP1, ALDH6A1, OAT
13 REACTOME THE CITRIC_ NDUFAF7, NDUFAB1, PDK4, PDK2, UQCRC1,
NDUFS1,
ACID TCA CYCLE AND R MPC1, CS, HAGH, NDUFB4, ME1, IDH3G, PDK3, SDHA,
ESPIRATORY ELECTRON_ ME2, L2HGDH, NDUFB2, PDPR, DLD, FH, ATP5F1D,
TRANSPORT NDUFB7, SLC16A8, ACO2, GSTZ1, NDUFAF5,
IDH3B,
SLC25A14, ETFB, OGDH, NDUFC1, UCP1, PDHX, NDUFS8,
ATP5F1B, LDHB, COX6A1, PPARD, NNT, TIMMDC1,
NDUFS7, COQ10B, COX7A2L, ATP5PB, SDHB, NDUFB3,
NDUFA8, DLST, NDUFAF4, ATP5F1E, GL01, NDUFA1,
DMAC2L, COX6131, TRAP1, COX7C, UQCR11, NDUFA5,
ECSIT, NDUFA10, COX4I1, COX7B, NDUFA2, PDHAl,
SC01, C0X16, LDHA, ATP5MC2, COQ10A, COX5B, SUCLA2,
TAC01, NDUFB5, NDUFAF1, LRPPRC, NDUFA9, ETFA,
UQCRC2, NDUFB10, SLC16A3, MPC2, SDHC, NDUFS6,
MDH2, NDUFB11, ADHFE1, NDUFB9, SURF1, DLAT,
NDUFC2, ME3, NUBPL, ATP5F1A, PDK1, SLC25A27,
ATP5MC3, ATP5PF, SLC16A1, NDUFAF6, UQCRB, NDUFS2,
ATP5MC1, NDUFV3, PDHA2, SUCLG1, COX18, NDUFAF2,
NDUFS4, UQCRQ, COX6C, PDP1, NDUFB6, ATPSF1C,
NDUFB8, COX11, IDH3A, LDHC, LDHAL6A, ATP5MG,
NDUFV1, ATP5PD, PDHB, NDUFS5, ATP5ME, UQCRFS1,
NDUFA3, TMEM126B, ETFDH, LDHAL6B, CYCS, BSG,
SUCLG2, PDP2, UQCRH, NDUFAll, UCP3, UCP2, COX8A,
ACAD9, NDUFAF3, NDUFV2, COX14, COX5A, CYCl, FAHD1,
D2HGDH, IDH2, NDUFB1, UQCR10, NDUFA12, NDUFA6,
NDUFA13, RXRA, NDUFA4, MT-ND6, MT-0O2, MT-CYB, MT-
ND2, MT-NDS, MT-001, MT-ND3, MT-ND4, MT-ND1, MT-
ATP6, MT-0O3, COX20, SDHD, VDAC1, NDUFS3, MT-ATP8,
ATP5MF, ATP5P0, NDUFA7
14 HALLMARK nLYCOLYST,q PC4K1, 7\LD07\, EN01, TPT1, PFKP,
ERMA, ALDOR,
VEGFA, MXI1, PKM, HK2, LDHA, EXT1, GUSB, PFKFB1,
PGAM1, PYGB, AK4, P4HA1, PMM2, FAM162A, SDC1,
EGLN3, PC, B4GALT7, FBP2, IGFBP3, CHPF, B3GAT3,
CHST12, HS2ST1, MPI, GNPDA1, AKR1A1, PPFIA4,
B3GAT1, CHPF2, G6PD, MDH2, CHST6, AC010618.1,
PGAM2, CHST1, GPC1, TSTA3, ALG1, GFPT1, PRPS1,
GOT1, MDH1, 5LC35A3, GALK1, EGFR, ANGPTL4, CITED2,
PLOD2, QS0X1, ME2, SPAG4, P45A2, GAPDHS, EN02,
GOT2, EXT2, SLC25A13, PDK3, CXCR4, GPC4, ECD, GNE,
B4GALT2, FUT8, MIOX, VCAN, GPC3, B3GALT6, HSPA5,
ME1, ADORA2B, UGP2, MIF, NANP, ZNF292, STC2,
TPST1, PGM2, GYS1, TKTL1, TGFA, CHST2, PHKA2,
GALE, LCT, IRS2, B4GALT1, EFNA3, LHX9, KDELR3,
TALD01, DPYSL4, VLDLR, CD44, AGL, SOX9, DDIT4,
IDUA, CASP6, GLCE, COPB2, DSC2, HS6ST2, PLOD1,
SDC2, GMPPB, NSDHL, RARS, SLC16A3, GLRX, SRD5A3,
SDC3, HDLBP, COL5A1, CLDN9, TFF3, STC1, GYS2,
SLC37A4, LEPP, SDHC, B3GNT3, ISG20, LDHC, ARPP19,
HOMER', PIE, CYBSA, HAX1, COG2, IL13RA1,
AC074143.1, CLDN3, AGRN, CLN6, TXN, PAM, CAPN5,
PKP2, ABCB6, DCN, GMPPA, BPNT1, ANG, GPR87,
GAL3ST1, ALDH7A1, NT5E, IDH1, PYGL, NDUFV3, NDST3,
PPP2CB, PSMC4, TPBG, TGFBI, GALK2, CTH, KIF2A,
CACNA1H, ANKZFl, RBCK1, ELF3, RPE, B4GALT4, RRAGD,
IER3, ALDH9A1, DLD, MERTK, GCLC, FKBP4, SOD1,
MED24, AK3, XYLT2, ARTN, PPIA, CHST4
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15 REACTOME CELL CYCLE MAD1L1, PRKAR2B, PAFAH1B1, E2F2,
PSMB1, CDK11A,
PSMC4, MNAT1, PHF20, VRK2, TUBG2, PSMA4, OFD1,
TPR, RSF1, ANAPC4, SDCCAG8, SYNE2, SP011, CUL1,
MSH4, PPP1R12A, WAPL, PPP2R5A, STAG3, TP53BP1,
KIF2A, PSME4, PPP2R5B, CSNK2A2, PRKACA, UBE2D1,
FBXW11, CLASP1, TUBA3D, PPP1R12B, DYNC1I2, FKBP6,
SMC1B, PPP2R5C, PCM1, CDC14A, HSP9OAA1, GSK3B,
SEH1L, PSMC5, UIMC1, DYNLL1, ANAPC5, PCBP4, NUDC,
PSME1, AL096870.1, AAAS, PSMD5, HSP90AB1, JAK2,
ABL1, PSMD8, MAPK1, SUN2, RBX1, EP300, PSMC6,
PSMA3, PSMC1, PSMB5, PSMA6, PSME2, REC8, NINL,
RAE1, TUBB1, PSMA7, DID01, PTK6, CSNK2A1, MAPRE1,
LPIN2, PSMD1D, RBBP7, EMD, MAPK3, PSMD7, UBE2I,
RBL2, NBN, RAB2A, PPP2CB, TUBB4A, AKT2, PIAS4,
BABAM1, PPP2R1A, CDK6, PSMA2, PSMD3, PSMD11,
YWHAE, CCND1, CEP164, NUP98, PSMD9, RNF8, E2F3,
CCND3, RAD1, RAD50, SKP1, PPP2CA, TFDP2, GORASP1,
PSMD14, POLE4, GORASP2, ORC4, SUM01, HDAC1, AKT3,
BLZFl, STAG1, CCND2, SET, NEK6, NEK9, MLH3, PDS5A,
OPTN, B9D2, CDKN1A, NUP153, PSMF1, MAX, CEP250,
PSMB2, HSPA2, NUP214, TUBA4A, AKAP9, SEMI,
TUBGCP6, YWHAH, HERC2, ARPP19, PSMA1, AJUBA,
CDKN1C, MAU2, CDC16, TUBGCP2, CLIP1, DKC1, SYNE1,
PSME3, NUP21D, H3F3B, TERF2, LPIN3, CCNA1, E2F5,
TEX1S, CDK7, YWNAQ, LPIN1, CCNH, CARLES1, MDM2,
DYNC1LI2, NUPL2, HUS1, PSMB7, MYC, DCTN3, TUBB2A,
TUBB2B, NUMA1, PPP2R1B, ACO27237.1, TUBGCP4,
RAB1A, ACTR1A, NUP54, US01, PHLDA1, SYCP3, NUP58,
R131, KIF2B, ESC01, TPS3, CSNK1D, ANAPC11, CEP131,
AKT1, PSMB6, PSMA5, NME7, COP1, ENSA, RPS27A,
DYNC1LI1, CENPC, SKP2, NHP2, HIST1H2BA, HDAC8,
MCPH1, CETN2, CDKN2B, CDKN2A, ATM, TEX12, UBC,
RAD9B, NEK7, MZT2B, TUBA3E, RAD17, SMARCAS,
RANBP2, AHCTF1, PPP2R5E, PRKCA, PSMA8, FBXL18,
SEC13, DYRK1A, BABAM2, HIST1H2BD, HIST1H4H,
DYNC1I1, PSMD4, PSMB4, TACK1, PMF1, LMNA, PSMC2,
ZNF385A, SYCE2, LEMD2, RBBP4, H3F3A, ABRAXAS1,
CLASP2, PSMD6, ANAPC10, NIPBL, PRDM9, TERT, SUN1,
YWHAZ, WRN, PSMC3, BTRC, ANAPC16, PRKCB, NDEL1,
TERF2IP, YWHAB, GOLGA2, PPP2R3B, RAB8A, =BAIA,
HIST3H3, E2F6, UBE2V2, YWHAC, UBE2E1, U22, SYCE1,
RAD9A, PHF8, KAT5, MZT2A, PSMD1, LEMD3, RAB1B,
ATR, PSMD2, DCTN2, CENPS, BANF1, PPP2R2D, POLD4,
RUVBL1, SFN, CTDNEP1, TUBB6, SSNA1, ANAPC2, CDC26,
AN1LE2, TOP3A, UBE2N, RPS27, TUBAL3, RCC2, NPM1,
CEP63, FBXL7, HIST2H3D, TUBAS, KMT5A, HIST2H2BE,
BRCC3, PSMD13, PPP1CC, SYCP2, POM121, HIST1H2AD,
HIST3H2BB, DYNC1H1, SRC, PSMD12, HIST4H4, TUBA3C,
SFIl, MDM4, CEP290, SYCP1, HIST2H3C, HIST2H2AA3,
RPA4, PSMB8, FKBPL, CSNK2B, DCTN1, MZT1, PSMB10,
E2F4, CNEP1R1, LCMT1, LIN52, NUP62, PPP1CB,
CSNK1E, PPME1, SYCE3, PPP2R2A, UBAS2, PSM1311,
H2BFS, PSMB9, TUBA4B, H2AFJ, CDK11B, LYN, TUBB8,
DYNLL2, LIN37, HIST2H4A, P0M121C, H2AFB1,
HIST1H3E, PSMB3
16 REACTOME G2_M CHECKP PSMB1, PSMC4, PSMA4, TP53BP1, PSME4,
PSMC5, UIMC1,
OINTS PSME1, PSMD5, PSMD8, PSMC6, PSMA3,
PSMC1, PSMB5,
PSMA6, PSME2, PSMA7, PSMD10, PSMD7, MEN, PIAS4,
BABAM1, PSMA2, PSMD3, PSMD11, YWHAE, PSMD9, RNF8,
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RAD1 , RAD50, PSMD14, ORC4, SUM01, PSMF1, PSMB2,
SEMI, YWHAH, HERC2, PSMA1, PSME3, YWHAQ, HUS1,
PSMB7, TP53, PSMB6, PSMA5, RPS27A, HIST1H2BA, ATM,
UBC, RAD9B, RAD17, PSMA8, BABAM2, HIST1H2BD,
HIST1H4H, PSMD4, PSMB4, PSMC2, ABRAXAS1, PSMD6,
YWHAZ, WRN, PSMC3, YWHAR, HIST3H3, UBE2V2, YWHAG,
UBB, RAD9A, KAT5, PSMD1, ATR, PSMD2, SFN, TOP3A,
UBE2N, HIST2H2BE, BRCC3, PSMD13, HIST3H2BB,
PSMD12, HIST4H4, PSMB8, PSMB10, UBA52, PSMB11,
H2BFS, PSMB9, HIST2H4A, PSMB3
17 REACTOME MITOTIC MET MAD1L1, PAFAH1B1, PSMB1, PSMC4, VRK2,
PSMA4,
APHASE AND ANAPHASE ANAPC4, WAPL, PPP2R5A, KIF2A, PSME4,
PPP2R5B,
USE2D1, CLASP1, TUBA3D, DYNC1I2, PPP2R5C, SEH1L,
PSMC5, DYNLL1, ANAPC5, NUDC, PSME1, PSMD5, PSMD8,
PSMC6, PSMA3, PSMC1, PSMB5, PSMA6, PSME2, TUBB1,
PSMA7, MAPRE1, PSMD10, END, PSMD7, PPP2CB, TUBB4A,
PPP2R1A, PSMA2, PSMD3, PSMD11, N13P98, PSMD9,
PPP2CA, PSMD14, STA01, PDS5A, B9D2, PSMF1, PSMB2,
TURA4A, SEMI, PSMA1, CDC16, CLIP1, PSME3,
DYNC1LI2, PSMB7, TUBB2A, TUBB2B, PPP2R1B, KIF2B,
ANAPC11, PSMB6, PSMAS, RPS27A, DYNC1LI1, CENPC,
HDAC8, UBC, TUBA3E, RANBP2, AHCTF1, PPP2R5E,
PSMA8, SEC13, DYNC1I1, PSMD4, PSMB4, TACK1, PMF1,
LMNA, PSMC2, LEMD2, CLASP2, PSMD6, ANAPC10, PSMC3,
ANAPC16, NDEL1, TUBA1A, UBE2E1, UBB, PSMD1, LEMD3,
PSMD2, CENPS, BANF1, TUBB6, ANAPC2, CDC26, ANKLE2,
RPS27, TUBAL3, RCC2, TUBA8, PSMD13, PPP1CC,
DYNC1H1, PSMD12, TUBA3C, PSMB8, PSMB10, PPP2R2A,
UBA52, PSMD11, PSMB9, TUBA4B, TUBB8, DYNLL2, PSMB3
18 REACTOME M PHASE MAD1L1, PRKAR2B, PAFAH1B1, PSMB1,
PSMC4, VRK2,
TUBG2, PSMA4, OFD1, TPR, ANAPC4, SDCCAG8, WAPL,
PPP2RS7\, KTF2A, PSME4, PPP2RS13, CSNK27\2, PRKACA,
UBE2D1, CLASP1, TUBA3D, DYNC1I2, PPP2R5C, PCM1,
HSP9OAA1, SEH1L, PSMC5, DYNLL1, ANAPC5, NUDC,
PSME1, AAAS, PSMD5, PSMD8, MAPK1, PSMC6, PSMA3,
PSMC1, PSM135, PSMA6, PSME2, NINL, RAE1, TUBB1,
PSMA7, CSNK2A1, MAPRE1, LPIN2, PSMD10, EMD, MAPK3,
PSMD7, RAB2A, PPP2CB, TUBB4A, PPP2R1A, PSMA2,
PSMD3, PSMD11, YWHAE, CEP164, NUP98, PSMD9,
PPP2CA, GORASP1, PSMD14, GORASP2, BLZFl, STAG1,
SET, NEK6, NEK9, PDS5A, B9D2, NUP153, PSMF1,
CEP250, PSMB2, NUP214, TUBA4A, AKAP9, SEMI,
TUBGCP6, ARPP19, PSMA1, MAU2, CDC16, TUEGCP2,
CLIP1, PSME3, NUP210, H3F3B, LPIN3, LPIN1,
DYNC1LI2, NUPL2, PSMB7, DCTN3, TUBB2A, TUBB2B,
NUMA1, PPP2R1B, ACO27237.1, TUBGCP4, RAB1A,
ACTR1A, NUP54, US01, NUP58, R131, KIF2B, CSNK1D,
ANAPC11, CEP131, PSMB6, PSMA5, NME7, ENSA, RPS27A,
DYNC1LI1, CENPC, HIST1H2BA, HDAC8, MCPH1, CETN2,
UBC, NEK7, MZT2B, TUBA3E, RANBP2, AHCTF1, PPP2R5E,
PRKCA, PSMA8, SEC13, HIST1H2BD, HIST1H4H, DYNC1I1,
PSMD4, PSMB4, TAOK1, PMF1, LMNA, PSMC2, LEMD2,
H3F3A, CLASP2, PSMD6, ANAPC10, NIPBL, PSMC3,
ANAPC16, PRKCB, NDEL1, GOLGA2, TUBA1A, HIST3H3,
YWHAG, UBE2E1, UBB, PHF8, MZT2A, P9MD1, LEMD3,
RAB1B, PSMD2, DCTN2, CENPS, BANF1, PPP2R2D,
CTDNEP1, TURR6, SSNA1, ANAPC2, CDC26, ANKLE2,
RPS27, TUBAL3, RCC2, CEP63, HIST2H3D, TUBA8,
KMT5A, HIST2H2BE, PSMD13, PPP1CC, POM121,
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HI ST1H2AD , HIST3H2BB, DYNC1H1, PSMD12, HIST4H4,
TUBA3C, SFIl, CEP290, HIST2H3C, HIST2H2AA3, PSMB8,
CSNK2B, DCTN1, MZT1, PSMB10, CNEP1R1, NUP62,
CSNK1E, PPP2R2A, UBA52, PSMB11, H2BFS, PSMB9,
TUBA4B, H2AFJ, TUBB8, DYNLL2, HIST2H4A, POM121C,
H2AFB1, HIST1H3E, PSMB3
19 REACTOME THE ROLE OF PSMB1, PSMC4, PSMA4, PSME4, TUBA3D,
HSP9OAA1,
_GTSE1 IN G2 M PROGR PSMC5, PSME1, PSMD5, HSP90AB1, PSMD8, PSMC6,
ESSION AFTER G2 CHEC PSMA3, PSMC1, PSMBS, PSMA6, PSME2, TUBB1, PSMA7,
KPOINT MAPRE1, PSMD10, PSMD7, TUBB4A, PSMA2,
PSMD3,
PSMD11, PSMD9, PSMD14, CDKN1A, PSMF1, PSMB2,
TUBA4A, SEMI, PSMA1, PSME3, PSMB7, TUBB2A, TUBB2B,
TP53, PSMB6, PSMAS, RPS27A, UBC, TUBA3E, PSMA8,
PSMD4, PSMI84, PSMC2, PSMD6, PSMC3, TUBA1A, UBB,
PSMD1, PSMD2, TUBB6, TUBAL3, TUBA8, PSMD13,
PSMD12, TUBA3C, PSMB8, FKBPL, PSMB10, UBA52,
PSMB11, PSMB9, TUBA4B, TUBES, PSMB3
20 Oliveira.TEM GYG1, GPR183, GZMM, MEP, ANXA2, ANXA1,
IL7R,
S100A10, TCF7, PIK3R1, MCUB, GLUL, AUTS2, PERP,
CD300A, FAM102A, CMTM7, P2RY8, MATK, TNFSF14,
Clorf21, LGALS3, SATB1, LINCO2446, DKK3, EPHA4,
SAMD3, FRMD4B, AOAH, S1PR1, HLA-DQA2, GPR171,
ITGA5, TRMO, ANTXR2, AIM2, IFIT2, TSPAN2
21 Caushi.CD8- NKG7, GNLY, GZMH, S1PR5, GZMB, CCL4,
CCL4L2, PRF1,
Effector(3) CTSW, Clorf21, KLRD1, CST7, KLRG1,
SPON2, CD8A,
PLEK, CCL3, CCL5, SAMD3, ZEB2, ADGRG1, MATK,
FCGR3A, ENC1, TBX21, LYAR, CD8B, KLRF1, LITAF,
ADAH, TYROBP, PRSS23, PIK3R1, GZMA, FLNA, S1PR1,
KLF3, EOMES, ADRB2, LINC00861, HLA-DPB1, KLRC3,
CCL3L1, XCL2, SLA2, SRRT, IFNG, YBX3, LGALS1,
HOPX, RUNX3, TNFSF9, SLAMF7, APOBEC3G, ARRDC3,
PTGER2, MCOLN2, P2RY8, IFNGR1, AL118516.1, TUBB4A,
GABARAPL1, PYHIN1, CD300A, PTGDR, FCRL6, MYBL1,
LILRB1, AC004687.1, PLAC8, AUTS2
22 SadeF CD8 4_MEMORY E LMNA, NR4A3, GPR183, CDKN1A, CCR7,
S1PR1, KDM6B,
FF ELL2, TIPARP, SCSD, PLK3, ODES, NR4A1,
REL, PBX4,
TNF, IL7R, RGCC, FOSL2, SIK1, CSRNP1, GPR132,
GLUL, KIAA1683, RALGAPA1, PRNP, PRMT10, SORL1,
FAM177A1, CHMP1B, ZC3H12A, TSC22D2, P2RY8, NEU1,
TCF7, ZNF683, MYADM, ATP2B1, CREM, OAT, NFE2L2,
DNAJB9, SKIL, DENND4A, SERTAD1, YPEL5, BCL6, EGR1,
PDE4B, ANXA1, SOD2, RNF125, GADD45B, SELK, RORA,
SELL, MXD1, IFRD1, PIK3R1, NECA, MPZL3, USP36,
INSIG1, LTD, NR4A2, SLC2A3, PERI, S100A10, AIM1,
MGAT4A, CDC42EP3, NDEL1, IDI1, EIF4A3, BIRC3,
TSPYL2, DCTN6, HSPH1, C0K17, DDX21, PPP1R15B,
ZNF331, BTG2, AMD1, SLC7A5, POLR3E, JMJD6, CHD1,
TAF13, VPS37B, GTF2B, PAF1, BCAS2, RGPD6, TUBA4A,
RASA3, GPCPD1, RASGEF1B, DNAJA1, FAM46C, PTP4A1,
=BAIA, ZFAND5, SLC38A2, PLIN2, HEXIM1, TMEM123,
JUND, MTRNR2L1, GABARAPL1, STAT4, ALG13, FOSE,
GPR65, SDCBP, HBP1, MAP3K8, RANBP2, FAM129A, FOS,
DDIT3, CCNH, RGPD5, ATP1B3, GLIPR1, PRDM2, EMD,
HSPD1, MORF4L2, IL21R, NFKBIA, LYAR, DNAJB6,
TMBIM1, PFKFB3, FAM65B, MED29, B4GALT1, NXF1,
BIRC2, ARHGAP26, SYAP1, DNTTIP2, ETF1, BTG1,
PBXIP1, MKNK2, DEDD2, AKIRIN1
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Caushi et al., Nature 2021;596:126-132. Jansenet et al., Nature 2019. Zhang et
al., Nature
2018;564:268-272. Oliveira et al., Nature 2022. Moshe Sade-Feldman et al.,
Cell 2018;175:998-
1013.e20. Krishna et al., Science 2020;370:1328-1334. Subramanian et al.,
Proc. Natl. Acad. Sci.
U. S. A. 2005;102:15545-15550.
[0609] Metabolic Phenotype and Cell-Cycle
[0610] The CD8 I TIL product obtained according to the methods
disclosed herein in MRM
was also found to have a less exhausted phenotype. TILs generated from culture
in MRM had
generally lower expression of genes associated with a exhaustion, which were
generally expressed
at higher levels by TILs generated using AIM V or a control media (FIGs. 20D-
20E), and the
proportion of exhausted cells was gerenerally lower in the CD8+ TIL product
obtained using MRM
as compared to control or AIM V (FIG. 20F; each data set representing an
individual donor). A
similar effect was also observed in the CD4+ TIL products (FIGs. 20G, 20H, and
20J).
[0611] T-cell metabolism and immunological function are closely
connected: stem-like T-
cells predominantly use oxidative phosphorylation (OXPHOS) for energy
production, whereas in
effector T-cells glycolysis is preferentially utilized (Pearce et al., 2013,
Bourgeois et al., 2018).
Therefore, OXPHOS is considered more favorable in T-cell products. In
addition, Vardhana et al.,
2020 demonstrated that constant antigen exposure leads to altered metabolism
thereby limiting
intratumoral T-cell proliferation and self-renewal. The hallmark of this
altered metabolism is a
rapid induction of mitochondrial oxidative stress that limits the ability of T-
cells to engage in
oxidative phosphorylation, resulting in bioenergetic limitations that are
sufficient to block T-cell
proliferation.
[0612] To assess the metabolic fitness of the products, bulk
RNA-Seq analysis was
performed on CD45+CD8+TIL sorted from MRNI and Control products. The
comparison between
MRIVI and Control products was performed using bulk RNA-seq data from 4
donors. Gene set
enrichment analysis revealed that genes upregulated in MRM TIL product were
significantly
enriched for the OXPHOS, respiratory electron transport, and cholesterol gene
sets when compared
to Control TIL product (FIGs. 21A-21B). Additionally, genes downregulated in
the MRM TIL
product were significantly enriched for the glycolysis gene set. In addition,
enrichment of cell cycle
associated gene sets was also seen in the MRM TIL product when compared to the
Control TIL
product indicating that MRM product is capable of enhanced proliferation.
[0613] Tumor-Reactive Clones
[0614] CITEseq + scTCRseq was conducted on Day() gMACS t cells
from 6 donors.
Clustering using the R package Seurat was done by first normalizing the cells
by library size using
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the function NormalizeData. Cells were classified as either CD4 and CD8 based
on asinh scaled
expression of the proteins CD4 and CD8. Cells with double positive or double
negative expression
of CD4 and CD8 were excluded. Additionally, cells with fewer than 500 counts
and no CD3 protein
expression were excluded. Then highly variable genes were selected using the
Function
Find VariableFeatures, with genes associated with cell cycle, TCRs, IG
complex, mitochondria and
ribosomes excluded. Additionally, genes strongly correlated (pearson
correlation > .25) with G2M
and S phases of the cell cycle were also excluded from the variable features
list. Normalized
expression of the remaining variable features was scaled and G2M and S scores
for each cell were
regressed out using the function ScaleData. After running PCA using the RunPCA
function we
used the function RunHarmony to integrate cells across the 6 donors. The
harmony vectors were
used to create UMAP using the RunUMAP function and also used to identify
clusters using the
FindNeighbors and FindClusters functions. Clusters 8 and 12 were subclustered
using the
FindSubcluster function because of heterogeneous expression of exhaustion
associated markers
(FIGs. 22A-22H).
[0615] Putative tumor reactive clusters were identified based on
expression of exhaustion
associated markers (CXC13, CD137(4-1BB), CD279(PD-1), and TIGIT(VSTM3)) which
have
been identified in the publications cited above.
[0616] The Day gMACs samples were also sequenced using Adaptive
bulkTCR
sequencing' s survey depth. In addition to using the exhaustion phenotype as a
method for
identifying putative tumor reactive clones, we use the top 100 most frequent
clones as measured
using the bulkTCR sequencing.
[0617] FIGs. 23B and 23D show the number of putative tumor
reactive clones identified
in each sample based on the exhausted phenotype in the day() 5' CITEseq data
and bulkTCR
frequency (top 100 clones). Using bulkTCR data from each product we evaluated
the number of
tumor reactive clones preserved in each process. While each donor has a
variable # of tumor
reactive clones initially, the three processes preserve similar numbers of
putative tumor reactive
clones.
[0618] By summing all the frequencies of the putative tumor
reactive clones identified in
the bulkTCR products we estimate the percentage of the product coming from
putative tumor
reactive clones. We see that the percent of product that is putative tumor
reactive clones is
relatively consistent between processes (FIGs. 23A-23C). The CD8 putative
tumor reactive clones
(FIG. 23A) constitute a larger percentage of the product than the CD4 putative
tumor reactive
clones (FIG. 23C).
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[0619] Next, we evaluated the presence of stemlike putative
tumor reactive cells by
identifying clones that have both a stemlike phenotype in the product and
putative tumor reactive
phenotype in the day() sample. The amount of clones identified in each donor
is consistent between
the different processes (FIG. 23E).
[0620] Using the putative tumor reactive clones we identified
using the exhaustion
phenotype and most frequent clones, we did pseudobulk differential expression
between the 3
processes, MRM, control, and AIM-V, to identify genes and functional programs
that are enriched
in the tumor reactive cells for the T1VIRIVI product. We used DESeq2 to model
the expression
difference between the products and used the test statistic output to as input
to fgsea to conduct
gene set enrichment analysis (GSEA). The GSEA showed that the TMRM putative
tumor reactive
cells up-regulate a memory phenotype when compared to both the AIM-V and TCM
putative tumor
reactive cells. Additionally, the MRM putative tumor reactive cells have lower
expression of
exhaustion associated gene sets (FIG. 24B). The heatmap shows significantly
differentially
expressed genes associated with exhaustion and effector memory phenotypes
(FIG. 24A). CD39
and CD69 protein is down-regulated and SELL and IL7R RNA are up-regulated in
MRM putative
tumor reactive clones (FIG. 24C). This indicates that the MRM putative tumor
reactive clones are
more stemlike than the other processes. Gating putative tumor reactive cells
on CD39 and CD69
protein expression to identify double negative (stem like) and double positive
(exhausted) cells
shows that the MRM putative tumor reactive cells have a lower proportion of
double positive
putative tumor reactive cells than Control and AIM V (FIGs. 24D-24F).
[0621] Autologous TIL Co-culture
[0622] Autologous tumor cell lines from patient tumor samples
were generated by cutting
a fraction of the tumor into 2 to 3 small pieces using a sterile scalpel. The
tumor fragments were
then incubated in 2.2 ml of pre-warmed plain RPMI/Glutamax with the addition
of 100u1 of
Enzyme H, 50u1 of Enzyme R and 12.5u1 of Enzyme A (Tumor Dissociation Kit,
Miltenyi, # 130-
095-929) in a 50m1 conical tube. The tumor and enzyme mix was incubated in a
37 C incubator
with occasional vortexing until media became turbid and tumor fractions became
more
disintegrated (1 to 2 hours). After the incubation time, 8 ml of pre-warmed
RPMI/Glutamax was
added to the 15m1 conical tube to dilute the enzymes and the tumor dissociate
was filtered through
a 70[tm filter into a fresh 50m1 conical tube. Cells were spun down at 300xg
for 7 min and then
resuspended in 10 ml of pre-warmed RPMI/Glutamax+ 10% FBS+ 1% Pen/Strep and
plated onto
a 10cm tissue culture dish and incubated at 37 C. One to two days post-
plating, the cells were
observed to assure tumor cell adherence and media was changed. Upon stromal
outgrowth (1-2
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weeks post-plating), the cells were re-plated onto an NCCD-coated 10cm dish
and incubated in
Primary Cancer Cell Medium D-ACF (Primary Cancer Culture System, PromoCell, #
C-28081).
Media was replaced every 5 days and cells were observed for tumor cell
outgrowth. Four to six
weeks later, when colonies of tumor cells were present, the cells were
replated onto a new NCCD-
coated dish by differential trypsinization. This was continued until no more
stromal cells were
present. At the end, the tumor cells were taken off the Primary Cancer Culture
System and cultured
in RPMI/Glutamax+ 10% FBS-I- 1% Pen/Strep until needed for assays or were
viably
cryopreserved.
[0623] Twenty-four hours prior to TIL co-culture experiments,
autologous tumor cells
were plated onto a 96 well E-plate (Agilent, #300600910) at 30,000 cells per
well and co-culture
experiment was performed using the xCELLigence RTCA manufacturer's recommended
protocol.
Briefly, 501.1.1 of pre-warmed RPMI/Glutamax (+10% FBS) was added into each
well of a 96-well
E-plate. An initial scan of the wells was performed in the xCELLigence RTCA MP
(Agilent) and
then tumor cells were seeded into the wells of the plate. The plate was
incubated at room
temperature for 30 min to allow the cells to settle. After the incubation, the
plate was placed back
into the xCELLigence and impedance measurements were obtained every 15 min.
The plate was
incubated overnight in the xCELLigence platform until a cell index of 1-1.5
was reached,
indicating 90-100% confluency. The next day, TILs that were rested overnight
in control media
supplemented with IL-2, were washed twice with RPMI/Glutamax (+10% FBS),
counted and
plated onto the tumor cells cells at an E:T ratio of 1:1, 5:1 and 10:1 in
RPMI/Glutamax (+10%
FBS) (FIG. 25A) or at an E:T of 10:1 (AIM-V, MRM, MRM no pre-REP boost with 4-
1BB ligand
and TRANSACTTm, and Control; FIG. 25B). Tumor cytolysis readings were obtained
by the
xCELLigence every 15 min until the end of the experiment (-30 hours).
[0624] Cytotoxicity was observed in a dose-dependent manner
(FIG. 25A) with all
processes, indicating the preservation of anti-tumor functionality (FIG. 25B).
No-boost MRM had
the most superior tumor killing compared to Control, MRM, and AIM-V (FIGs.
25B). These data
indicate that while recognition was seen with all media conditions, MRM-
expanded TIL
outperformed AIM-V in recognition. Additionally, the no-boost MRM condition
exhibited
substantially better tumor reactivity than either control.
[0625] Product cells from MRM, MRM no boost, AIM-V, and control
were then co-
cultured with corresponding autologous tumor cell line for 4 hours and then
sequenced along with
control products. Clones that were identified as tumor reactive were
identified by 4-1BB
(TNFRSF9) expression, which was defined as greater than background control
expression (shown
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for 41BB as > 2; FIGs. 26A-26C). The amount of tumor reactive clones
identified in each co-
cultured product was consistent with FIG. 25A, where MRM no boost showed the
most killing also
had the most tumor reactive clones. Differential expression between the tumor
reactive clones in
the control product identified an enriched effector memory (GNLY, ZNF683,
PRF1) and reduced
exhaustion phenotype (LAG3) in MRM, similar to the differential expression
shown with putative
tumor reactive clones (FIG. 26D). Differential expression was done using the
FindMarkers
function in Seurat.
Example 15: Characterization of TILs Cultured in MRM
[0626] Fresh or viably cryopreserved single cell suspensions
prepared from metastatic
melanoma, NSCLC and colorectal metastatic tumors underwent TIL expansion using
standard,
control, and MRM processes. Both process variants included 2 steps: (A) A pre-
rapid expansion
protocol (REP) culture and (B) a REP culture. Historically, the pre-REP
expansion of TIL has been
performed utilizing high concentrations of IL2 (60001U/ml) over 3 to 5 weeks.
High levels of IL2
are thought to be required for promoting the expansion of dysfunctional TIL.
This traditional pre-
REP TIL expansion only includes one activation signal, resulting in a long
expansion duration to
achieve sufficient cell numbers for initiating the secondary REP expansion.
The MRM TIL
expansion process described herein incorporates a pre-REP boosting step, in
which TIL are
stimulated with TRANSACTTm and 4-1BB ligand on day 4 of culture to promote T
cell
proliferation. Standard and control TIL did not undergo this boosting step and
were expanded in
IL2-containing media. The MRM process resulted in a higher rate of success in
expanding Tit
compared to control processes. Pre-REP TIC, expansion greater than 1.5-fold
was considered a
successful expansion. Across all tumors studied, the overall success rate of
pre-REP expansion of
TIL cultures was 41% (7/17) in AIM-V media, 75% (12/17) TCM media, and 100%
(17/17) for
the MRM process (FIGs. 27A-27C). Further analysis was performed to assess the
performance of
each expansion process in each of the studied tumor indications (melanoma,
NSCLC and CRM)
(FIGs. 27D-27F). The overall success rate of pre-REP TIL expansion was higher
in the settings of
metastatic melanoma and NSCLC compared with CRM, likely due to higher T cell
infiltration of
these cancer types. Importantly, in each tumor type, the MRM process exhibited
improved
performance in successful pre-REP TIL expansions.
[0627] REP fold expansion was similar between control and MRIVI
process, both of which
were higher than the standard (FIG. 27G). Irradiated PBMC (feeder cells) from
healthy donors
were used in the REP process to enhance TIL proliferation while maintaining
cell viability. As
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these allogeneic feeder cells represent potential contamination risks in the
final product, it is
important to ensure these cells are unable to proliferate after irradiation.
Irradiated feeder cells and
non-irradiated healthy donor pooled PBMCs were labelled with cell trace violet
(CTV) dye to track
proliferation upon a-CD3 stimulation. Cells were stimulated using 30ng/m1 a-
CD3 (OKT3 clone)
in media containing 1500IU/m1IL2. Non-irradiated healthy donor PBMC were able
to proliferate,
while irradiated feeder cells were unable to proliferate based on CTV dilution
assay (FIGs. 2711-
271).
[0628] Gating strategy
[0629] To perform phenotypic analysis of the products, cells
were stained for flow
cytometry analysis to measure cell surface expression of relevant protein
markers. Lymphocytes
were first gated by FSC-A vs SSC-A, then singlets by FSC-A vs FSC-H, then live
cells by
exclusion dye against F SC-A, then CD3+ T cells by CD3 vs FSC-A. In some
product analyses,
CD4+ and CD8+ T cells were sub gated and compared separately between
conditions. The CD8+
T cells were analyzed for sternness characteristics by plotting CD39 against
CD69 and gating on
the double positive population (CD39+CD69+) and double negative population
(CD39-CD69-).
CD39-CD69- T cells are stem-like cells as defined by previous literature.
CD39+CD69+ T cells
are highly differentiated cells. For co-stimulatory positive stern-like cell
populations in the CD8+
T cell subset, CD62L and CD27 or CD27 and CD127 were plotted against each
other.
CD62L+CD27+CD8+ and CD27+CD127+ CD8+ T cells were defined as co-stimulatory
positive
stem-like cells (FIG. 28). Favorable phenotypic attributes included
CD3910/CD6910
,
CD27high/CD62Lhigh, CD27high/CD127high, CD39dim/CD691' and CD39high/CD691'.
[0630] TheMRM process promotes a CD8+ T cell dominated product
[0631] Mature human alpha-beta T cells can be broadly classified
based on their exclusive
co-receptor expression; either CD4 expressing (CD4+) or CD8 expressing (CD8+).
CD4+ T cells
recognize antigen presented on HLA Class II by professional antigen presenting
cells (pAPCs) and
can be classified into several different subsets, including Th2 subsets, CD4+
helper T cells which
promote CD8+ T cell function and regulatory T cells that suppress immune
responses. CD8+ T
cells predominantly drive target cell killing cytolysis through specific
recognition of cognate
antigens presented by HLA Class I molecules. In the context of tumor
immunotherapy, while direct
recognition of tumor antigens by CD4+ T cells is sometimes limited by a lack
of tumor expression
of HLA Class IT, CD8+ T cells are capable of' recognizing tumor antigen
presented in the context
of HLA Class I directly on tumor cells, as well as (pAPCs). Therefore,
infusion of high numbers
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of CD8+ T cells with the capacity to directly kill tumor cells may be linked
with improved clinical
anti-tumor responses, as has been documented in human melanoma patients (Iran
et al., 2014).
[0632] We assessed the ratio of CD4+ and CD8+ T cells in our TIL
products using flow
cytometric analysis. While the ratio of CD4+ and CD8+ T cells varied by
patient sample, we
observed that MRM expansion of T1Ls promoted an increased abundance of CD8+ T
cells (FIG.
29A) and a corresponding decrease in the abundance of CD4 I T cells (FIG. 29B)
in the MRM
product relative to the standard and control expansion processes.
[0633] The MRM process better maintains stem like receptor
positive TILs
[0634] Upon antigen-recognition, naive T cell undergo activation
and a path of
differentiation to acquire functions for lasting effector immune response.
Some T cells differentiate
into short term effector cells, driving a robust immune response before dying
off. Other T cells
acquire memory phenotypes, which allow them to survive longer term and become
endowed with
relatively rapid recall responses should they re-encounter antigen. There is a
path of differentiation
within the memory T cell pool, resulting in multiple memory T cell subsets.
There exist a number
of hypotheses regarding the lineage of these memory T cell subsets, but the
younger or more stem-
like T memory cells are generally thought to progressively differentiation
into the terminally
differentiate subset, after which they die off Each subset has distinct
attributes and surface
markers, summarized in Table 4.
Table 4: Summary of Memmory T Cell Atribtutes and Surface Markers.
T effector memory
T stem cell memory T central memory T effector memory
cells expressing
cells (TSCMs) cells (TCMs) cells (TEMs)
CD45RA (TEMRAs)
Attribute
Self-renewal ++++ ++
Multipotency ++++
Persistence ++++ +++
Surface Marker
TCF7 ++++ +++ ++
CD62L ++++ +++ ++
CD27 ++++ +++ ++
CD127
CCR7 ++++ +++ ++
CD45RA ++
CD45R0 ++
PD1 ++
+++
[0635] Factors such as ICI{ signal strength, antigen abundance
and exposure, and the
inflammatory milieu will impact composition T cell memory pool. Given that
most T memory cells
in the tumor are experiencing repeated antigen exposure, we would expect the
most stem-like
subset of memory T cell in our TIL culture to be TCMs.
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[0636] Within each subset, a range of sternness can be
maintained, i.e., TCMs can exhibit
higher or lower levels of CD27. However, the stem-like quality of cells is
best observed when
using a combination of stem-like markers. For example, CD62L alone can be a
marker on both
memory bystander or naïve T cells, but used in combination with CD45RO, it can
mark more stem-
like TCM cells. We assess the proportion of these stem-like receptor positive
cells to compare the
stem-like attributes of our MRM TIL product versus the control TIL product.
[0637] The enhanced presence of certain stem-like receptor
positive populations in TIL has
been correlated with favorable clinical responses. For example, in patients
with checkpoint therapy
treated melanoma, CD27 and CD62L expression on CD8+ TIE, was higher in
patients with
complete remissions and partial responses compared to non-responding patients
(van den Berg et
al, 2020). Similarly, another study in metastatic melanoma patients receiving
TIL ACT showed
taht the degree of response (complete, partial, no response) was strongly
correlated with expression
of CD27 on CD8+ T cell in the infusion product (Rosenberg et al, 2011). CD127,
aka IL7 receptor,
was found to be more highly expressed in the neoantigen-specific T cells of
NSCLC patients with
a maj or pathological response versus those of NSCLC patients with no major
pathological response
(Caushi et. al, 2021).
[0638] We found that MR1\4 TIL product harbored a significantly
higher percentage of
CD62L+CD27+ (FIGs. 30B-30C) and CD27+CD127+ (FIG. 30D) CD8 T cells.
Furthermore,
MR1V1 TIL product also had higher CD27+ alone on CD8 T cells (FIG. 30A). Taken
together, the
heightened expression of these stem-like receptors on the CD8 T cells in our
MIRM TIL product
compared to the control product would suggest that our infusion product is
poised to improve
clinical responses over standard TIL products.
[0639] The MRM process enriches less differentiated TILs
[0640] The co-expression of CD39 and CD69 on CD8 TIL has
recently been identified as
a readout for their sternness, persistence, and, ultimately, favorable
clinical responses to ACT
therapy in melanoma patients. Analysis of the TIL ACT product from responder
and non-responder
patients revealed that TIL product in responders was enriched in CD39-CD69-
CD8 T cells. In
turn, stratifying the patients by abundance of CD39-CD69- CD8 T cells
effectively separated the
patients by survival, with higher CD39-CD69- CD8 T cells identifying the
surviving patients.
Further analysis of this population exhibited sternness properties, including
self-renewal capacity
persistence post-infusion. On the other hand, CD39+CD69+ TIL exhibited a more
differentiated
and exhausted phenotype. Tumor antigen responsive T cells were detected in
both populations, but
were notably higher in the CD39+CD69+ subset (Krishna, 2020).
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[0641] We turned to flow analysis to assess whether the MRM TIL
product contained a
higher percentage of CD39-CD69- CD8 T cells, and in turn, a lower percentage
of CD39+CD69+
CD8 T cells compare to the control product. MRM TIL product exhibited at least
three-fold more
CD39-CD69- CD8 T cells compared to the control product (FIGs. 31A-31B). On the
contrary,
MRM product contained more than two-fold fewer CD39+CD69+ CD8 T cells (FIG.
31C).
[0642] The effect of the MRM TIL production in not restricted to
CD8 T cells. We analyzed
the CD4+ T cells in both control and MRM products and found a similar pattern
as for CD8+ T
cells, whereby MRM enriched CD39-CD69- subset (FIG. 32A) and reduced
CD39+CD69+
population (FIG. 32B) compared to the control product. Since CD4 T cells bear
tumor-reactive
clones, and provide T cell help to improve CD8+ T cell cytolysis, their
presence and sternness
attributes in the MR1\4 TIL product could improve the anti-tumor responses by
the majority CD8
T cell population.
[0643] The MR1\4 product retains TIL polyclonality
[0644] Expansion success and enrichment of stern-like co-
receptor positive TILs by MRM
process prompted us to explore its impact on the T-cell repertoire. Initial
tumor materials can have
diverse TCR repertoire at different frequency. Due to existence of multiple
tumor reactive T cells
in the initial tumor material it is necessary to maintain diverse TCR
repertoire during ex-vivo
expansion. It is worth noting that each T cell clone has different capability
to proliferate. One of
the metrics used to understand and interpret the diversity of these cells is
clonality. To assess the
polyclonality of MRM TIL after expansion, next-generation sequencing of the
CDR3 variable
region of the TCR in MRM, standard and control TIL was performed. The Simpson
clonality
coefficient was calculated for each sample to quantitatively measure T-cell
polyclonality. In this
calculation, a value of 0 represents a completely evenly distributed sample
and 1 represents a
monoclonal sample. Therefore, Simpson clonality coefficient values nearer to 0
are more desirable
for a TIL product. The Simpson clonality was comparable between both the
control and MRM
processes (FIG. 33), suggesting that the increase in CD8+ T cells with stem-
like attributes driven
by MRM TIL expansion did not come at the expense of a loss of clonal diversity
in the final TIL
population.
Example 16: Analysis of TCR Diversity of Tits Expanded with MRM for Large
Scale and
Research Scale
[0645] To assess the clonal diversity of TILs from NSCLC,
melanoma, and colon cancer
expanded in MRM at research and large scale, according to the methods
disclosed above, tumor
fragments and TILs obtained from the tumor fragments (e.g., dissociated tumor
fragments) were
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expanded in metabolic reprogramming media (MRM). Total genomic DNA was
isolated from
tumor and TIL samples using DNeasy Blood and Tissue Kit (QIAGEN) and sequenced
using
Immuno-seq for TCRI3 and CDR3 regions (Adaptive Biotechnologies, Seattle, WA).
[0646] The diversity metrics were assessed by Simpsons clonality
AlIpi2 where pi is the
proportional abundance of clone i in a given sample. Simpsons clonality and
productive
rearrangements were examined in dissociated tumor fragments ("tumor") and TILs
cultured in
MRM ("MRM") for NSCLC, melanoma, and colon cancer (FIG. 35A). As shown in FIG.
35A,
TILs expanded in 1VIRI\4 at research scale exhibited preserved polyclonality
(as evidenced by the
comparable Simpson clonality coefficient) when compared to the clonal
diversity of the source
tumor tissue. As shown in FIG 35B, TIL cultured using the MEM process
described herein to
produce large-scale products exhibit even more polyclonalality than TILs
cultured using MRM at
research scale.
Example 17: Analysis of Cytolytic Activity and Pro-Inflammatory and
Immunosuppressive
Cytokine Secretion
[0647] In addition, to assess the specificity and tumor killing
of TILs expanded with the
MRIV1 process, autologous tumor cells were co-cultured with MRM expanded TILs.
An autologous
tumor cell line was obtained as described above. Cells were plated at an E:T
ratio of 1:1, 1:5, and
10:1; tumor cells alone were used as a control. TILs produced with MRM
exhibited potent anti-
tumor function as measured in vitro using an autologous tumor cell line,
including dose-dependent
cytolytic activities and proinflammatory cytokine secretion in response to
stimulation by anti-
CD3/CD28 (FIG. 36A-36B). The ability to produce immunosuppressive cytokines
upon antigen
stimulation with anti-CD3/CD28 was assessed and showed a significant
difference between TILs
expanded with MRM and TIL expanded with the control process. As shown in FIG.
36C, Tits
expanded and cultured in MRM produced significantly lower amounts of
immunosuppressive 1L-
4, IL-5, and 1L-13 as compared to those TILs produced using the control
process.
[0648] The above results further highlight that the MRM process
described herein (for both
research and large scale and for tumors of different cancers) can be useful in
producing TILs that
are highly diverse and exhibit potent anti-tumor activity.
Example 18: Analysis of Polyclonality and Anti-Tumor Activity of TILs Cultured
in MRM
[0649] TIL that are highly enriched with tumor-reactive T cell
clones have been shown to
mediate responses to treatment (van den Berg JH, Heemskerk B, van Rooij N, et
al. J Immunother
Cancer. 2020;8:e000848, Tran E, Turcotte S, Gros A, et al. Clin Cancer Res.
2014;344:641-645,
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Zacharakis N, Chinnasamy H, Black M, et al. Nat Med. 2018;24:724-730).
Conventional TIL
products consist of a mixture of extensively differentiated T cells and low
levels of stem-like cells;
higher proportions of stem-like T cells have been associated with improved
outcomes in patients
treated with TIL (Krishna S, Lowery FJ, Copeland AR. Science. 2020;370:1328-
1334). To further
assess whether the culturing process described herein (e.g., in M_RM) can
improve one or more
properties of TILs, the following methods were used.
Identifying, Tracking and Analyzing Putative Tumor Reactive Clones
[0650] Using validated methods for analyzing putative tumor
reactive clones (Krishna S,
Lowery FJ, Copeland AR. Science 2020;370:1328-1334. Oliviera G, Stromhaug K,
Klaeger S, et
al. Nature. 2021;596:119-125), clones that are both high frequency and display
exhausted
characteristics in the dissociated tumor suspension of Day 0 tumor samples
(from melanoma, lung,
or colorectal cancer) were identified as putative tumor reactive (FIG. 34).
Identifying clones with
an exhausted pheneotype captured those clones that are most likely to be tumor
reactive, although
these clones could vary in their differentiation states in the final expanded
TIL (e.g., product). The
top 100 high frequency TCR clones were identified using bulk RNA-seq.
Exhausted cell TCR
clones were identified using single cell RNA-seq and single-cell TCR-seq. A
subset of 100 high
frequency clones that are also "exhausted" were identified as putative tumor
reactive cells. Bulk
TCR-seq was used to determine the presence and proportion of putative tumor
reactive clones in
the expanded TILs. Single-cell RNA and TCR-seq was then used to evaluate the
phenotype of the
tumor reactive clones in the expanded TILs.
Analysis of Putative Tumor Reactive Clones
[0651] To demonstrate tumor reactivity of putative tumor
reactive clones, putative tumor
reactive TCRs were transduced into healthy donor CD8+ T cells and the
transduced cells were co-
cultured with autologous tumor cells corresponding to the selected TCRs. Tumor
only was used as
a control. Tumor reactivity was measured by cytolysis and IFNy secretion 24
hours post-co-
cu ltu re .
[0652] In addition, tumor reactivity was demonstrated in
putative tumor reactive clones
identified in MRM expanded TIL product produced at research scale and large
scale. The
expanded TIL product was co-cultured with either corresponding dissociated
tumor suspension or
corresponding tumor cell line. Tumor reactivity was measured by 41BB and IFNy
expression in
single-cell RNA/TCR-seq.
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Example 19: Analysis of the Effect of MRM on Preserving Putative Tumor-
Reactive Clones
Identified in Initial Tumor Samples
[0653] Putative tumor reactive clones from each tumor (melanoma,
lung, and colorectal
cancer) expanded in MRIVI were identified from single cell suspensions of
dissociated tumor using
bulk TCR-seq as described above to identify high frequency clones. Using
single-cell RNA/TCR-
seq, tumor reactive clones (clones that have an exhausted phenotype) from
melanoma, lung and
colon cancer were identified by CXCL13, 4-1BB, PD-1, and TIGIT (FIG. 37A-37C).
Cells
identified by tumor reactive phenotype and high frequency in bulkTCR-seq were
characterized as
putative tumor reactive and biased towards CD8+ (FIG. 37D).
[0654] To demonstrate tumor reactivity of identified putative
tumor-reactive clones from
TIL expanded in MR1\4, as described above, the top three putative tumor-
reactive TCRs were
cloned into healthy donor T cells and co-cultured with the corresponding
autologous tumor cell
line. As shown in FIG. 38A, at a E:T ratio of 5:1, two of the three TCRs
showed 100% cytolysis.
The cytolysis data correlated with the specific secretion of IFNy as shown in
FIG. 38B.
[0655] Putative tumor reactive clones from TIL cultured and
expanded in MRM at research
scale and large scale were verified as tumor-reactive by co-culturing the
expanded TIL with a
corresponding target of either autologous tumor cell line or dissocoiated
tumor suspension and
measuring activation based on a 4-1BB+/TFNy+ phenotype The results shown in
FTG. 39
demonstrate that putative tumor-reactive clones expanded in MIRM using both
the large scale and
research scale process are tumor reactive when captured at high frequencies.
[0656] Furthermore, in accordance with the verified putative
tumor reactivily approach
described above, bulkTCR-seq analysis of TILs expanded using (a) the control
process, (b) MRM
process at research scale, or (c) MRM process at large scale from melanoma,
colon and NSCLC
tumor types was performed according to the methods described above. Although
bulkTCR-seq
showed that the MRM process for expanding TILs at reaseach scale and the
control process
preserve a comparable number of putative tumor-reactive clones, the large
scale MRM process
preserved an average of 94% of the identified putative tumor reactive clones
(data not shown).
[0657] Putative tumor reactive clones were identified in Day 0
dissociated tumor samples
by tumor reactive phenotype (CXCL13, 4-1BB, PD-1, and TIGIT (Pasetto et al.
CIR 2016, Lowrey
et al. Science 2022, Oliveira et al. Nature 2021). These putative tumor
reactive clones were then
analyzed by scRNAseq as described herein to identify the number of clones
preserved after
expansion using the control process or the MRM large-scale process, as shown
in Table 5.
Table 5: Number of Putative Reactive Clones
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Sample Identified Preserved in Preserved in
from Day 0 Control MRM large-
scale
LY158 95 77 87
LY137 58 33 51
LY142 48 28 45
LY170 77 66 74
LY189 62 43 58
LY193 68 46 63
LY222 93 90
LYITD12 51 51
[0658] The above results collectively demonstrate that the MRM
process described herein
preserves the putative tumor-reactive clones that exist within the initial TIL
population and that
these putative tumor-reactive clones are indeed tumor reactive.
Example 20: Analysis of the Effect of MRM on the Sternness of Putative Tumor-
Reactive
Cells Identified in Initial Tumor Samples
[0659] Next, to further assess the effect that the MRM process
has on improving various
properties of TILs, TILs were expanded and cultured using (a) the control
process, (b) the MIRM
process at research scale, or (c) the MRIVI process at large scale. Single-
cell RNA/TCR-seq analysis
was performed to characterize the phenotype of the putative tumor reactive
from 23 TIL products
(9 control, 9 MRM research scale, and 6 MRM large scale). The Uniform Manifold
Approximation
and Projection (UMAP) for all Tits expanded under conditions (a), (b), and (c)
is shown in FIG.
40A. The stem-like clusters for all TILs expanded under all conditions were
identified by
expression of SELL+, CD39-, and CD69- and gene sets listed in Table 3 and are
presented in FIG.
40B as a gradient of expression levels. FIGs. 40C-40D show that the putative
tumor reactive clones
were generally enriched in the non-stemlike clusters, as expected And, as
shown by the differential
expression of the many different genes associated with sternness or exhaustion
in FIGs. 41A and
41B, respectively, the MRM expanded putative tumor reactive Tits for both
large scale and small
scale exhibited increased sternness and reduced exhaustion when compared to
the control_ As
further support, GSEA identified up-regulation of sternness associated
genesets and down
regulation of exhaustion associated genes in TILs expanded using MRM at both
research (FIG.
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41C) and large scale (FIG. 41D) as demonstrated by the Normalized Enrichment
Scores (NES) for
the various gene sets. And, as shown in FIG. 42, the percentage of CD8+
stemlike cells was higher
for TILs cultured and expanded in MRIVI at research scale and large scale
compared to TIL cultured
with the control process.
[0660] Collectively, the above results further demonstrate that
the global effect that the
MRIVI process provided herein has on TILs (e.g., increased stemness and
reduced exhaustion), is
seen specifically within the verified tumor reactive cell population of the
product. The results
provided here confirm that MRM processes described herein are useful in
generating TIL products
with improved properties, including tumor reactivity and stemness.
Example 21: Analysis of the Effect of MR1VI on Immune Checkpoint Inhibitor-
Treated and
Naïve Tumors
[0661] Advancement of immune checkpoint inhibitors has had
significant effect on cancer
therapeutics. However, increasing studies have suggested that certain tumors
can be resistant to
immune checkpoint therapy or become more resistant with treatment. See, e.g.,
Levi et al., Clin
Cancer Res 28(14): 3042-3052 (Jul. 2022). To demonstrate that the cell
compositions described
herein (e.g., TILs expanded and cultured in MRM) can be successfully expanded
and have a
comparable therapeutic effect on tumors that are refractory to immune
checkpoint inhibitors, TILs
from either naive tumors (i.e., not previously treated with an immune
checkpoint inhibitor) or
tumors previously treated with one or more immune checkpoint inhibitors were
expanded and
cultured using the control process or the MRM process described herein. Then,
both the stemness
and polyclonality of the resulting TILs were assessed using one or more of the
methods described
in the earlier examples.
[0662] As shown in FIGs. 43B and 43C, with the MRM process, the
TILs exhibited
increased sternness (as evidenced by increased percentage of CD39-CD69- cells)
and were more
skewed towards CD8+ T cells, as compared to the control process. This was true
regardless of
whether the TILs were from naïve tumors or tumors that were previously treated
with an immune
checkpoint inhibitor. And, as shown in FIG. 43A, as compared to the control
process, with the
MRM process, all ICB samples produced sufficient pre-Rep yields for further
expansion versus
only one of the samples in the control group.
***
[0663] It is to be appreciated that the Detailed Description
section, and not the Summary
and Abstract sections, is intended to be used to interpret the claims. The
Summary and Abstract
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sections may set forth one or more but not all exemplary embodiments of the
present disclosure as
contemplated by the inventor(s), and thus, are not intended to limit the
present disclosure and the
appended claims in any way.
[0664] The present disclosure has been described above with the
aid of functional building
blocks illustrating the implementation of specified functions and
relationships thereof. The
boundaries of these functional building blocks have been arbitrarily defined
herein for the
convenience of the description. Alternate boundaries can be defined so long as
the specified
functions and relationships thereof are appropriately performed.
[0665] The foregoing description of the specific embodiments
will so fully reveal the
general nature of the disclosure that others can, by applying knowledge within
the skill of the art,
readily modify and/or adapt for various applications such specific
embodiments, without undue
experimentation, without departing from the general concept of the present
disclosure. Therefore,
such adaptations and modifications are intended to be within the meaning and
range of equivalents
of the disclosed embodiments, based on the teaching and guidance presented
herein. It is to be
understood that the phraseology or terminology herein is for the purpose of
description and not of
limitation, such that the terminology or phraseology of the present
specification is to be interpreted
by the skilled artisan in light of the teachings and guidance.
[0666] The breadth and scope of the present disclosure should
not be limited by any of the
above-described exemplary embodiments, but should be defined only in
accordance with the
following claims and their equivalents.
[0667] The contents of all cited references (including
literature references, U.S. or foreign
patents or patent applications, and websites) that are cited throughout this
application are hereby
expressly incorporated by reference as if written herein in their entireties
for any purpose, as are
the references cited therein. Where any inconsistencies arise, material
literally disclosed herein
controls.
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