CA3234821A1

CA3234821A1 - Methods for culturing immune cells

Info

Publication number: CA3234821A1
Application number: CA3234821A
Authority: CA
Inventors: Suman Kumar VODNALA; Yogin PATEL; Nicholas P. Restifo; Benjamin Harris
Original assignee: Lyell Immunopharma Inc
Current assignee: Lyell Immunopharma Inc
Priority date: 2021-10-28
Filing date: 2022-10-27
Publication date: 2023-05-04
Also published as: AU2022377637A1; WO2023077034A1

Abstract

The preset disclosure provides methods of culturing TILs in a medium comprising at least about 30 mM to at least about 100 mM potassium ion. In some aspects, the methods disclosed herein enhance expansion of CD8+ TILs, relative to CD4+ TILs. In some aspects, the methods further increase the number of less-differentiated cells, e.g., less-differentiated TILs, in the population of cells. In some aspects, the methods disclosed herein enrich for tumor-reactive, e.g., tumor specific, TILs such that clonal diversity is preserved. In some aspects, the cells, e.g., the TILs, are administered to a subject in need thereof.

Description

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-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 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

[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 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 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, 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 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%, 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%, 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 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 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.

[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%, 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 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.

[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 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.

[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 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 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.

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 (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.

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).

[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 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 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, 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.

[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 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 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.

[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.

[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, (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.

[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 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 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, 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

[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.

[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 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 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.

[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 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 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.

[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 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.

[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.

[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 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

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+-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, 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 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 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.

[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

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).

[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.

[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 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 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 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.

[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+

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+

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 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 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 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.

[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.

[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 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 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 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 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 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 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 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 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 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 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 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 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 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.

[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 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 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 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.

[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 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.

[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., 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 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) 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 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 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.

[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 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.

[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

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 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 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 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 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.

[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

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

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.

[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 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.

[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

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 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-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 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 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 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 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 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, 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 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

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.

[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 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 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 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).

[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 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.

[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-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 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 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 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 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 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.

[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 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 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, 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 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 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 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.

[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 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 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 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 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 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 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 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.

[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 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.

[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

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 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

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.

[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.

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, 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.

[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 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 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%.

[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 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%, 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'.

[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-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 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 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 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 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 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).

[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 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 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 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 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) 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.

[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, 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, 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 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 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.

[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 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.

[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 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, 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 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 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 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, 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-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-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 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 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.

[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 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 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 (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, 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 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 Media K NaC1 Glucose Ca Osmolality Tonicity*
(mM) (mM) (mM) (mM) (mOsmol) (mOsmol) Basal Media 4 118.47 -24mM -2.8mM 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 Hyper K 50 80.5 17.7 1.8 -254.7 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) Hypotonic 250 70 55 Isotonic 280 70 70 Hypertonic 320 60 100 so 80 * Tonicity is calculated according to the formula: Tonicity = ([K] + [MICA]) x 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), (BD-#562381), CD127 (Bio legend- 4351324), CD45RA (BD-4560673).

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-(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-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, 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 (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 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 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

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.

[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

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 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 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 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.

[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.

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, 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, WO 2021,077034 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, 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, 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, WO 2021,077034 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, 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, WO 2021,077034 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, WO 2021,077034 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 WO 2021,077034 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, WO 2021,077034 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, WO 2021,077034 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 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 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).

[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 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 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 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 , 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 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 ++++ +++ ++

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.

[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).

[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 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, 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.

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 Sample Identified Preserved in Preserved in from Day 0 Control MRM large-scale

[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.

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 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.

Claims

WHAT IS CLAIMED IS:

1. A method of culturing tumor infiltrating lymphocytes (TILs) ex 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.

2. The method of claim 1, wherein the heterogeneous population of TILs is enriched in CD8+
TILs after being placed in the MRM.

3. 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.

4. A method of preparing a CD8+-enriched population of TILs, comprising culturing a heterogeneous population of TILs ex vivo or in vitro in an MR1VI comprising potassium ion at a concentration of about 30 mM to about 100 mM.

5. The method of any one of claims 1 to 4, wherein the heterogeneous population of TILs comprises CD4+ TILs and CD8+ TILs.

6. The method of any one of claims 1 to 5, wherein the heterogeneous population of TILs is obtained from one or more tumor sample obtained from a subject.

7. The method of claim 6, wherein the tumor sample subjected to an initial TIL culture.

8. The method of claim 7, wherein the initial TIL culture comprises culturing the tumor sample in the MRM.

9. The method of claim 8, wherein the MRM further comprises IL-2 during the initial TIL
culture.

10. The method of claim 9, wherein the MRM further comprises IL-7, IL-15, IL-21, or any combination thereof during the initial TIL culture.

11. The method of any one of claims 8 to 10, wherein the MR1VI comprises IL-2 and IL-21 during the initial TIL culture.

12. The method of any one of claims 7 to 11, wherein the initial TIL
culture lasts at least about 14-19 days.

13. The method of any one of claims 7 to 12, wherein the initial TIL
culture lasts at least about 14 days.

14. The method of any one of claims 7 to 13, wherein 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.

15. The method of any one of claims 7 to 14, wherein the TILs are stimulated following the initial T1L culture.

16. The method of claim 15, wherein the TILs are stimulated by culturing the TILs with a CD3 agonist and/or a CD28 agonist.

17. The method of any one of claims 6 to 16, wherein the tumor sample comprises a tumor biopsy.

18. The method of any one of claims 6 to 17, wherein the tumor sample is fragmented prior to culturing.

19. The method of any one of claims 1 to 18, wherein following culture of the heterogeneous population of TILs, at least about 30%, atleast about 35%, atleast 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.

20. The method of any one of claims 1 to 19, wherein following culture of the heterogeneous population of TILs, at least about 50% of the TILs in the population are CDS+
TILs.

21. The method of any one of claims 1 to 20, wherein the MRM further comprises sodium ion, calcium ion, glucose, or any combination thereof

22. The method of any one of claims 1 to 21 wherein the MRM further comprises a cell expansion agent.

23. The method of claim 22, wherein the cell expansion agent comprises a GSK3B inhibitor, an ACLY inhibitor, a PI3K inhibitor, an AKT inhibitor, or any combination thereof

24. The method of claim 23, wherein the PI3K inhibitor comprises LY294002, pictilisib, CAL101, IC87114, or any combination thereof.

25. The method of claim 24, wherein the AKT inhibitor comprises MK2206, A443654, AKTi-VIII, or any combination thereof.

26. The method of any one of claims 3 to 25, wherein 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.

27. The method of any one of claims 3 to 26, wherein 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 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 mM to about 50 mM.

28. The method of any one of claims 1 to 27, wherein the concentration of potassium ion is about 40 mM to about 90 mM.

29. The method of any one of claims 1 to 28, wherein the concentration of potassium ion is about 50 mM to about 90 mM.

30. The method of any one of claims 1 to 29, wherein the concentration of potassium ion is about 50 mM to about 80 mM.

31. The method of any one of claims 1 to 30, wherein the MRM further comprises sodium ion.

32. The method of claim 31, wherein the concentration of the sodium ion is from about 25 mM
to about 100 mM.

33. The method of claim 31 or 32, wherein 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 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 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 mM, or about 75 mM to about 80 mM.

34. The method of any one of claims 31 to 33, wherein 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.

35. The method of any one of claims 31 to 34, wherein the concentration of the sodium ion is about 55 mM.

36. The method of any one of claims 31 to 35, wherein the concentration of the sodium ion is about 60 mM.

37. The method of any one of claims 31 to 36, wherein the concentration of the sodium ion is about 65 mM.

38. The method of any one of claims 1 to 37, wherein the MRM further comprises glucose.

39. The method of claim 38, wherein the concentration of glucose is more than about 10 mM.

40. The method of claim 38 or 39, wherein 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 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 mM, about 17 mM to about 20 mM, about 17 mM to about 19 mM, or about 17 mM to about 18 mM.

41. The method of any one of claims 38 to 40, wherein the concentration of glucose is about 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.

42. The method of any one of claims 1 to 41, wherein the MRM further comprises calcium ion.

43. The method of claim 42, wherein the concentration of calcium ion is more than about 0.4 mM.

44. The method of claim 42 or 43, wherein 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.

45. The method of any one of claims 42 to 44, wherein 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.

46. The method of any one of claims 1 to 45, wherein 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).

47. A method of expanding TILs obtained from a human subject comprising:
a. culturing the TILs in an initial TIL culture media;
b. culturing the TILs in a secondary TIL culture media;
c. 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 TEL expansion media are MRM.

48. The method of claim 47, wherein the initial TIL culture media and the secondary TIL
expansion media are hyperkalemic and the third TIL expansion media are not hyperkalemic.

49. The method of claim 47 or 48, wherein the initial TIL culture media further comprises IL-2.

50. The method of claim 49, wherein the initial Tit culture media further comprises IL-21.

51. The method of any one of claims 47 to 50, wherein the initial Tit culture media 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., FUNGIN.TM.), and/or sodium pyruvate.

52. The method of any one of claims 47 to 51, wherein the TILs are cultured in the initial TIL
culture media for at least about 1 week, at least about 2 weeks, or at least about 3 weeks.

53. The method of any one of claims 47 to 52, wherein the TILs 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, or at least about 10x106 cells per fragment.

54. The method of any one of claims 47 to 53, wherein the TILs are stimulated with a CD3 agonist, a CD28 agonisst, or both in or prior to the secondary TIL culture media in (b).

55. The method of any claims 47 to 54, wherein the TILs are further stimulated with a CD27 agonist in or prior to the secondary TIL culture media.

56. The method of any one of claims 47 to 55, wherein the TILs are further stimulated with a 4-1BB agonist in or prior to the secondary T1L culture media.

57. The method of any one of claims 53 to 56, wherein the TILs 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.

58. The method of any one of claims 47 to 57, wherein the TILs 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, atleast about 8x107, atleast about 9x107, atleast about 10x 107, 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.

59. The method of any one of claims 47 to 58, wherein the TILs are stimulated with a CD3 agonist, a CD28 agonist, a CD27 agonist, and/or a 4-1BB agonist in the third TIL culture media.

60. The method of claim 59, wherein the third TIL culture media is not hyperkalemic.

61. The method of claim 59 or 60, wherein the TILs 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, 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.

62. A method of increasing tumor reactive TILs comprising:
a. culturing one or more tumor fragments in initial TIL culture media, which are hyperkalemic and comprise IL-2 and optionally IL-21, up to about 14 to 19 days thereby obtaining TILs from the tumor fragment;
b. 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;

c. 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.

63. The method of any one of claims 1 to 62, wherein 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.

64. The method of any one of claims 1 to 63, wherein the population of TILs comprises an increased proportion of CD8- CD62L TILs following culture in the MRM, relative to the proportion of CD8+ CD62L+ TILs following culture in a control medium that is not hyperkal em i c.

65. The method of any one of claims 1 to 64, wherein the population of TILs comprises an increased proporti on of CD8+ PD1+ T11.s following culture in the MRM, relative to the proportion of CD8+ PD1+ TILs following culture in a control medium that is not hyperkalemic.

66. The method of any one of claims 1 to 65, further comprising administering the population of T1Ls in a subject having a tumor.

67. The method of claim 66, wherein the tumor is refractory to a checkpoint inhibitor.

68. The method of claim 67, wherein the checkpoint Uthibitor comprises a PD-1 antagonist, a CTLA-4 antagonist, a TIM3 antagonist, a GITR antagonist, a KIR antagonist, a antagonist, or any combination thereof.

69. The method of claim 67, wherein 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.

70. The method of any one of claims 66 to 69, wherein the tumor is relapsed.

71. The method of any one of claims 66 to 70, wherein the tumor is metastatic.

72. A composition of immune cells, comprising one or more CD8+ TIL cultured according to the method of any one claims 1 to 65.

73. The composition of claim 72, 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.

74. 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.

75. The composition of any one of claims 72 to 74, wherein at least about 50% of the cells are CD8+ TILs.

76. The composition of any one of claims 72 to 75, wherein the cells exhibit increased expression of TCF7 following culture in the MRM, relative to TCF7 expression in a population of immune cells following culture in a control medium that is not hyperkalemic.

77. The composition of any one of claims 72 to 76, wherein 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 CD8-7CD62I: TILs.

78. The composition of any one of claims 72 to 77, wherein 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 PDT'.

79. The composition of any one of claims 72 to 78, wherein 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 CD39 .

80. The composition of any one of claims 72 to 79, wherein 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+.

81. The composition of any one of claims 72 to 80, wherein 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+.

82. The composition of any one of claims 72 to 81, wherein 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+.

83. The composition of any one of claims 72 to 82, wherein 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 Tit culture are PD1+ CD27+.

84. The composition of any one of claims 72 to 83, wherein 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+.

85. The composition of any one of claims 72 to 84, wherein 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+ PDF' TCF7+.

86. The composition of any one of claims 72 to 85, comprising 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.

87. The composition of any one of claims 72 to 86, comprising 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 CD8 cells.

88. A method of treating a 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 the method of any one of claims 1 to 65.

89. The method of claim 88, wherein the tumor is refractory to a checkpoint inhibitor.

90. The method of claim 89, wherein the checkpoint inhibitor comprises a PD-1 antagonist, a CTLA-4 antagonist, a TIM3 antagonist, a GITR antagonist, a KIR antagonist, a antagonist, or any combination thereof.

91. The method of claim 89, wherein 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.

92. The method of any one of claims 88 to 91, wherein the tumor is relapsed.

93. The method of any one of claims 88 to 92, wherein the tumor is metastatic.

94. The method of any one of claims 88 to 93, wherein the population of T1Ls is enriched for CD8+ TILs.

95. The method of any one of claims 88 to 94, 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 TILs in the population of TILs are CD8+ TILs.

96. The method of any one of claims 88 to 95, wherein at least about 50% of the TILs in the population of TILs are CD8 TILs.

97. A method treating a tumor in a subject in need thereof, comprising administering to a subject the composition of any one of claims 72 to 87.

98. The method of claim 97, wherein the tumor is refractory to a checkpoint inhibitor.

99. The method of claim 98, wherein the checkpoint inhibitor comprises a PD-1 antagonist, a CTLA-4 antagonist, a TIM3 antagonist, a GITR antagonist, a KIR antagonist, a antagonist, or any combination thereof.

100. The method of claim 99, wherein 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.

101. The method of any one of claims 97 to 100, wherein the tumor is relapsed.

102. The method of any one of claims 97 to 101, wherein the tumor is metastatic.

103. The method of any one of claims 88 to 102, wherein the tumor comprises a solid tumor.

104. The method of any one of claims 88 to 103, wherein the tumor 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 endocervi cal adenocarcinoma, esophageal carcinoma, bladder urotheli al carcinoma, breast invasive carcinoma, kidney renal papillary cell carcinoma, colon adenocarcinoma, or any combination thereof.

105. The method of any one of claims 88 to 104, wherein a therapeutically effective amount of the population of TILs is 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 1019, or at least about 1 0 x 1019, or at least about 15 x 1010, or at least about 20 x 1019, or at least about 25 x101 , or at least about 30 x 1010 cells to the subject.

106. The method of any one of claims 88 to 105, wherein a therapeutically effective amount of the population of TILs is 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.

107. The method of any one of claims 88 to 106, further comprising administering a checkpoint inhibitor.

108. The method of claim 107, wherein the checkpoint inhibitor is administered to the subject after administering the population of cells.

109. The method of claim 107 or 108, wherein the checkpoint inhibitor comprises a CTLA-4 antagonist, a PD1 antagonist, a TIM-3 antagonist, or a combination thereof.

110. The method of any one of claims 107 to 109, wherein the checkpoint inhibitor comprises an anti-CTLA-4 antibody, an anti-PD1 antibody, an anti-PD-Ll antibody, an anti-antibody, or a combination thereof.

111. The method of any one of claims 107 to 110, wherein the checkpoint inhibitor is administered to the subject after administering the population of TILs.

112. The method of any one of claims 88 to 106, further comprising administering a checkpoint inhibitor.

113. The method of claim 112, wherein the checkpoint activator comprises an 0X40 agonist, a LAG-3 agonist, a 4-1BB (CD137) agonist, a GITR agonist, a TIIVI3 agonist, or a combination thereof.

114. The method of claim 112 or 113, wherein 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

115. The method of any one of claims 88 to 114, further comprising administering a cytokine.

116. The method of claim 115, wherein the cytokine is administered to the subject after administering the population of TILs.

117. The method of claim 115 or 116, wherein the cytokine is IL-2.

118. The method of any one of claims 88 to 117, further comprising administering a lymphodepleting therapy to the subject prior to administering the population of cells.

119 The m ethod of cl aim 118, wherei n the lymph odepl eting therapy compri ses cyclophosphamide, fludarabine, or both cyclophosphamide and fludarabine.

120. The method of any one of claims 1 to 71 and 88 to 119, wherein the heterogeneous population of TlLs has increased clonal diversity after being placed in the 1VERIVI, as compared to the clonal diversity of a heterogenous population of TILs placed in a control medium.

121. The method of any one of claims 1 to 71 and 88 to 119, wherein the heterogeneous population of TILs after being placed in the MRM 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.

122. The method of any one of claims 1 to 71 and 88 to 119, wherein the heterogeneous population of TILs after being placed in the MRM 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.

123. The method of any one of claims 1 to 71 and 88 to 122, wherein the heterogeneous population of TILs after being placed in the MRM has a clonal diversity score of less than about 0.3 as measured by Simpsons clonality.

124. The method of any one of claims 1 to 71 and 88 to 122, wherein 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.

125. The method of any one of claims 1 to 71 and 88 to 122, wherein the heterogeneous population of TILs after being placed in the MRM has a clonal diversity score of less than about 0.2 as measured by Simpsons clonality.

126. The method of any one of claims 1 to 71 and 88 to 122, wherein the heterogeneous population of TILs after being placed in the MRM has a clonal diversity score of less than about 0.1 as measured by Simpsons clonality.

127. The composition of any one of claims 72 to 87, wherein 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%, 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.

128. The composition of any one of claims 72 to 87 and 127, wherein the CD8+
TILs 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.

129. The composition of any one of claims 72 to 87, 127, and 128, wherein the CD8+ TILs have a clonal diversity score of less than about 0.3 as measured by Simpsons clonality.

130. The composition of any one of claims 72 to 87 and 127 to 129, wherein the CDR+ TTT,s have a clonal diversity score of less than about 0.25 as measured by Simpsons clonality.

131. The composition of any one of claims 72 to 87 and 127 to 130, wherein the CD8+ Tits have a clonal diversity score of less than about 0.2 as measured by Simpsons clonality.

132. The composition of any one of claims 72 to 87 and 127 to 131, wherein the CDR+ Tits have a clonal diversity score of less than about 0.1 as measured by Simpsons clonality.

133. 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.

134. 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.

135. The population of expanded TILs of claim 133 or 134, wherein the clonal diversity score is less than about 0.3 as measured by Simpsons clonality.

136. The population of expanded TILs of any one of claims 133 to 135, wherein the clonal diversity score is less than about 0.25 as measured by Simpsons clonality.

137. The population of expanded TILs of any one of claims 133 to 136, wherein the clonal diversity score is less than about 0.2 as measured by Simpsons clonality.

138. The population of expanded TILs of any one of claims 133 to 137, wherein the clonal diversity score is less than about 0.1 as measured by Simpsons clonality.

139. The population of expanded TILs of any one of claims 133 to 138, comprising 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

140. The population of expanded TILs of any one of claims 133 to 139, comprising 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 CD8+ cells.

141. The population of expanded TILs of any one of claims 133 to 140, 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 expanded TILs are CD8+ TILs.

142. The population of expanded TILs of any one of claims 133 to 141, wherein at least about 50% of the expanded TILs are CD8+ TILs.

143. The population of expanded TILs of any one of claims 133 to 142, wherein the expanded TILs exhibit increased expression of TCF7 following culture in the MRM, relative to TCF7 expression in a population of immune cells following culture in a control medium that is not hyperkalemic.

144. The population of expanded TILs of any one of claims 133 to 143, wherein 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.

145. The population of expanded TILs of any one of claims 141 to 144, wherein 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+.

146. The population of expanded TILs of any one of claims 141 to 145, wherein 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+.

147. The population of expanded TTI,s of any one of claims 141 to 146, wherein 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 CDS+ TILs are CD27 .

148. The population of expanded TILs of any one of claims 141 to 147, wherein 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 .

149. The population of expanded TILs of any one of claims 141 to 148, wherein 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 .

150. The population of expanded TILs of any one of claims 141 to 149, wherein 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+
CD27 .

151. The population of expanded TILs of any one of claims 141 to 150, wherein 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+.

152. The population of expanded TILs of any one of claims 141 to 151, wherein 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+
CD28+
CD103+ PD1+ TCF7 .

153. A method of treating a cancer in a subject in need thereof, comprising administering the composition of any one of claims 127 to 132 or the population of expanded TILs of any one of claims 133 to 152.

154. The method of claim 153, wherein the tumor is refractory to a checkpoint inhibitor.

155. The method of claim 154, wherein the checkpoint inhibitor comprises a PD-1 antagonist, a CTLA-4 antagonist, a TIM3 antagonist, a GITR antagonist, a KIR antagonist, a antagonist, or any combination thereof.

156. The method of claim 154, wherein 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.

157. The method of any one of claims 153 to 156, wherein the tumor is relapsed.

158. The method of any one of claims 1 53 to 157, wherein the tumor is metastatic.

159. The composition of any one of claims 72 to 87 and 127 to 132 or the population of expanded TILs of any one of claims 133 to 152, comprising at least one immune cell expressing one or more stem-like markers and one or more effector-like markers

160. The composition of any one of claims 72 to 87, 127 to 132, and 159 or the population of expanded TILs of any one of claims 133 to 152 and 159, wherein the stem-like markers comprise CD4.5RA+, CD62L+, CCR7+, CD27+, CD28+, BACH2+, LEF1+, TCF7+, or any combination thereof

161. The composition of any one of claims 72 to 87, 127 to 132, 159, and 160 or the population of expanded TILs of any one of claims 133 to 152, 159, and 160, wherein the effector-like markers comprise pSTAT5+, STAT5+, pSTAT3+, STAT3+, or any combination thereof

162. The composition of any one of claims 72 to 87, 127 to 132, 159, 160, and 161 or the population of expanded TILs of any one of claims 133 to 152, 159, 160, and 161, wherein 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 expressing one or more stem-like markers and one or more effector-like markers.

163. A TIL comprising one or more stem-like markers and one or more effector-like markers.

164. The TIL of claim 163, wherein the stern-like markers comprise CD45RA+, CD62L+, CCR7+, CD27+, CD28+, BACH2+, LEF1+, TCF7+, or any combination thereof

165. The TIL of claim 163 or 164, wherein the effector-like markers comprise pSTAT5+, STAT5+, pSTAT3+, STAT3+, or any combination thereof

166. A population of expanded TILs comprising the TIL of any one of claims 163 to 165.

167. The population of expanded TILs of claim 166, wherein 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 T1Ls cells comprise the TIL of any one of claims 163 to 166.

168 A pharmaceutical composition comprising the TIL of any one of claims 163 to 165 or the population of expanded TILs of claim 166 or 167 and a pharmaceutically acceptable carrier.

169. A method of treating a disease or condition in a subject in need thereof comprising administering the TIL of any one of claims 163 to 165, the population of expanded TILs of claim 166 or 167, or the pharmaceutical composition of claim 168 to the subject.

170. The method of claim 169, wherein the disease or condition is a tumor.

171. The method of claim 170, wherein the tumor is refractory to a checkpoint inhibitor.

172. The method of claim 171, wherein the checkpoint inhibitor comprises a PD-1 antagonist, a CTLA-4 antagonist, a TIM3 antagonist, a GITR antagonist, a KIR antagonist, a antagonist, or any combination thereof.

173. The method of claim 171, wherein 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.

174. The method of any one of claims 171 to 173, wherein the tumor is relapsed.

175. The method of any one of claims 171 to 174, wherein the tumor is metastatic.

176. The method of any one of claims 169 to 175, wherein the tumor is selected from the group consisting of melanoma, colorectal cancer, pancreatic cancer, head and neck cancer, cervical cancer, ovarian cancer, and any combination thereof.

177. A method of preparing the TIL of any one of claims 163 to 165, comprising culturing the immune cell in culture medium under suitable conditions.

178. The method of any one of claims 1 to 71, 88 to 126, 153 to 158, and 169 to 177, the composition of any one of claims 72 to 87, 127 to 132, and 159 to 162, or the population of expanded TILs or any one of claims 133 to 152, 166, and 167, wherein the MRM
comprises NaC1, wherein total concentration of potassium ion and NaC1 is between 110 mM and 140 mM.

179. The method of any one of claims 1 to 71, 88 to 126, 153 to 158, and 169 to 178, the composition of any one of claims 72 to 87, 127 to 132, and 159 to 162, or the population of expanded TILs of any one of claims 133 to 152, 166, and 167, wherein the population of TILs comprises an increased proportion of CD39-/CD69- TILs following culture in the MR1\4, relative to the proportion of CD39-/CD69- TILs following culture in a control medium.

180. The method of any one of claims 1 to 71, 88 to 126, 153 to 158, and 169 to 179, the composition of any one of claims 72 to 87, 127 to 132, 159 to 162, and 178 to 179, or the population of expanded TILs of any one of claims 133 to 152, 166, 167, and 178 to 179, wherein 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 CD39-/CD69-.

181. The method of any one of claims 7 to 71, 88 to 126, 153 to 158, and 169 to 179, wherein the initial culture lasts at least about 11 days.

182. The method of any one of claims 47 to 71, 88 to 126, 153 to 158, and 169 to 179, wherein the culturing of the TILs in the secondary TIL culture medium is carried out in one or more gas-permeable containers.

183. The method of any one of claims 47 to 71, 88 to 126, 153 to 158, and 169 to 179, wherein the culturing of the TILs in the third (or final) TIL culture medium is not carried out in one or more gas-permeable containers.

184. The method of any one of claims 47 to 71, 88 to 126, 153 to 158, and 169 to 179, wherein the TILs are split between the culturing of the TILs in the secondary TIL
culture medium and the culturing of the Tits in the third (or final) TIL culture medium.

185. A method 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").

186. The method of claim 185, wherein no CD3 agonist and no antigen-presenting cells are added during the dynamic-REP step.

187. The method of claim 185 or 186, wherein the agitation comprises rocking.

188. The method of any one of claims 185 to 187, wherein the dynamic-REP step comprises perfusi on

189. The method of claim 188, wherein 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.

190. The method of claim 188 or 189, wherein the perfusion comprises continuous medium exchange at a rate of about 25% of the working volume of the culture every 24 hours.

191. The method of claim 188 or 189, wherein the perfusion comprises continuous medium exchange at a rate of about 50% of the working volume of the culture every 24 hours.

192. The method of claim 190 or 191, wherein 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 dynarnic-REP culture.

193. The method of any one of claims 189 to 192, wherein the continuous medium exchange comprises addition to the culture of fresh MRM, wherein the fresh MRM does not comprise an anti-CD-3 antibody.

194. The method of any one of claims 189 to 193, wherein the continuous medium exchange comprises addition to the culture of fresh MRM, wherein the fresh MRM does not comprise antigen-pre senting cells.

195. The method of any one of claims 189 to 194, wherein 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.

196. The method of any one of claims 189 to 195, wherein the concentration of CD3 agonist in the dynamic-REP culture i s 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.

197. The method of any one of claims 189 to 196, wherein 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.

198. The method of any one of claims 189 to 197, wherein 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.

199. The method of any one of claims 189 to 198, wherein 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.

200. The method of any one of claims 189 to 199, wherein 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.

201. The method of any one of claims 189 to 200, wherein 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.

202. The method of any one of claims 189 to 201, wherein 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.

203. The method of any one of claims 189 to 202, wherein 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.

204. The method of any one of claims 189 to 203, wherein 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.

205. The method of any one of claims 185 to 204, wherein the anti-CD3 antibody comprises OKT-3.

206. The method of any one of claims 185 to 205, wherein the antigen-presenting cells comprise irradiated PBMCs.

207. The method of any one of claims 185 to 206, wherein the TILs are cryopreserved following the static-REP and prior to the dynamic-REP.

208. The method of any one of claims 185 to 207, wherein the TILs are cryopreserved following the dynamic-REP.

209. The method of any one of claims 185 to 208, wherein the TILs are subjected to the static-REP for about 5 days.

210. The method of any one of claims 185 to 208, wherein the TILs are subj ected to the dynamic-REP for about 9 days to about 12 days.

211. The method of anyone of claims 185 to 208, wherein the TILs are subjected to the static-REP for about 5 days followed by the dynamic-REP for about 9 days.

212. The method of anyone of claims 185 to 208, wherein the TILs are subjected to the static-REP for about 5 days followed by the dynamic-REP continues for about 12 days.

213. The method of any one of claims 185 to 212, wherein the medium of the static-REP culture comprises IL-2.

214. The method of any one of claims 185 to 213, wherein 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 TU, at least about 1900 TU, or at least about 2000 IU IL-2.

215. The method of any one of claims 185 to 214, wherein the medium of the static-REP culture comprises about 1500 IU IL-2.

216. The method of any one of claims 185 to 215, wherein the medium of the static-REP culture comprises IL-21.

217. The method of any one of claims 185 to 216, wherein 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.

218. The method of any one of claims 185 to 217, wherein the medium of the static-REP culture comprises about 10 ng/mL IL-21.

219. The method of any one of claims 185 to 218, wherein the medium of the static-REP culture comprises IL-15.

220. The method of any one of claims 185 to 219, wherein 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.

221. The method of any one of claims 185 to 220, wherein the medium of the static-REP culture comprises about 0.4 ng/mL IL-15.

222. The method of any one of claims 185 to 221, wherein 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.

223. The method of any one of claims 185 to 222, wherein the medium of the perfused fresh medium of the dynamic-REP culture comprises IL-2.

224. The method of any one of claims 185 to 223, wherein the perfused fresh medium of the dynamic-REP culture comprises at least about 1000 IU, at least about 1100 IU, at least about 1200 1U, at least about 1300 1U, at least about 1400 IU, at least about 1500 1U, at least about 1600 IU, at least about 1700 1U, at least about 1800 IU, at least about 1900 1U, or at least about 2000 IU IL-2.

225. The method of any one of claims 185 to 224, wherein the perfused fresh medium of the dynamic-REP culture comprises about 1500 IU IL-2.

226. The method of any one of claims 185 to 225, wherein the perfused fresh medium of the dynamic-REP culture comprises IL-21.

227. The method of any one of claims 185 to 226, wherein 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.

228. The method of any one of claims 185 to 227, wherein the perfused fresh medium of the dynamic-REP culture comprises about 10 ng/mL IL-21.

229. The method of any one of claims 185 to 228, wherein the perfused fresh medium of the dynamic-REP culture comprises IL-15.

230. The method of any one of claims 185 to 229, wherein 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.

231. The method of any one of claims 185 to 230, wherein the perfused fresh medium of the dynamic-REP culture comprises about 0.4 ng/mL IL-15.

232. The method of any one of claims 185 to 231, wherein 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.

233. The method of any one of claims 185 to 232, wherein prior to the static-REP, the TILs are subjected to an initial TIL expansion.

234. The method of claim 233, wherein the initial TIL expansion comprises culturing a tumor sample or a portion thereof in MRM, wherein the MRM further comprises IL-2.

235. The method of claim 233 or 234, wherein the MRM further comprises IL-7, IL-15, IL-21, or any combination thereof during the initial TIL expansion.

236. The method of any one of claims 233 to 235, wherein the MRM comprises IL-2 and IL-21 during the initial TIL culture.

237. The method of any one of claims 233 to 236, wherein the MRM comprises about 6000 IU/mL IL-2 and about 30 ng/mL IL-21 during the initial TIE- culture.

238. The method of any one of claims 233 to 237, wherein the initial TlL
culture lasts about 11 days to about 15 days.

239. The method of any one of claims 233 to 238, wherein the initial TIL
culture lasts at least about 11 days.

240. The method of any one of claims 233 to 238, wherein the initial TIL
culture lasts at least about 13 days.

241. The method of any one of claims 233 to 238, wherein the initial TIL
culture lasts at least about 15 days.

242. The method of any one of claims 233 to 241, wherein the initial TIL
culture lasts until cell yield in the initial TIL culture reaches at least about 10x106-50x106 cells.

243. The method of any one of claims 233 to 242, wherein 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.

244. The method of any one of claims 233 to 243, wherein the initial TIL
culture lasts until cell yield in the initial culture reaches at least about 30x106 cells.

245. The method of any one of claims 233 to 244, wherein the initial TIL
expansion further comprises contacting the TILs with TRANSACTTM.

246. The method of claim 245, wherein 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.

247. The method of claim 245 or 246, wherein the TILs are contacted with TRANSACTTm on about day 4 of the initial TM culture.

248. The method of any one of claims 233 to 247, wherein the initial TM
expansion further comprises contacting the TILs with 4-1BB ligand

249. The method of claim 248, wherein 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.

250. The method of claim 248 or 249, wherein the TILs are contacted with 4-1BB
ligand on about day 4 of the initial TlL culture.

251. The method of any one of claims 233 to 250, wherein the initial TIL
expansion comprises contacting the TILs with TRANSACTTM and 4-1BB ligand on about day 4 of the initial TM culture.

252. A population of TILs obtained by the method of any one of claims 185 to 251.

253. A method of treating a subject in need thereof comprising administering to the subject the population of TILs of claim 252.

254. The method of claim 253, wherein the subject has a tumor.

255. The method of claim 254, wherein the tumor comprises a solid tumor.

256. The method of claim 254 or 255, wherein the tumor 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, bladder urothelial carcinoma, breast invasive carcinoma, kidney renal papillary cell carcinoma, colon adenocarcinoma, or any combination thereof.

257. The method of claim 256, wherein the tumor is refractory to a checkpoint inhibitor.

258. The method of claim 257, wherein the checkpoint inhibitor comprises a PD-1 antagonist, a CTLA-4 antagonist, a TIM3 antagonist, a GITR antagonist, a KIR antagonist, a antagonist, or any combination thereof.

259. The method of claim 257, wherein 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.

260. The method of any one of claims 253 to 259, wherein the tumor is relapsed.

261. The method of any one of claims 253 to 260, wherein the tumor is metastatic.

262. The population of TILs of claim 252, wherein about 20% to about 80% of the CD8+ TILs are stemlike TILs.

263. The population of TILs of claim 252, wherein at least 50% to 90% of the CD8+ cells are putative tumor reactive TILs.

264. The population of TILs of claim 252, wherein at least 35% to 90% of the TILs are tumor reactive TILs.