Methods for culturing immune cells

Abstract

The preset disclosure provides methods of culturing TILs by first culturing a tumor sample in a first expansion medium, wherein the first expansion medium comprises a CD3 agonist and APCs at a first ratio (a "first REP step"); and culturing the TILs from first REP step in a second expansion medium, wherein the second expansion medium comprises a CD3 agonist and APCs at a second ratio (a "second REP step"). 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 CELLSCROSS-REFERENCE TO RELATED APPLICATIONS

[0001] This application claims priority benefit of U.S. Provisional Application Nos. 63 / 510,601, filed June 27, 2023; 63 / 593,129, filed on October 25, 2023; and 63 / 551,053, filed on February 7, 2024, each of which is incorporated by reference herein in its entiretyFIELD

[0002] The present disclosure relates to methods of culturing immune cells, e.g., tumor infiltrating lymphocytes (TILs). 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

[0003] 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.

[0004] 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 heterogeneous, 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.

[0005] 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 heterogeneous 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.

[0006] 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

[0007] Some aspects of the present disclosure are directed to a method of culturing tumor infiltrating lymphocytes (TILs) ex vivo or in vitro for a therapy to a subject in need thereof comprising: (i) dissociating a tumor sample obtained from the subject comprising TILs to generate a single cell suspension; (ii) culturing the single cell suspension in a first expansion medium, wherein the first expansion medium comprises a CD3 agonist and APCs at a first ratio (a "first REP step"); and (iii) culturing the TILs from the first REP step in a second expansion medium, wherein the second expansion medium comprises a CD3 agonist and APCs at a second ratio (a "second REP step").

[0008] Some aspects of the present disclosure are directed to a method of culturing TILs ex vivo or in vitro for a therapy in a subject in need thereof comprising: (i) culturing a dissociated tumor sample comprising TILs in a first expansion medium, wherein the first expansion medium comprises a CD3 agonist and APCs at a first ratio (a "first REP step"); and (ii) culturing the TILs from the first REP step in a second expansion medium, wherein the second expansion medium comprises a CD3 agonist, and APCs at a second ratio (a "second REP step"). In some aspects, the dissociated tumor sample is a single cell suspension.

[0009] Some aspects of the present disclosure are directed to a method of culturing TILs ex vivo or in vitro for a therapy in a subject in need thereof comprising culturing a population of TILs obtained from a tumor sample of the subject in an expansion medium comprising a CD3 agonist and antigen-presenting cells (APCs); wherein, prior to or after culturing in the expansion medium, the TILs are not cultured in a medium that does not comprising APCs.

[0010] In some aspects, prior to culturing in the expansion medium, the population of TILs is cultured in a medium comprising APCs. In some aspects, after culturing in the expansion medium, the population of TILs is cultured in a medium comprising APCs.

[0011] In some aspects, the TILs are cultured in the expansion medium for less than 11 days. In some aspects, the TILs are cultured in the expansion medium for less than 10 days. In some aspects, the TILs are cultured in the expansion medium for less than 9 days.

[0012] In some aspects, prior to culturing in the expansion medium, the population of TILs is cultured for less than 11 days in the medium comprising APCs. In some aspects, prior to culture in the expansion medium, the population of TILs is cultured for less than 10 days in the medium comprising APCs.

[0013] In some aspects, prior to culture in the expansion medium, the population of TILs is cultured for less than 9 days in the medium comprising APCs. In some aspects, prior to culture in the expansion medium, the population of TILs is cultured for less than 8 days in the medium comprising APCs.

[0014] In some aspects, the population of TILs is obtained from a dissociated tumor sample.

[0015] In some aspects, the TILs are cultured in the first REP step for less than 11 days. In some aspects, the TILs are cultured in the first REP step for less than 10 days. In some aspects, the TILs are cultured in the first REP step for less than 9 days. In some aspects, the TILs are cultured in the first REP step for less than 8 days.

[0016] In some aspects, the number of TILs present in the dissociated tumor sample is increased by 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 following culture in the first REP step. In some aspects, the number of TILs present in the dissociated tumor sample is increased by at least about 20-fold following culture in the first REP step.

[0017] In some aspects, the number of TILs following culture in the first REP step is at least about 5 x 107, at least about 1 x 108, at least about 2 x 108, at least about 3 x 108, at least about 4 x 108, at least about 5 x 108, at least about 6 x 108, at least about 7 x 108, at least about 8 x 108, at least about 9 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, or at least about 2 x 1010TILs.

[0018] In some aspects, the TILs are cultured in the second REP step for less than 11 days. In some aspects, the TILs are cultured in the second REP step for less than 10 days. In some aspects, the TILs are cultured in the second REP step for less than 9 days. In some aspects, the TILs are cultured in the second REP step for less than 8 days.

[0019] In some aspects, the number of TILs present following the first REP step is increased by 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 following culture in the second REP step. In some aspects, the number of TILs present following the first REP step is increased by at least about 20-fold following culture in the second REP step.

[0020] In some aspects, the number of TILs following culture in the second REP step is 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 IO10, at least about 2 x IO10, at least about 3 x IO10, at least about 4 x IO10, at least about 5 x IO10, at least about 6 x IO10, at least about 7 x IO10, at least about 8 x IO10, at least about 9 x IO10, or at least about 1 x 1011TILs.

[0021] In some aspects, the TILs are cultured in the first REP step for 7 to 10 days, and the TILs are cultured in the second REP step for 7 to 10 days, wherein the total number of TILs following culture in the second REP step is at least about 1 x 109TILs. In some aspects, the TILs are cultured in the first REP step for 7 days, and the TILs are cultured in the second REP step for 7 days, wherein the total number of TILs following culture in the second REP step is at least about 1 x 109TILs. In some aspects, the TILs are cultured in the first REP step for 10 days or less, and the TILs are cultured in the second REP step for 10 days or less, wherein the total number of TILs following culture in the second REP step is at least about 1 x 109TILs.

[0022] In some aspects, the first expansion medium comprises a dissociated tumor cell to APC ratio of about 1 :5, about 1 :6, about 1 :7, about 1 :8, about 1 :9, about 1 : 10, about 1 : 11, about 1 : 12, about 1 :13, about 1 : 14, about 1 : 15, about 1 : 16, about 1 : 17, about 1 : 18, about 1 : 19, about 1 :20, about 1 :25, about 1 :30, about 1 :35, about 1 :40, about 1 :45, or about 1 :50. In some aspects, the first expansion medium comprises a dissociated tumor cell to APC ratio of about 1 :10.

[0023] In some aspects, the second expansion medium comprises a dissociated tumor cell to APC ratio of about 1 :5, about 1 :6, about 1 :7, about 1 :8, about 1 :9, about 1 : 10, about 1 : 11, about 1 : 12, about 1 :13, about 1 : 14, about 1 : 15, about 1 : 16, about 1 : 17, about 1 : 18, about 1 : 19, about 1 :20, about 1 :25, about 1 :30, about 1 :35, about 1 :40, about 1 :45, or about 1 :50. In some aspects,the second expansion medium comprises a first REP expanded TIL to APC ratio of about 1 : 1, about 1 :2, about 1 :3, about 1 :4, about 1 :5, about 1 :6, about 1 :7, about 1 :8, about 1 :9, or about 1 : 10. In some aspects, the second expansion medium comprises a first REP expanded TIL to APC ratio of about 1 :3. In some aspects, the second expansion medium comprises a first REP expanded TIL to APC ratio of about 1 :4. In some aspects, the second expansion medium comprises a first REP expanded TIL to APC ratio of about 1 :5. In some aspects, the first expansion medium comprises a dissociated tumor cell to APC ratio of about 1 :10, and wherein the second expansion medium comprises a first REP expanded TIL to APC ratio of about 1 :3. In some aspects, the first expansion medium comprises a dissociated tumor cell to APC ratio of about 1 : 10, and wherein the second expansion medium comprises a first REP expanded TIL to APC ratio of about 1 :4. In some aspects, the first expansion medium comprises a dissociated tumor cell to APC ratio of about 1 : 10, and wherein the second expansion medium comprises a first REP expanded TIL to APC ratio of about 1 :5.

[0024] In some aspects, the subject is previously received one or more therapy for treating the tumor. In some aspects, the subject is relapsed or refractory to one or more prior therapy for treating the tumor. In some aspects, the one or more therapy for treating the tumor is selected from a chemotherapy, an immunotherapy, a radiotherapy, a surgery, or any combination thereof.

[0025] 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-PDl 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.

[0026] In some aspects, the tumor is metastatic. In some aspects, the tumor comprises a solid tumor. In some aspects, the solid tumor is 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.

[0027] In some aspects, the first expansion medium, the second expansion medium, or both comprises potassium ion. In some aspects, the first expansion medium, the second expansion medium, or both comprises potassium ion higher than 4 mM.

[0028] In some aspects, the first expansion medium, the second expansion medium, or both further comprises sodium ion, calcium ion, glucose, or any combination thereof.

[0029] In some aspects, the first expansion medium, the second expansion medium, or both 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.

[0030] 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.

[0031] In some aspects, the first expansion medium, the second expansion medium, or both 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 60mM 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.

[0032] 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.

[0033] In some aspects, the first expansion medium, the second expansion medium, or both 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.

[0034] In some aspects, the first expansion medium, the second expansion medium, or both 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.

[0035] In some aspects, the first expansion medium, the second expansion medium, or both comprise 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). In some aspects, the first expansion medium, the second expansion medium, or both comprise 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 25 mM glucose; (iii) about 0.5 mM to about 2.8 mM calcium ion; or (iv) any combination of (i)-(iii).

[0036] In some aspects, the first expansion medium, the second expansion medium, or both further comprises IL2. In some aspects, the concentration of IL2 is from about 1000 lU / ml to at least about 2000 lU / ml. In some aspects, the concentration of IL2 is at least about 1000 lU / ml, at least about 1100 lU / ml, at least about 1200 lU / ml, at least about 1300 lU / ml, at least about 1400 lU / ml, at least about 1500 lU / ml, at least about 1600 lU / ml, at least about 1700 lU / ml, at least about 1800 lU / ml, at least about 1900 lU / ml, or at least about 2000 lU / ml. In some aspects, the concentration of IL2 is about 1500 lU / ml.

[0037] In some aspects, the first expansion medium, the second expansion medium, or both further comprises IL21. In some aspects, the concentration of IL21 is from about 1 ng / ml to at least about 100 ng / ml. v the concentration of IL21 is 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 20 ng / ml, at least about 25 ng / ml, at least about 30 ng / ml, at least about 35 ng / ml, at least about 40 ng / ml, at least about 45 ng / ml, 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, or at least about 100 ng / ml. In some aspects, the concentration of IL21 is about 10 ng / ml.

[0038] In some aspects, the first expansion medium, the second expansion medium, or both further comprises IL15. In some aspects, the concentration of IL15 is from about 0.1 ng / ml to at least about 10 ng / ml. In some aspects, the concentration of IL15 is 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 1.1 ng / ml, at least about 1.2 ng / ml, at least about 1.3 ng / ml, at least about 1.4 ng / ml, at least about 1.5 ng / ml, at least about 2.0 ng / ml, at least about 2.5 ng / ml, at least about 3.0 ng / ml, at least about 3.5 ng / ml, at least about 4.0 ng / ml, at least about 4.5 ng / ml, at least about 5.0 ng / ml, at least about 6.0 ng / ml, at least about 7.0 ng / ml, at least about 8.0 ng / ml, at leastabout 9.0 ng / ml, or at least about 10.0 ng / ml. In some aspects, the concentration of IL15 is about 0.4 ng / ml.

[0039] In some aspects, first expansion medium, the second expansion medium, or both further comprises IL7.

[0040] In some aspects, the CD3 agonist comprises OKT3. In some aspects, the concentration of OKT3 is from about 1 to about 100 ng / ml. In some aspects, the concentration of OKT3 is at least about 1 ng / ml, at least about 5 ng / ml, at least about 10 ng / ml, at least about 15 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, at least about 40 ng / ml, at least about 45 ng / ml, 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, or at least about100 ng / ml. In some aspects, the concentration of OKT3 is about 20 ng / ml.

[0041] In some aspects, the second REP step comprises a first phase followed by a second phase, wherein the first phase of the second REP step comprises a first phase medium and the second phase of the second REP step comprises a second phase medium, and wherein the first phase medium and the second phase medium are not the same.

[0042] In some aspects, the first phase medium comprises about 45 mM potassium ion to about 90 mM potassium ion. In some aspects, the first phase medium comprises 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 potassium ion.

[0043] In some aspects, fresh medium is added to the TILs and the first phase medium to generate the second phase medium. In some aspects, the first phase medium is diluted by the fresh medium at a ratio of 1 : 1, 1 :2, 1 :3, 1 :4, 1 :5, 2: 1, 3: 1, or 4: 1 (first phase mediunrfresh medium). In some aspects, the first phase medium is diluted by the fresh medium at a ratio of 1 : 1 (first phase medium: fresh medium). In some aspects, the first phase medium is diluted by the fresh medium at a ratio of 1 :2 (first phase mediunrfresh medium). In some aspects, the first phase medium is diluted by the fresh medium at a ratio of 1 :3 (first phase mediunrfresh medium).

[0044] In some aspects, the first phase medium is diluted by the fresh medium to a final concentration of about 75%, about 70%, about 65%, about 60%, about 55%, about 50%, about 45%, about 40%, about 35%, about 30%, about 25%, about 20%, about 15%, about 10%, or about 5% first phase medium. In some aspects, the first phase medium is diluted by the fresh medium to a final concentration of about 50% first phase medium. In some aspects, the first phase medium is diluted by the fresh medium to a final concentration of about 40% first phase medium. In some aspects, the first phase medium is diluted by the fresh medium to a final concentration of about30% first phase medium. In some aspects, the first phase medium is diluted by the fresh medium to a final concentration of about 25% first phase medium. In some aspects, the first phase medium is diluted by the fresh medium to a final concentration of about 20% first phase medium. In some aspects, the first phase medium is diluted by the fresh medium to a final concentration of about 15% first phase medium. In some aspects, the first phase medium is diluted by the fresh medium to a final concentration of about 10% first phase medium. In some aspects, the first phase medium is diluted by the fresh medium to a final concentration of about 5% first phase medium. In some aspects, the first phase medium is diluted by the fresh medium to a final concentration of less than about 5% first phase medium.

[0045] In some aspects, the concentration of potassium ion in the second phase medium is about 75%, about 70%, about 65%, about 60%, about 55%, about 50%, about 45%, about 40%, about 35%, about 30%, about 25%, about 20%, about 15%, about 10%, about 5%, or less than about 5% the concentration of potassium in the first phase medium. In some aspects, the concentration of potassium ion in the second phase medium is about 50% the concentration of potassium in the first phase medium. In some aspects, the concentration of potassium ion in the second phase medium is about 40% the concentration of potassium in the first phase medium. In some aspects, the concentration of potassium ion in the second phase medium is about 30% the concentration of potassium in the first phase medium. In some aspects, the concentration of potassium ion in the second phase medium is about 25% the concentration of potassium in the first phase medium. In some aspects, the concentration of potassium ion in the second phase medium is about 20% the concentration of potassium in the first phase medium. In some aspects, the concentration of potassium ion in the second phase medium is about 15% the concentration of potassium in the first phase medium. In some aspects, the concentration of potassium ion in the second phase medium is about 10% the concentration of potassium in the first phase medium. In some aspects, the concentration of potassium ion in the second phase medium is about 5% the concentration of potassium in the first phase medium. In some aspects, the concentration of potassium ion in the second phase medium is about 5% the concentration of potassium in the first phase medium.

[0046] In some aspects, the first phase of the second REP comprises culturing the TILs in a first container, and the second phase of the second REP comprises culturing the TILs in a second container, wherein the second container comprises a bioreactor. In some aspects, the first container does not comprise a bioreactor.

[0047] In some aspects, the duration of the first phase of the second REP step is about 1 day to about 5 days. In some aspects, the duration of the first phase of the second REP step is about 1 day, about 2 days, about 3 days, about 4 days, or about 5 days.

[0048] In some aspects, the duration of the second phase of the second REP step is about 3 days to about 13 days. In some aspects, the duration of the second phase of the second REP step is about 3 days, about 4 days, about 5 days, about 6 days, about 7 days, about 8 days, about 9 days, about 10 days, about 11 days, about 12 days, or about 13 days.

[0049] In some aspects, the duration of the first phase of the second REP step is about 3 days, and the duration of the second phase of the second REP step is about 7 days. In some aspects, the duration of the first phase of the second REP step is about 3 days, and the duration of the second phase of the second REP step is about 8 days. In some aspects, the duration of the first phase of the second REP step is about 3 days, and the duration of the second phase of the second REP step is about 9 days. In some aspects, the duration of the first phase of the second REP step is about 4 days, and the duration of the second phase of the second REP step is about 6 days. In some aspects, the duration of the first phase of the second REP step is about 4 days, and the duration of the second phase of the second REP step is about 7 days. In some aspects, the duration of the first phase of the second REP step is about 4 days, and the duration of the second phase of the second REP step is about 8 days. In some aspects, the duration of the first phase of the second REP step is about 4 days, and the duration of the second phase of the second REP step is about 9 days.

[0050] In some aspects, the second REP step comprises a static-REP step followed by dynamic-REP step, wherein the static-REP step comprises culturing the TILs in a medium comprising greater than 4 mM potassium ion, a CD3 agonist, and antigen-presenting cells; and wherein the dynamic-REP step comprises adding the TILs from the static-REP step to fresh medium, wherein the fresh medium does not comprise a CD3 agonist or antigen-presenting cells, and wherein agitation is applied to the culture. In some aspects, the dynamic-REP step comprises inoculating the contents of the static-REP into a rocking motion bioreactor, wherein the rocking motion bioreactor comprises fresh medium.

[0051] In some aspects, the medium of the static-REP comprises about 45 mM potassium ion to about 90 mM potassium ion. In some aspects, the medium of the static-REP comprises 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 potassium ion.

[0052] In some aspects, the medium of the static-REP is diluted by the fresh medium of the dynamic-REP step at a ratio of 1 : 1, 1 :2, 1 :3, 1 :4, 1 :5, 2: 1, 3: 1, or 4: 1 (static-REP :fresh medium).In some aspects, the medium of the static-REP is diluted by the fresh medium of the dynamic-REP step at a ratio of 1 : 1 (static-REP:fresh medium). In some aspects, the medium of the static-REP is diluted by the fresh medium of the dynamic-REP step at a ratio of 1 :2 (static-REP:fresh medium). In some aspects, the medium of the static-REP is diluted by the fresh medium of the dynamic-REP step at a ratio of 1 :3 (static-REP:fresh medium).

[0053] In some aspects, the medium of the static-REP is diluted by the fresh medium to a final concentration of about 75%, about 70%, about 65%, about 60%, about 55%, about 50%, about 45%, about 40%, about 35%, about 30%, about 25%, about 20%, about 15%, about 10%, or about 5% static-REP medium. In some aspects, the medium of the static-REP is diluted by the fresh medium to a final concentration of about 50% first phase medium. In some aspects, the medium of the static-REP is diluted by the fresh medium to a final concentration of about 40% static-REP medium. In some aspects, the medium of the static-REP is diluted by the fresh medium to a final concentration of about 30% static-REP medium. In some aspects, the medium of the static-REP is diluted by the fresh medium to a final concentration of about 25% static-REP medium. In some aspects, the medium of the static-REP is diluted by the fresh medium to a final concentration of about 20% static-REP medium. In some aspects, the medium of the static-REP is diluted by the fresh medium to a final concentration of about 15% static-REP medium. In some aspects, the medium of the static-REP is diluted by the fresh medium to a final concentration of about 10% static-REP medium. In some aspects, the medium of the static-REP is diluted by the fresh medium to a final concentration of about 5% static-REP medium. In some aspects, the medium of the static- REP is diluted by the fresh medium to a final concentration of less than about 5% static-REP medium.

[0054] In some aspects, the concentration of potassium ion in the medium of the dynamic- REP is about 75%, about 70%, about 65%, about 60%, about 55%, about 50%, about 45%, about 40%, about 35%, about 30%, about 25%, about 20%, about 15%, about 10%, about 5%, or less than about 5% the concentration of potassium in the medium of the static-REP. In some aspects, the concentration of potassium ion in the medium of the dynamic-REP is about 50% the concentration of potassium in the medium of the static-REP. In some aspects, the concentration of potassium ion in the medium of the dynamic-REP is about 40% the concentration of potassium in the medium of the static-REP. In some aspects, the concentration of potassium ion in the medium of the dynamic-REP is about 30% the concentration of potassium in the medium of the static-REP. In some aspects, the concentration of potassium ion in the medium of the dynamic-REP is about 25% the concentration of potassium in the medium of the static-REP. In some aspects, theconcentration of potassium ion in the medium of the dynamic-REP is about 20% the concentration of potassium in the medium of the static-REP. In some aspects, the concentration of potassium ion in the medium of the dynamic-REP is about 15% the concentration of potassium in the medium of the static-REP. In some aspects, the concentration of potassium ion in the medium of the dynamic- REP is about 10% the concentration of potassium in the medium of the static-REP. In some aspects, the concentration of potassium ion in the medium of the dynamic-REP is about 5% the concentration of potassium in the medium of the static-REP. In some aspects, the concentration of potassium ion in the medium of the dynamic-REP is less than about 5% the concentration of potassium in the medium of the static-REP.

[0055] Some aspects of the present disclosure are directed to a population of TILs expanded according to a method disclosed herein.

[0056] Some aspects of the present disclosure are directed to a method of treating a cancer in a subject in need thereof, comprising administering a population of expanded TILs disclosed herein. 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-PDl 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.

[0057] In some aspects, the tumor is relapsed. In some aspects, the tumor is metastatic.BRIEF DESCRIPTION OF THE DRAWINGS / FIGURES

[0058] FIG. 1 is a box-plot representation of cell expansion using the MRM process, as previously described (see, e.g., WO / 2022 / 109501, which is incorporated by reference herein in its entirety), and the MRM 2.0 process, described herein. The MRM 2.0 REP1 phase of the process consistently achieved higher fold expansion of TILs compare to PreREP of the MRM process from different tumor types (Metastatic melanoma, NSCLC and metastatic colon tumors). Fold expansion was calculated based on the number of cells seeded compared to the number of cells at the end of PreREP or REPL

[0059] FIG. 2 is a box-plot representation of cell expansion using the MRM process and the MRM 2.0 process for cells obtained from a naive metastatic melanoma tumor. The MRM 2.0 REP1 phase of the process consistently achieved higher fold expansion of TILs compare to PreREP of the MRM process from check-point naive metastatic melanoma patient tumor. Fold expansionwas calculated based on the number of cells seeded compared to the number of cells at the end of PreREP or REP1.

[0060] FIG. 3 is a box-plot representation of cell expansion using the MRM process and the MRM 2.0 process for cells obtained from a checkpoint blockade refractor melanoma tumor. The MRM 2.0 REP1 phase of the process consistently achieved higher fold expansion of TILs compare to PreREP of MRM process from check-point refractory metastatic melanoma patient tumor. Fold expansion was calculated based on the number of cells seeded compared to the number of cells at the end of PreREP or REP1.

[0061] FIG. 4 is a box-plot representation of cell expansion using the MRM process and the MRM 2.0 process for cells obtained from a non-small cell lung cancer (NSCLC) tumor. The MRM 2.0 REP1 phase of the process consistently achieved higher fold expansion of TILs compare to PreREP of the MRM process from NSCLC patient tumor. Fold expansion was calculated based on the number of cells seeded compared to the number of cells at the end of PreREP or REPL

[0062] FIG. 5 is a box-plot representation of cell expansion using the MRM process and the MRM 2.0 process for cells obtained from a colorectal cancer (CRC / CRLM) tumor. The MRM 2.0 REP1 phase of the process consistently achieved higher fold expansion of TILs compare to PreREP of the MRM process from metastatic colon patient tumor. Fold expansion was calculated based on the number of cells seeded compared to the number of cells at the end of PreREP or REPL

[0063] FIG. 6 is a box-plot representation of the projected cell yield for manufacturing scale using the MRM 2.0 process for cells obtained from various tumor types (metastatic melanoma naive (1), check-point blockade refractory metastatic melanoma (2), metastatic CRC (3), and NSCLC (4)) at research scale. A minimum dose of 1010cells can be achieved from all listed indications using the MRM 2.0 process at a starting cell number of at least 40 million. The projected total yield indicates that the MRM 2.0 process can achieve a median cell yield of 60 x 109in multiple indications. Projected cell yield was calculated using the MRM 2.0 REP1 and REP2 research scale fold expansion. Projected cell yield for REP1 was calculated using the REP1 fold expansion obtained at research scale multiplied by the number of live cells that would likely be seeded from the same patient tumor. REP1 projected yield was multiplied by the research scale REP2 fold expansion to calculate the final projected yield.

[0064] FIG. 7 is a box plot demonstrating %CD8+ T cells for the MRM and MRM 2.0 process final products (P=0.0097). 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.

[0065] FIGs. 8A-8B are bar graphs illustrating TIL fold expansion following a first REP step (REP1; FIG. 8 A) and a second REP step (REP2; FIG. 8B) for cells obtained from an immune check-point blockade refractory melanoma patient using TCM (control medium), MRM 2.0 (disclosed herein), or MRM CD3 only (MRM with only anti-CD3 antibodies and no feeder cells). The REP1 and REP2 fold expansions demonstrate that the addition of feeder cells in REP1 allows for a higher cell yield in REP2 (FIGs. 8A-8B). FIGs. 8C-8E are FACS plots demonstrating MRM media favors expansion of CD8 T cells in the MRM 2.0 process (FIG. 8D) and MRM medium lacking feeder cells (FIG. 8E) as compared to control (FIG. 8C).

[0066] FIGs. 9A-9B are bar graphs illustrating fold expansion following a first REP step (REP1 ; FIG. 9A) and a second REP step (REP2; FIG. 9B) for cells obtained from a NSCLC patient using TCM (control medium), MRM 2.0 (disclosed herein), or MRM CD3 only (MRM with only anti-CD3 antibodies and no feeder cells). These REP1 and REP2 fold expansions demonstrate that without feeder cells in REP1, the cell yield in REP2 can drop significantly. (FIGs. 9A-9B). FIGs. 9C-9E are FACS plot demonstrating MRM media favors expansion of CD8 T cells in the MRM 2.0 process (FIG. 9D) and MRM medium lacking feeder cells (FIG. 9E) as compared to control (FIG. 9C).

[0067] FIGs. 10A-10B: Based on flow cytometric surface protein expression, CD8+ TIL were plotted on CD8 versus CD27, and the percentage frequency of total CD8+ TIL were illustrated by box plot for MRM and MRM 2.0 final cell products generated from naive and immune checkpoint inhibitor refractory metastatic melanoma, NSCLC, and metastatic colon cancer patients (FIG. 10 A); and CD8+ TIL were plotted on CD69 versus CD39 and the percentage frequency of CD39-CD69- cells were illustrated by box plot for MRM and MRM 2.0 final cell products generated from naive and immune checkpoint inhibitor refractory metastatic melanoma, NSCLC, and metastatic colon cancer patients.

[0068] FIGs. 11A-11D are box plot representations demonstrating the percentage frequency, based on flow cytometric surface protein expression, of CD8+ TIL plotted on CD8 versus CD27 (FIG. 11 A), CD8+ TIL plotted on CD62L versus CD27 (FIG. 11B), and CD8+ TIL plotted on CD39 versus CD69 (FIG. 11C-1 ID), for MRM and MRM 2.0 final products generated from immune check-point blockade treated melanoma patients. CD39-CD69- cells represent stemlike cells, which are correlated with positive clinical outcome in ACT-TIL. CD39+CD69+ TILs represent highly differentiated cells which are associated with poor persistence in ACT-TIL.

[0069] FIGs. 12A-12D are box plot representations demonstrating the percentage frequency, based on flow cytometric surface protein expression, of CD8+ TIL plotted on CD8versus CD27 (FIG. 12 A), CD8+ TIL plotted on CD62L versus CD27 (FIG. 12B), and CD8+ TIL plotted on CD39 versus CD69 (FIG. 12C-12D), for MRM and MRM 2.0 final products generated from immune check-point blockade treated melanoma patients.

[0070] FIGs. 13A-13D are box plot representations demonstrating the percentage frequency, based on flow cytometric surface protein expression, of CD8+ TIL plotted on CD8 versus CD27 (FIG. 13 A), CD8+ TIL plotted on CD62L versus CD27 (FIG. 13B), and CD8+ TIL plotted on CD39 versus CD69 (FIG. 13C-13D), for MRM and MRM 2.0 final products generated from NSCLC / lung cancer patients.

[0071] FIGs. 14A-14D are box plot representations demonstrating the percentage frequency, based on flow cytometric surface protein expression, of CD8+ TIL plotted on CD8 versus CD27 (FIG. 14 A), CD8+ TIL plotted on CD62L versus CD27 (FIG. 14B), and CD8+ TIL plotted on CD39 versus CD69 (FIG. 14C-14D), for MRM and MRM 2.0 final products generated from metastatic colon cancer patients.

[0072] FIGs. 15A-15D are box plots illustrating cell phenotypes based on flow cytometric surface protein expression for MRM and MRM 2.0 final products manufactured from immune check-point blockade treated melanoma patients (FIG. 15 A), check-point naive melanoma patients (FIG. 15B), NSCLC / Lung patients (FIG. 15C), and metastatic colon patients (FIG. 15D). CD8+ TIL were plotted on CD8 versus PD1, and the percentage frequency of parent of the CD8+ TIL are illustrated by box plot.

[0073] FIG. 16 is a box plot demonstrating Simpson clonality calculated from TCR[3 sequencing of final products manufactured using MRM and MRM 2.0 process. Simpson clonality demonstrates TCR repertoire diversity.

[0074] FIGs. 17A-17C are graphical representations of TCR beta sequencing that was performed on three patients (FIGs. 17A-17C, respectively), illustrating the Top 200 clone distribution of TIL in baseline tumor (Day 0-tumor) and products generated from the initial tumor using MRM (process 1) and MRM 2.0 (Process 2). Each TCR template was normalized to 30,000 (to account for the final product comprising TILs) and plotted. The graphs demonstrate consistent preservation of TCR templates in the TIL products generated using the MRM and MRM 2.0 process. Final TIL products were manufactured from immune checkpoint blockade (ICB) treated patients.

[0075] FIG. 18 is a schematic of the experimental design for identifying, tracking, and analyzing putative tumor-reactive clones, as previously described (see, e.g., WO / 2023 / 077034, which is incorporated by reference herein in its entirety).

[0076] FIGs. 19A and 19B are graphs illustrating 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. 19A shows the ability of the different modified T cells to kill autologous tumor cells (at 5: 1 effectortarget ration) using an in vitro killing assay. Tumor only group was used as a control. FIG. 19B shows IFN-y production by the different modified T cells after in vitro stimulation with the autologous tumor cells i.e., "TR TCR I," "TR TCR 2," and "TR TCR 3 "). Tumor only and unstimulated cells (i.e., "TIL only") were used as control.

[0077] FIG. 20A and FIG. 20B are two representations of the same graph showing the TCR beta sequencing illustrating the distribution (clone size) of the top 100 clones in the Day 0 Tumor, MRM TIL product and the MRM 2.0 TIL product. FIG. 20 A demonstrates that the top 100 putative tumor-reactive clones (by frequency) found in the Day 0 Tumor sample are preserved during the MRM process and slightly better (e.g., clone size) in the MRM 2.0 process. The bold squares in FIG. 20B represent the confirmed tumor-reactive clones from FIG. 19A and 19B (with TCR 1, TCR 2, and TCR 3) within the preserved population of putative tumor-reactive clones shown.

[0078] FIG. 21 is a graphical representation of cytolysis of the TIL products generated from metastatic melanoma using MRM and MRM 2.0, as measured by co-culturing autologous tumor cells at a ratio of 1 : 1.

[0079] FIGs. 22A-22G are graphical representations of MRM 2.0 TIL tumor specificity. FIGs. 22A-22D show representative flow cytometry plots of MRM 2.0 TIL incubated with (FIGs. 22A and 22C) or without (FIGs. 22B and 22D) anti-MHC-I blocking antibody and autologous tumor cells for two NSCLC samples. FIG. 22E shows a tSNE plot of CD3+ cells from four tumor samples (three NSCLC and one metastatic breast cancer) demonstrating clustering based on expression of 13 markers involved in T cell differentiation, sternness, activation, and exhaustion. FIG. 22F shows a graph of CD8+ T cell fold change in INF-y secretion (pg / mL) by MRM 2.0 TIL in autologous tumor cell co-cultures with and without anti-MHC-I blocking antibodies. FIG. 22G shows a graph of the 4-1BB+ population fold change of MRM 2.0 TIL in autologous tumor cell co-cultures with and without anti-MHC-I blocking antibodies.

[0080] FIG. 23 is a schematic summary of a tumor-reactive enrichment TIL production processes disclosed herein.

[0081] FIG. 24 is a schematic representation of fluorescence activated cell sorting (FACS) based on expression of a specific marker, according to an aspect of the present description.

[0082] FIGs. 25A-25B are schematic representations of sorting cells based on magnetic selection. FIG. 25 A shows the process of magnetic selection. FIG. 25B shows an example of how cells can be labeled and selected using phycoerythrin (PE) conjugated antibodies and magnetic bead conjugated antibodies.

[0083] FIGs. 26A-26B are graphical representations of analysis of different surface markers by flow cytometry of single cell suspensions of tumors from multiple indications. FIG. 26 A shows the percentage of CD8+PD1+CD39+ and CD8+PD1-CD39- TIL expressing HLA-DR. FIG. 26B shows the percentage of CD8+PD1+CD39+ and CD8+PD1-CD39- TIL expressing HLA-DR, displayed by tumor type.

[0084] FIGs. 27A-27B are graphical representations of T cell enrichment without altering HLA-DR expression. FIG. 27A shows the percentage of T cells in the tumor single cell suspension on Day 0 and Day 4 (presorted cells), as measured by percent of CD3+ cells of live cells. FIG. 27B shows the percentage of HLA-DR+ T cells in the tumor single cell suspension on Day 0 and Day 4 (presorted cells) as measured by percent of HLA-DR+ cells of CD3+ cells. Statistical analysis was measured by paired, two-tailed T test.

[0085] FIGs. 28A-28C are illustrations of FACS analyses of final TIL product from bulk (FIG. 28 A), HLA-DR+ (FIG. 28B), or HLA-DR- (FIG. 28C) TILs in a metastatic melanoma tumor analyzed for the presence of TIL cells that recognize pooled tetramers melanoma associated antigens (MARTI, MAGEA2, gplOO, tyrosinase).

[0086] FIGs. 29A-29E are graphical representations of enrichment of high frequency clones from tumor single cell suspension. FIGs. 29A-29D illustrate clone size based on the frequency of unique templates (TCR clones), normalized to 30K templates, from the top 200 TCR clones from the tumor single cells suspension for four samples. FIG. 29E shows the sum of frequency of the top 200 productive TCR clones from the tumor single cell suspension found in the corresponding TIL products. TIL products represent two metastatic melanoma, one breast and one CRLM tumor.

[0087] FIG. 30 is a schematic summary of tumor-reactive enrichment TIL production processes disclosed herein.

[0088] FIG. 31 is a schematic representation of fluorescence activated cell sorting (FACS) based on expression of a specific marker, according to an aspect of the present description.

[0089] FIGs. 32A-32B are schematic representations of sorting cells based on magnetic selection. FIG. 32A shows the process of magnetic selection. FIG. 32B shows an example of how cells can be labeled and de-selected using phycoerythrin (PE) conjugated antibodies and magnetic bead conjugated antibodies.

[0090] FIGs. 33A-33B are graphical representations of analysis of different surface markers by flow cytometry of single cell suspensions of tumors from multiple indications. FIG. 33A shows the percentage of CD8+PD1+CD39+ and CD8+PD1-CD39- TIL expressing CD127. FIG. 33B shows the percentage of CD8+PD1+CD39+ and CD8+PD1-CD39- TIL expressing CD127, displayed by tumor type.

[0091] FIGs. 34A-34B are graphical representations of T cell enrichment without altering CD127 expression. FIG. 34A shows the percentage of T cells in the tumor single cell suspension on Day 0 and Day 4 (presorted cells), as measured by percent of CD3+ cells of live cells. FIG. 34B shows the percentage of CD127- T cells in the tumor single cell suspension on Day 0 and Day 4 (presorted cells) as measured by percent of CD 127- cells of CD3+ cells. Statistical analysis was measured by paired, two-tailed T test.

[0092] FIG. 35 is a graphical representation of CD127- selected TIL expansion after 16 days as compared to Bulk Unsorted or CD 127+ TIL, across various indications.

[0093] FIGs. 36A and 36B are graphical representations of enrichment of high frequency clones from tumor single cell suspensions. FIG. 36A illustrates clone size based on the frequency of unique templates (TCR clones), normalized to 30K templates, from the top 200 TCR clones from the tumor single cells suspension for four samples. FIG. 36B shows the sum of frequency of the top 200 productive TCR clones from the tumor single cell suspension, sorted either by PD1 versus CD39 expression or by CD127 expression, found in the corresponding TIL products. TIL products represent two different metastatic melanoma tumors.

[0094] FIG. 37A is a graphical representation of CD127- TIL product IFN-y secretion in an antigen-dependent manner when co-cultured with autologous tumor cells, as compared to bulk, and bulk or CD127- with an MHCI block. Each symbol represents an individual tumor coculture. FIG. 37B is a graphical representation of CD127- TIL product CD8+41BB+ expression in CD3+ cells in an antigen-dependent manner when co-cultured with autologous tumor cells, as compared to bulk, and bulk or CD127- with an MHCI block.

[0095] FIG. 38A-38G are line graphs illustrating stem-like qualities of CS127- de-selected TIL products produced using sorted CD127- and CD127+ TIL from a NSCLC tumor and cultured using either a control process (TCM) or a metabolic reprogramming medium (MRM) process.Expression is shown for each of CD39+CD69+ (FIG. 38A), CD39-CD69- (FIG. 38B), CD62L+ (FIG. 38C), CD62L+CD27+ (FIG. 38D), CD7+ (FIG. 38E), LAG3+NKG2A+ (FIG. 38F), and Ki67+ (FIG. 38G).

[0096] FIGs. 39A-39B are graphical representations of REP2 fold expansion in a multiexpansion stage (static and reactor) REP2 by day, for two runs, at 100% MRM, 50% MRM, and 25% MRM.DETAILED DESCRIPTION

[0097] 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 oligocl onal 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 CD8+TILs and / or increasing multipotency and / or pluripotency of the cultured TILs in a shorter period of time than conventional methods. 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.

[0098] Some aspects of the present disclosure are directed to methods of culturing TILs ex vivo or in vitro comprising (i) dissociating a tumor sample obtained from the subject comprising TILs to generate a single cell suspension; (ii) culturing the single cell suspension in a first expansion medium, wherein the first expansion medium comprises a CD3 agonist and APCs at a first ratio (a "first REP step"); and (iii) culturing the TILs from the first REP step in a second expansion medium, wherein the second expansion medium comprises a CD3 agonist and APCs at a second ratio (a "second REP step").

[0099] Some aspects of the present disclosure are directed to methods of culturing TILs ex vivo or in vitro for a therapy in a subject in need thereof comprising: (i) culturing a dissociated tumor sample comprising TILs in a first expansion medium, wherein the first expansion medium comprises a CD3 agonist and APCs at a first ratio (a "first REP step"); and (ii) culturing the TILs from the first REP step in a second expansion medium, wherein the second expansion medium comprises a CD3 agonist, and APCs at a second ratio (a "second REP step"). In some aspects, the dissociated tumor sample is a single cell suspension.

[0100] Some aspects of the present disclosure are directed to methods of culturing TILs ex vivo or in vitro for a therapy in a subject in need thereof comprising culturing a population of TILs obtained from a tumor sample of the subject in an expansion medium comprising a CD3 agonist and antigen-presenting cells (APCs); wherein, prior to or after culturing in the expansion medium, the TILs are not cultured in a medium that does not comprise APCs. In some aspects, prior to culturing in the expansion medium, the population of TILs is cultured in a medium comprising APCs. In some aspects, after culturing in the expansion medium, the population of TILs is cultured in a medium comprising APCs.

[0101] Some of the aspects of the present disclosure are directed to methods of increasing the number of tumor-reactive tumor infiltrating lymphocytes (TILs) ex vivo or in vitro in a composition for a TIL therapy comprising selecting for TILs that express an HLA polypeptide ("population of selected TILs") from a heterogeneous population of TILs obtained from a tumor. Some aspects of the present disclosure are directed to methods of generating an expanded population of tumor-reactive TILs comprising selecting for TILs that express an HLA polypeptide ("population of selected TILs") from a heterogeneous population of TILs obtained from a tumor.

[0102] In some aspects, the method further comprises obtaining the heterogeneous population of TILs from a tumor. In some aspects, the tumor is obtained from a subject. In some aspects, the tumor is dissociated to generate the heterogeneous population of TILs. In some aspects, the dissociated tumor sample is a single cell suspension. In some aspects, the tumor is cultured in a medium comprising IL-2 to generate the heterogeneous population of TILs. In some aspects, prior to the selecting, the heterogeneous population of TILs is cultured in a medium comprising IL-2 ("initial expansion"). In some aspects, the method further comprises culturing the selected population of TILs in a medium comprising IL-2.

[0103] In some aspects, the method further comprises culturing the population of selected TILs in a first expansion medium comprising a cell-support matrix, a cytokine, and a T cell activator (a "first REP step"). In some aspects, the method further comprises culturing the population of selected TILs after the first REP step in a second expansion medium comprising a cell-support matrix, cytokine, and a T cell activator (a "second REP step").

[0104] Some aspects of the present disclosure are directed to methods of culturing tumor infiltrating lymphocytes (TILs) ex vivo or in vitro for a therapy for a subject in need thereof comprising: (i) dissociating a tumor sample obtained from the subject comprising TILs to generate a single cell suspension comprising a heterogeneous population of TILs; (ii) selecting for TILs that express an HLA polypeptide ("population of selected TILs") from the heterogeneous populationof TILs; (iii) culturing the population of selected TILs in a first expansion medium, wherein the first expansion medium comprises a T cell activator and a cell support matrix at a first ratio (a "first REP step"); and (iv) culturing the TILs from the first REP step in a second expansion medium, wherein the second expansion medium comprises a T cell activator and a cell support matrix at a second ratio (a "second REP step").

[0105] Some aspects of the present disclosure are directed to methods of culturing tumor infiltrating lymphocytes (TILs) ex vivo or in vitro for a therapy for a subject in need thereof comprising: (i) dissociating a tumor sample obtained from the subject comprising TILs to generate a single cell suspension comprising a heterogeneous population of TILs; (ii) culturing the heterogeneous population of TILs in a first expansion medium, wherein the first expansion medium comprises a T cell activator and a cell support matrix at a first ratio (a "first REP step"); (iii) selecting for TILs that express an HLA polypeptide ("population of selected TILs") from the heterogeneous population of TILs; and (iv) culturing the population of selected TILs from the first REP step in a second expansion medium, wherein the second expansion medium comprises a T cell activator and a cell support matrix at a second ratio (a "second REP step"). In some aspects, the method further comprises culturing the heterogeneous population of TILs in a medium comprising IL-2 before the first REP step.

[0106] Some aspects of the present disclosure are directed to methods of culturing tumor infiltrating lymphocytes (TILs) ex vivo or in vitro for a therapy for a subject in need thereof comprising: (i) dissociating a tumor sample obtained from the subject comprising TILs to generate a single cell suspension comprising a heterogeneous population of TILs; (ii) culturing the heterogeneous population of TILs in an initial expansion medium, wherein the initial expansion medium comprises IL-2; (iii) selecting for TILs that express an HLA polypeptide ("population of selected TILs") from the heterogeneous population of TILs; (iv) culturing the population of selected TILs in a first expansion medium, wherein the first expansion medium comprises a T cell activator and a cell support matrix at a first ratio (a "first REP step"); and (v) culturing the TILs from the first REP step in a second expansion medium, wherein the second expansion medium comprises a T cell activator and a cell support matrix at a second ratio (a "second REP step").

[0107] Some aspects of the present disclosure are directed to methods of culturing tumor infiltrating lymphocytes (TILs) ex vivo or in vitro for a therapy for a subject in need thereof comprising: (i) dissociating a tumor sample obtained from the subject comprising TILs to generate a single cell suspension comprising a heterogeneous population of TILs; (ii) culturing the heterogeneous population of TILs in an initial expansion medium, wherein the initial expansionmedium comprises IL-2; (iii) selecting for TILs that express an HLA polypeptide ("population of selected TILs") from the heterogeneous population of TILs; (iv) culturing the population of selected TILs in a first expansion medium, wherein the first expansion medium comprises a T cell activator and a cell support matrix at a first ratio (a "first REP step"); and (v) culturing the TILs from the first REP step in a second expansion medium, wherein the second expansion medium comprises a T cell activator and a cell support matrix at a second ratio (a "second REP step").

[0108] Some of the aspects of the present disclosure are directed to methods of increasing the number of tumor-reactive tumor infiltrating lymphocytes (TILs) ex vivo or in vitro in a composition for a TIL therapy comprising removing or de-selecting TILs that express CD 127 ("population of de-selected TILs") from a heterogeneous population of TILs obtained from a tumor. Some aspects of the present disclosure are directed to methods of generating an expanded population of tumor-reactive TILs comprising removing or de-selecting TILs that express CD127 ("population of de-selected TILs") from a heterogeneous population of TILs obtained from a tumor.

[0109] In some aspects, the method further comprises obtaining the heterogeneous population of TILs from a tumor. In some aspects, the tumor is obtained from a subject. In some aspects, the tumor is dissociated to generate the heterogeneous population of TILs. In some aspects, the dissociated tumor sample is a single cell suspension. In some aspects, the tumor is cultured in a medium comprising IL-2 to generate the heterogeneous population of TILs. In some aspects, prior to the removing or de-selecting, the heterogeneous population of TILs is cultured in a medium comprising IL-2 ("initial expansion"). In some aspects, the method further comprises culturing the population of CD127- TILs (“population of selected TIL”) without the population of de-selected CD 127+ TILs in a medium comprising IL-2.

[0110] In some aspects, the method further comprises culturing the population of selected TILs in a first expansion medium comprising a cell-support matrix, a cytokine, and a T cell activator (a "first REP step"). In some aspects, the method further comprises culturing the population of selected TILs after the first REP step in a second expansion medium comprising a cell-support matrix, cytokine, and a T cell activator (a "second REP step").

[0111] Some aspects of the present disclosure are directed to methods of culturing tumor infiltrating lymphocytes (TILs) ex vivo or in vitro for a therapy for a subject in need thereof comprising: (i) dissociating a tumor sample obtained from the subject comprising TILs to generate a single cell suspension comprising a heterogeneous population of TILs; (ii) removing or deselecting for TILs that express CD127 ("population of de-selected TILs") from the heterogeneouspopulation of TILs; (iii) culturing the population of TILs without the population of de-selected TILs in a first expansion medium, wherein the first expansion medium comprises a T cell activator and a cell support matrix at a first ratio of cell support matrix to TIL (a "first REP step"); and (iv) culturing the TILs from the first REP step in a second expansion medium, wherein the second expansion medium comprises a T cell activator and a cell support matrix at a second ratio of cell support matrix to TIL (a "second REP step").

[0112] Some aspects of the present disclosure are directed to methods of culturing tumor infiltrating lymphocytes (TILs) ex vivo or in vitro for a therapy for a subject in need thereof comprising: (i) dissociating a tumor sample obtained from the subject comprising TILs to generate a single cell suspension comprising a heterogeneous population of TILs; (ii) culturing the heterogeneous population of TILs in an initial expansion medium, wherein the initial expansion medium comprises IL-2; (iii) removing or de-selecting for TILs that express CD127 ("population of de-selected TILs") from the heterogeneous population of TILs; (iv) culturing the population of TILs without the population of de-selected TILs (“population of selected TILs”) in a first expansion medium, wherein the first expansion medium comprises a T cell activator and a cell support matrix at a first ratio of cell support matrix to TIL (a "first REP step"); and (v) culturing the TILs from the first REP step in a second expansion medium, wherein the second expansion medium comprises a T cell activator and a cell support matrix at a second ratio of cell support matrix to TIL (a "second REP step").

[0113] Some aspects of the present disclosure are directed to methods of culturing tumor infiltrating lymphocytes (TILs) ex vivo or in vitro for a therapy for a subject in need thereof comprising: (i) dissociating a tumor sample obtained from the subject comprising TILs to generate a single cell suspension comprising a heterogeneous population of TILs; (ii) culturing the heterogeneous population of TILs in an initial expansion medium, wherein the initial expansion medium comprises IL-2; (iii) removing or de-selecting for TILs that express CD127 ("population of de-selected TILs") from the heterogeneous population of TILs; (iv) culturing the population of selected TILs without the population of de-selected TILs (“population of selected TILs”) in a first expansion medium, wherein the first expansion medium comprises a T cell activator and a cell support matrix at a first ratio of cell support matrix to TIL (a "first REP step"); and (v) culturing the TILs from the first REP step in a second expansion medium, wherein the second expansion medium comprises a T cell activator and a cell support matrix at a second ratio of cell support matrix to TIL (a "second REP step").

[0114] 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.

[0115] 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

[0116] 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.

[0117] 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.

[0118] 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.

[0119] 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.

[0120] 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.

[0121] 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.

[0122] Abbreviations used herein are defined throughout the present disclosure. Various aspects of the disclosure are described in further detail in the following subsections.

[0123] 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, z.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.

[0124] 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).

[0125] 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.

[0126] 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., CTS™ OPTIMIZER™. 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.

[0127] As used herein, a "control process" or a "MRM process" refers to a method disclosed herein, wherein the TIL culturing and expansion process includes a control medium, or an MRM medium, respectively. As such, a "control process" refers to the culturing of TILs in a control medium disclosed herein, such as AIM V or TCM without a modified concentration of potassium (z.e., less than 40 mM, e.g., 5 mM, potassium ion), and a "MRM process" refers to the culturing of TILs in a MRM medium disclosed herein.

[0128] 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 (NK) 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.

[0129] 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. In some aspects, the population of cells comprises a population comprising one or more cells that have been selected based on a particular cell characteristic. In some aspects, the population of cells comprises a population comprising one or more cells that do not include cells that have been selected based on a particular cell characteristic.

[0130] 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.

[0131] 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 aP, 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.

[0132] 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 Th 1 cell. In some aspects, the immune cell is a Th2 cell. In some aspects, the immune cell is a Tcl7 cell. In some aspects, the immune cell is a Th 17 cell. In some aspects, the immune cell is a Tregcell.

[0133] 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 primaryexposure. 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.

[0134] 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+, CD27+, 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 CD8+ 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.

[0135] 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.

[0136] 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.

[0137] 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, GHR, GRB2, IFNA1, IFNA10, IFNA13, IFNA14, IFNA16, IFNA17, IFNA2, IFNA21, IFNA4, IFNA5, IFNA6, IFNA7, IFNA8, IFNAR1, IFNAR2, IFNB1, IFNE, IFNG, IFNGR1, IFNGR2, IFNK, IFNL1, IFNL2, IFNL3, IFNLR1, IFNW1, IL10, IL10RA, IL10RB, IL11, IL11RA, IL12A, IL12B, IL12RB1, IL12RB2, IL13, IL13RA1, IL13RA2, IL15, IL15RA, IL19, 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, PIK3R1, PIK3R2, PIK3R3, PIK3R5, PIM1, PRL, PRLR, PTPN11, PTPN6, SOCS1, SOCS2, SOCS3, SOCS4, SOCS5, SOCS7, S0S1, S0S2, 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 777:837-51 (2002); Tripathi et al., J. Immunology 755:2116-24 (2010); and / or Johnnidis et al., Science Immunology 6:eabe3702 (Jan. 15, 2021), each of which is incorporated by reference herein in its entirety.

[0138] 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 fromMTCH2, RAB6C, KIAA0195, SETD2, C2orf24, NRD1, GNA13, COPA, SELT, TNIP1, CBFA2T2, LRP10, PRKCI, BRE, ANKS1A, PNPLA6, ARL6IP1, WDFY1, MAPK1, GPR153, SHKBP1, MAP1LC3B2, PIP4K2A, HCN3, GTPBP1, TLN1, C4orB4, KIF3B, TCIRG1, PPP3CA, ATG4D, TYMP, TRAF6, C17orf76, 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, RASGRP1, Clorf58, Cl lorf63, C6orfl29, 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, MYO5A, KIR2DL3, DLG3, MXD4, RALGDS, S1PR5, WSB2, CCR3, TIP ARP, SP140, CD151, SOX13, KRTAP5-2, NF1, PEA15, PARP8, RNF166, UEVLD, LIMK1, CACNB1, TMX4, SLC6A6, LB Al, SV2A, LLGL2, IRF1, PPP2R5C, CD99, RAPGEF1, PPP4R1, OSBPL7, FOXP4, SLA2, TBC1D2B, ST7, JAZF1, 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, GPRIN3,, ADCY9, CD300A, SCD5, ABB, PTPN22, LGALS1, SYTL3, BMPR1A, TBK1, PMAIP1, RASGEF1A,, GCNT1, GABARAPL1, STOM, CALHM2, ABCA2, PPP1R16B, SYNE2, PAM, C12orf75, CLCF1, MXRA7, APOBEC3C, CLSTN3, ACOT9, HIP1, LAG3, TNFAIP3, DCBLD1, KLF6, CACNB3, RNF19A, RAB27A, FADS3, DLG5, APOBEC3D, TNFRSF1B, ACTN4, TBKBP1, ATXN1, ARAP2, ARHGEF12, FAM53B, MAN1A1, FAM38A, PLXNC1, GRLF1, SRGN, HLA-DRB5, B4GALT5, WIPI1, PTPRJ, SLFN11, DUSP2, ANXA5, AHNAK, NEO1, 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, KATNAL1, FAM46C, ZC3HAV1L, ANXA2P2, CTNNA1, NPC1, C3AR1, CRIM1, SH2D2A, ERN1, YPEL1, TBX21, SLC1A4, FASLG, PHACTR2, GALNT3, ADRB2, PIK3AP1, TLR3, PLEKHA5, DUSP10, GNAO1, PTGDR, FRMD4B, ANXA2, EOMES, CADM1, MAF, TPRG1, NBEAL2, PPP2R2B, PELO, SLC4A4, KLRF1, FOSL2, RGS2, TGFBR3, PRF1, MYO1F, GAB3, C17orf66, MICAL2, CYTH3, TOX, HLA-DRA, SYNE1, WEE1, PYHIN1, F2R, PLD1, THBS1, CD58, FAS, NETO2, CXCR6, ST6GALNAC2, DUSP4, AUTS2, Clorf21, KLRG1, TNIP3, GZMA, PRR5L, PRDM1, ST8SIA6, PLXND1, PTPRM, GFPT2, MYBL1, SLAMF7, FLJ16686,, GNLY, ZEB2, CST7, IL18RAP,CCL5, KLRD1, KLRB1, and any combination thereof (see, e.g., Gattinoni, L., et al., Nat Med 17(10): 1290-97 (2011).

[0139] As used herein, the term "effector memory T cells" or "TEM cells" refer to memory T cells that express CD45RO 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.

[0140] 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.

[0141] 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 activated TEFF cell and an immune response.

[0142] 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.

[0143] 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 fragment into 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 fragment 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 dissociated 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.

[0144] "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 anincrease 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.

[0145] As used herein, the term "rapid expansion protocol" or "REP" refers to a process of expanding a population of immune cells, e.g., TILs, by culturing the immune cells, e.g., TILs, in the presence of one or more agents that stimulate the immune cells. In some aspects, the one or more agents that stimulate the immune cells comprise (i) a CD3 agonist, (ii) antigen presenting cells (APCs), or (iii) both a CD3 agonist and APCs. See, e.g., Dudley, et al., Science 298:850-54 (2002); Dudley, et al., J. Clin. Oncol. 23:2346-57 (2005); Dudley, et al., J. Clin. Oncol. 26:5233- 39 (2008); Riddell, et al., Science 257:238-41 (1992); and Dudley, et al., J. Immunother. 26:332- 42 (2003), each of which is incorporated by reference herein in its entirety. Any CD3 agonist can be used in a REP step disclosed herein. In some aspects, the CD3 agonist comprises OKT3. The term "APCs," as used herein, refers to natural cells and to synthetic equivalents (i.e., artificial antigen presenting cells, or aAPCs), such as vesicles and / or microparticles. 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, 4 IBB ligand, and 0X40 ligand. K562 cells do not express HLA-A, HLA-B, or HLA-DR molecules, which makes them a powerful tool for T cell expansion when transduced with the above mentioned costimulatory ligands (See, e.g., Suhoski et al., Molecular therapy, 2007).

[0146] 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. In some aspects, the anti-CD3 antibody comprises visilizumab. In some aspects, the anti-CD3 antibody comprises otelixizumab. In some aspects, the anti-CD3 antibody comprises teplizumab.

[0147] 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-availableforms such as OKT-3 (30 ng / mL, MACS GMP 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.

[0148] 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 TILs. The yield can be determined using any methods, including, but not limited to, estimating the yield based on a representative sample.

[0149] 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 CD45ROlow. In some aspects, a "less-differentiated" or "stemlike" 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., etal., J. Clin. Invest. 115: 1616-1626 (2005); Gattinoni, L., etal. Nat Med 15(7):808-814 (2009); Lynn, R.C., et al., Nature 576(7786): 293-300 (2019); Gattinoni, L., et al, J. Clin. Invest. 115: 1616-1626 (2005); Gattinoni, L., et al. Nat Med 15(7):808-814 (2009); and Gattinoni, L., et al., Nat Med 17(10): 1290-1297 (2011).

[0150] Sternness 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, DSC1, TSHZ2, MYB, LEF1, TIMD4, MAL, KRT73, SESN3, CDCA7L, LOC283174, TCF7, SLC16A10, LASS6,UBE2E2, IL7R, GCNT4, TAF4B, SULT1B1, SELP, KRT72, STXBP1, TCEA3, FCGBP, CXCR5, GPA33, NELL2, APBA2, SELL, VIPR1, FAM153B, PPFIBP2, FCER1G, GJB6, 0CM2, GCET2, LRRN1, IL6ST, LRRC16A, IGSF9B, EFHA2, LOC129293, APP, PKIA, ZC3H12D, CHMP7, KIAA0748, SLC22A17, FLJ13197, NRCAM, C5orfl3, GIPC3, WNT7A, FAM117B, BEND5, LGMN, FAM63A, FAM153B, ARHGEF11, RBM11, RIC3, LDLRAP1, PELI1, PTK2, KCTD12, LM07, CEP68, SDK2, MC0LN3, ZNF238, EDAR, FAM153C, FAAH2, BCL9, C17orf48, MAP1D, ZSWIM1, S0RBS3, IL4R, SERPINF1, C16orf45, SPTBN1, KCNQ1, LDHB, BZW2, NBEA, GAL3ST4, CRTC3, MAP3K1, HLA-DOA, RAB43, SGTB, CNN3, CWH43, KLHL3, PIM2, RGMB, C16orf74, AEBP1, SNORD115-11, 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, IGF1R, KRT72, SLC16A10, GJB6, LRRN1, PRAGMIN, GIPC3, FLNB, ARRB1, SLC7A8, NUCB2, LRRC7, MYO15B, MAL, AEBP1, SDK2, BZW2, GAL3ST4, PITPNM2, ZNF496, FAM117B, C16orf74, TDRD6, TSPAN32, C18orf22, C3orf44, LOC129293, ZC3H12D, MLXIP, C7orfl0, STXBP1, KCNQ1, FLJ13197, LDLRAP1, RAB43, RIN3, SLC22A17, AGBL3, TCEA3, NCRNA00185, FAM153B, FAM153C, VIPR1, MMP19, HBS1L, EEF2K, SNORA5C, UBASH3A, FLJ43390, RP6-213H19.1, INPP5A, PIM2, TNFRSF10D, SNRK, LOC100128288, PIGV, LOC100129858, SPTBN1, PROS1, MMP28, HES1, CACHD1, NSUN5C, LEF1, TTTY14, SNORA54, HSF2, C16orf67, NSUN5B, KIAA1257, NRG2, CAD, TARBP 1, STRADB, MT IF, TMEM41B, PDHX, KDM6B, LOCI 00288322, UXS1, LGMN, NANOS2, PYGB, RASGRP2, C14orf80, XPO6, SLC24A6, FAM113A, MRM1, FBXW8, NDUFS2, 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, LEF1, AP3M2, SLC2A3, ICAM2, PLAC8, SCML1, IL7R, ABLIM1, RASGRP2, TRABD2A, SATB1, ALG13, ARID5A, BACH2, PABPC1, GPCPD1, NELL2, TAF4B, FCMR, ARRDC2, Clorfl62, FAM177A1, ANKRD12, TXK, SORL1, AQP3, ADTRP, FXYD7, CD28, P2RY8, CRYBG1, TNFSF8, BEX2, PGAP1, PTGER4, MAML2, BEX3, PCSK1N, INPP4B, AC119396.1, CXCR5, LINC00402, CCR4, IL6R, ZBTB10, ITGA6, ARMH1, RILPL2, FOXP1, TESPA1, YPEL5, LPAR6, CMSS1, RIPOR2, ZNF331, EMP3, GIMAP7, WDR74, RIC3, CYSLTR1, ITGB1, CD5, SAMHD1, SERINC5, and any combination thereof (see e.g., Caushi et al., Nature 596: 126-132 (2021)).

[0151] 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.

[0152] The term "clonotype," as used herein, refers to a population of T cells with unique DNA sequences that result from TCRa or TCR13 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.

[0153] 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+] + [NaCl]) X 2 = 280).

[0154] 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).

[0155] 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 byincreasing 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 NaCl concentration, multiplied by two.

[0156] As used herein, the terms "potassium," "potassium ion," "potassium cation," and "K+" 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.

[0157] 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.

[0158] 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., CaCh) 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.

[0159] 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 mediumcomprises 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 expansion medium (e.g., MRM) comprises potassium ion at a concentration of about 50 mM. In some aspects, the expansion medium (e.g., MRM) comprises potassium ion at a concentration of about 55 mM. In some aspects, the expansion medium (e.g., MRM) comprises potassium ion at a concentration of about 60 mM. In some aspects, the expansion medium (e.g., MRM) comprises potassium ion at a concentration of about 65 mM. In some aspects, the expansion medium (e.g., MRM) comprises potassium ion at a concentration of about 70 mM. In certain aspects, the expansion medium (e.g., MRM) comprises about 40 mM to about 80 mM NaCl, 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 expansion medium (e.g., MRM) comprises potassium ion at a concentration of about 70 mM. In certain aspects, the expansion medium (e.g., MRM) comprises about 40 mM to about 80 mM NaCl, about 40 mM to about 90 mM KC1, about 0.5 mM to about 2.8 mM calcium, and about 10 mM to about 25 mM glucose. In some aspects, the expansion medium (e.g., MRM) further comprises an osmolality of about 250 to about 340 mOsmol.

[0160] 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 (IMDM), M199, OPTMIZER™ CTS™ T-Cell Expansion Basal Medium (ThermoFisher), OPTMIZER™ Complete, IMMUNOCULT™ XF (STEMCELL™ Technologies), IMMUNOCULT™ XF, AIM V, TEXMACS™ medium, TRANSACT™ TIL expansion medium, TIL rapid expansion protocolmedium, 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 X-VIVO™ 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 OPTMIZER™ Complete supplemented with ICSR, AIM V supplemented with ICSR, IMMUNOCULT™ XF supplemented with ICSR, RPMI supplemented with ICSR, TEXMACS™ 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 NaCl free CTS™ OPTIMIZER™. 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 RPML1640 medium. In some aspects, the basal media comprises NaCl free CTS™ OpTimizer™. In some aspects, the basal media comprises one or more sodium salt in addition to the NaCl that is added to control the tonicity, e.g., NaCl added in combination with potassium ion.

[0161] 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)-l, 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-a, IFN-P, 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 - Q9HBE4) 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 IgGl and IgG3 in B-cells. IL-21 may also play a role in proliferation and maturation of natural killer (NK) cells in synergy with IL-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.

[0162] 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.

[0163] The term "preferentially," as used herein, refers to the predominant outcome. For example, if the methods disclosed herein preferentially promote expansion of CD8+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 TILs are CD8+, rather the term suggests that CD8+TILs are expanded to a greater extent than CD8' TILs.

[0164] 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.

[0165] 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.

[0166] 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.

[0167] 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).

[0168] 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.

[0169] 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).

[0170] 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.

[0171] 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+TIL, 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 CD8+TIL. In some aspects, a TIL is a CD4+TIL.

[0172] As used herein, the term "anti -turn or immune response" refers to an immune response against a tumor antigen.

[0173] 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., TILs, cultured as described herein to control tumor growth.

[0174] 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.

[0175] 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 (z.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.

[0176] 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 in vitro assays.

[0177] 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).

[0178] 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.

[0179] 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).

[0180] 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 P 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 MHCs. The TCR complex is associated with 6 polypeptides forming 2 heterodimers, CD3ys and CD35s, and 1 homodimer CD3which 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.

[0181] As used herein, the terms "ug" and "uM" are used interchangeably with "pg" and "pM," respectively.

[0182] 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.

[0183] As used herein, the terms "large scale," "MRM large scale process," "large scale TIL" or "manufacturing scale" refer to TILs cultured and / expanded in MRM with higher target TIL yields and final product volumes compared to a research scale MRM process. In some aspects, different ratios of TIL to PBMCs can be used compared to the research scale MRM process. For instance, in some aspects, the ratio of TIL to PBMCs for a large scale MRM 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.

[0184] As used here, the term "DO" refers to the starting point of a given process. For example, in some aspects of the methods disclosed herein, "DO" refers to the beginning of the entire culture process, such as the day on which a single cell suspension disclosed herein is first contacted with IL-2. If no particular process is defined, then "DO" refers to the initiation of the entire culture process. As such, if the dissociated tumor sample, z.e., the single cell suspension is contacted with IL-2 at 9:00 am on a Monday, DO runs from 9:00 am to 11 :59 pm on that Monday. "DI " then refers to the next full day, e.g., from 12:00 am until 11 :59 pm on Tuesday; "D2" then refers to the next full day, e.g., from 12:00 am until 11 :59 pm on Wednesday; and so forth. The term "D#" can be used interchangeably with the term "day #", wherein the "#" is any whole number. Unless otherwise specified, any action taken on a particular day can occur at any time between 12:00 am and 11 :59 pm on that day.

[0185] The term “selection,” as used herein, refers to a process of preferentially enriching for particular cell types or cells with specific phenotypic features, either by preferential separation or by expansion, or by removal of non-preferred cells. In some aspects, selection can be carried out by the addition of cell culture components, such as nutrients or metabolites, preferentially usedby the selected cells. In some aspects, the selected cells can be sorted based on physically expressed markers. In some aspects, sorting can be carried out using fluorescence activated cell sorting (FACS), magnetic beads, buoyancy activated cell sorting (BACS), lateral flow cassettes or any similar method.

[0186] As used herein, the term "cell support matrix" refers to a structure that supports the growth, expansion, survival, and / or activity of a cell during culture. Non-limiting examples of a "cell support matrix" include antigen presenting cells (APCs) and TRANSACT™. The term "APCs," as used herein, refers to natural cells (e.g., irradiated PBMC feeder cells) and to synthetic equivalents (z.e., artificial antigen presenting cells (aAPCs), engineered APCs (eAPCs), and APC mimics), such as vesicles and / or microparticles (e.g., feeder cells and irradiated PBMCs), artificial APCs (aAPCs), synthetic APCs, an APC mimetic, and combinations thereof. In some aspects, the cell support matrix comprises dynabeads. In some aspects, the cell support matrix comprises biotinylated antigen presenting molecules coated onto a streptavidin plate.

[0187] Various aspects described herein are described in further detail in the following subsections.II. Methods of the Disclosure

[0188] The present disclosure is directed to methods of culturing immune cells, e.g., TILs, ex vivo or in vitro. The cell culturing methods of the present disclosure are capable of enhancing the expansion of CD8+TILs and / or increasing multipotency and / or pluripotency of the cultured TILs in a shorter period of time than conventional methods. 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.

[0189] Some aspects of the present disclosure are directed to methods of culturing TILs ex vivo or in vitro comprising (i) dissociating a tumor sample obtained from the subject comprising TILs to generate a single cell suspension; (ii) culturing the single cell suspension in a first expansion medium, wherein the first expansion medium comprises a CD3 agonist and APCs at a first ratio (a "first REP step"); and (iii) culturing the TILs from the first REP step in a second expansion medium, wherein the second expansion medium comprises a CD3 agonist and APCs at a second ratio (a "second REP step").

[0190] Some aspects of the present disclosure are directed to methods of culturing TILs ex vivo or in vitro for a therapy in a subject in need thereof comprising: (i) culturing a dissociated tumor sample comprising TILs in a first expansion medium, wherein the first expansion medium comprises a CD3 agonist and APCs at a first ratio (a "first REP step"); and (ii) culturing the TILs from the first REP step in a second expansion medium, wherein the second expansion medium comprises a CD3 agonist, and APCs at a second ratio (a "second REP step"). In some aspects, the dissociated tumor sample is a single cell suspension.

[0191] Some aspects of the present disclosure are directed to methods of culturing TILs ex vivo or in vitro for a therapy in a subject in need thereof comprising culturing a population of TILs obtained from a tumor sample of the subject in an expansion medium comprising a CD3 agonist and antigen-presenting cells (APCs); wherein, prior to or after culturing in the expansion medium, the TILs are not cultured in a medium that does not comprise APCs. In some aspects, prior to culturing in the expansion medium, the population of TILs is cultured in a medium comprising APCs. In some aspects, after culturing in the expansion medium, the population of TILs is cultured in a medium comprising APCs.

[0192] Some of the aspects of the present disclosure are directed to methods of increasing the number of tumor-reactive tumor infiltrating lymphocytes (TILs) ex vivo or in vitro in a composition for a TIL therapy comprising selecting for TILs that express an HLA polypeptide ("population of selected TILs") from a heterogeneous population of TILs obtained from a tumor. Some aspects of the present disclosure are directed to methods of generating an expanded population of tumor-reactive TILs comprising selecting for TILs that express an HLA polypeptide ("population of selected TILs") from a heterogeneous population of TILs obtained from a tumor.

[0193] In some aspects, the method further comprises obtaining the heterogeneous population of TILs from a tumor. In some aspects, the tumor is obtained from a subject. In some aspects, the tumor is dissociated to generate the heterogeneous population of TILs. In some aspects, the dissociated tumor sample is a single cell suspension. In some aspects, the tumor is cultured in a medium comprising IL-2 to generate the heterogeneous population of TILs. In some aspects, prior to the selecting, the heterogeneous population of TILs is cultured in a medium comprising IL-2 ("initial expansion"). In some aspects, the method further comprises culturing the selected population of TILs in a medium comprising IL-2.

[0194] In some aspects, the method further comprises culturing the population of selected TILs in a first expansion medium comprising a cell-support matrix, a cytokine, and a T cell activator (a "first REP step"). In some aspects, the method further comprises culturing thepopulation of selected TILs after the first REP step in a second expansion medium comprising a cell-support matrix, cytokine, and a T cell activator (a "second REP step").

[0195] Some aspects of the present disclosure are directed to methods of culturing tumor infiltrating lymphocytes (TILs) ex vivo or in vitro for a therapy for a subject in need thereof comprising: (i) dissociating a tumor sample obtained from the subject comprising TILs to generate a single cell suspension comprising a heterogeneous population of TILs; (ii) selecting for TILs that express an HLA polypeptide ("population of selected TILs") from the heterogeneous population of TILs; (iii) culturing the population of selected TILs in a first expansion medium, wherein the first expansion medium comprises a T cell activator and a cell support matrix at a first ratio (a "first REP step"); and (iv) culturing the TILs from the first REP step in a second expansion medium, wherein the second expansion medium comprises a T cell activator and a cell support matrix at a second ratio (a "second REP step").

[0196] Some aspects of the present disclosure are directed to methods of culturing tumor infiltrating lymphocytes (TILs) ex vivo or in vitro for a therapy for a subject in need thereof comprising: (i) dissociating a tumor sample obtained from the subject comprising TILs to generate a single cell suspension comprising a heterogeneous population of TILs; (ii) culturing the heterogeneous population of TILs in a first expansion medium, wherein the first expansion medium comprises a T cell activator and a cell support matrix at a first ratio (a "first REP step"); (iii) selecting for TILs that express an HLA polypeptide ("population of selected TILs") from the heterogeneous population of TILs; and (iv) culturing the population of selected TILs from the first REP step in a second expansion medium, wherein the second expansion medium comprises a T cell activator and a cell support matrix at a second ratio (a "second REP step"). In some aspects, the method further comprises culturing the heterogeneous population of TILs in a medium comprising IL-2 before the first REP step.

[0197] Some aspects of the present disclosure are directed to methods of culturing tumor infiltrating lymphocytes (TILs) ex vivo or in vitro for a therapy for a subject in need thereof comprising: (i) dissociating a tumor sample obtained from the subject comprising TILs to generate a single cell suspension comprising a heterogeneous population of TILs; (ii) culturing the heterogeneous population of TILs in an initial expansion medium, wherein the initial expansion medium comprises IL-2; (iii) selecting for TILs that express an HLA polypeptide ("population of selected TILs") from the heterogeneous population of TILs; (iv) culturing the population of selected TILs in a first expansion medium, wherein the first expansion medium comprises a T cell activator and a cell support matrix at a first ratio (a "first REP step"); and (v) culturing the TILsfrom the first REP step in a second expansion medium, wherein the second expansion medium comprises a T cell activator and a cell support matrix at a second ratio (a "second REP step").

[0198] Some aspects of the present disclosure are directed to methods of culturing tumor infiltrating lymphocytes (TILs) ex vivo or in vitro for a therapy for a subject in need thereof comprising: (i) dissociating a tumor sample obtained from the subject comprising TILs to generate a single cell suspension comprising a heterogeneous population of TILs; (ii) culturing the heterogeneous population of TILs in an initial expansion medium, wherein the initial expansion medium comprises IL-2; (iii) selecting for TILs that express an HLA polypeptide ("population of selected TILs") from the heterogeneous population of TILs; (iv) culturing the population of selected TILs in a first expansion medium, wherein the first expansion medium comprises a T cell activator and a cell support matrix at a first ratio (a "first REP step"); and (v) culturing the TILs from the first REP step in a second expansion medium, wherein the second expansion medium comprises a T cell activator and a cell support matrix at a second ratio (a "second REP step").

[0199] Some of the aspects of the present disclosure are directed to methods of increasing the number of tumor-reactive tumor infiltrating lymphocytes (TILs) ex vivo or in vitro in a composition for a TIL therapy comprising selecting for TILs that express an HLA polypeptide ("population of selected TILs") from a heterogeneous population of TILs obtained from a tumor. Some aspects of the present disclosure are directed to methods of generating an expanded population of tumor-reactive TILs comprising selecting for TILs that express an HLA polypeptide ("population of selected TILs") from a heterogeneous population of TILs obtained from a tumor.

[0200] In some aspects, the method further comprises obtaining the heterogeneous population of TILs from a tumor. In some aspects, the tumor is obtained from a subject. In some aspects, the tumor is dissociated to generate the heterogeneous population of TILs. In some aspects, the dissociated tumor sample is a single cell suspension. In some aspects, the tumor is cultured in a medium comprising IL-2 to generate the heterogeneous population of TILs. In some aspects, prior to the selecting, the heterogeneous population of TILs is cultured in a medium comprising IL-2 ("initial expansion"). In some aspects, the method further comprises culturing the selected population of TILs in a medium comprising IL-2.

[0201] In some aspects, the method further comprises culturing the population of selected TILs in a first expansion medium comprising a cell-support matrix, a cytokine, and a T cell activator (a "first REP step"). In some aspects, the method further comprises culturing the population of selected TILs after the first REP step in a second expansion medium comprising a cell-support matrix, cytokine, and a T cell activator (a "second REP step").

[0202] Some aspects of the present disclosure are directed to methods of culturing tumor infiltrating lymphocytes (TILs) ex vivo or in vitro for a therapy for a subject in need thereof comprising: (i) dissociating a tumor sample obtained from the subject comprising TILs to generate a single cell suspension comprising a heterogeneous population of TILs; (ii) selecting for TILs that express an HLA polypeptide ("population of selected TILs") from the heterogeneous population of TILs; (iii) culturing the population of selected TILs in a first expansion medium, wherein the first expansion medium comprises a T cell activator and a cell support matrix at a first ratio (a "first REP step"); and (iv) culturing the TILs from the first REP step in a second expansion medium, wherein the second expansion medium comprises a T cell activator and a cell support matrix at a second ratio (a "second REP step").

[0203] Some aspects of the present disclosure are directed to methods of culturing tumor infiltrating lymphocytes (TILs) ex vivo or in vitro for a therapy for a subject in need thereof comprising: (i) dissociating a tumor sample obtained from the subject comprising TILs to generate a single cell suspension comprising a heterogeneous population of TILs; (ii) culturing the heterogeneous population of TILs in a first expansion medium, wherein the first expansion medium comprises a T cell activator and a cell support matrix at a first ratio (a "first REP step"); (iii) selecting for TILs that express an HLA polypeptide ("population of selected TILs") from the heterogeneous population of TILs; and (iv) culturing the population of selected TILs from the first REP step in a second expansion medium, wherein the second expansion medium comprises a T cell activator and a cell support matrix at a second ratio (a "second REP step"). In some aspects, the method further comprises culturing the heterogeneous population of TILs in a medium comprising IL-2 before the first REP step.

[0204] Some aspects of the present disclosure are directed to methods of culturing tumor infiltrating lymphocytes (TILs) ex vivo or in vitro for a therapy for a subject in need thereof comprising: (i) dissociating a tumor sample obtained from the subject comprising TILs to generate a single cell suspension comprising a heterogeneous population of TILs; (ii) culturing the heterogeneous population of TILs in an initial expansion medium, wherein the initial expansion medium comprises IL-2; (iii) selecting for TILs that express an HLA polypeptide ("population of selected TILs") from the heterogeneous population of TILs; (iv) culturing the population of selected TILs in a first expansion medium, wherein the first expansion medium comprises a T cell activator and a cell support matrix at a first ratio (a "first REP step"); and (v) culturing the TILs from the first REP step in a second expansion medium, wherein the second expansion medium comprises a T cell activator and a cell support matrix at a second ratio (a "second REP step").

[0205] Some aspects of the present disclosure are directed to methods of culturing tumor infiltrating lymphocytes (TILs) ex vivo or in vitro for a therapy for a subject in need thereof comprising: (i) dissociating a tumor sample obtained from the subject comprising TILs to generate a single cell suspension comprising a heterogeneous population of TILs; (ii) culturing the heterogeneous population of TILs in an initial expansion medium, wherein the initial expansion medium comprises IL-2; (iii) selecting for TILs that express an HLA polypeptide ("population of selected TILs") from the heterogeneous population of TILs; (iv) culturing the population of selected TILs in a first expansion medium, wherein the first expansion medium comprises a T cell activator and a cell support matrix at a first ratio (a "first REP step"); and (v) culturing the TILs from the first REP step in a second expansion medium, wherein the second expansion medium comprises a T cell activator and a cell support matrix at a second ratio (a "second REP step").

[0206] Some of the aspects of the present disclosure are directed to methods of increasing the number of tumor-reactive tumor infiltrating lymphocytes (TILs) ex vivo or in vitro in a composition for a TIL therapy comprising removing or de-selecting TILs that express CD 127 ("population of de-selected TILs") from a heterogeneous population of TILs obtained from a tumor. Some aspects of the present disclosure are directed to methods of generating an expanded population of tumor-reactive TILs comprising removing or de-selecting TILs that express CD127 ("population of de-selected TILs") from a heterogeneous population of TILs obtained from a tumor.

[0207] In some aspects, the method further comprises obtaining the heterogeneous population of TILs from a tumor. In some aspects, the tumor is obtained from a subject. In some aspects, the tumor is dissociated to generate the heterogeneous population of TILs. In some aspects, the dissociated tumor sample is a single cell suspension. In some aspects, the tumor is cultured in a medium comprising IL-2 to generate the heterogeneous population of TILs. In some aspects, prior to the removing or de-selecting, the heterogeneous population of TILs is cultured in a medium comprising IL-2 ("initial expansion"). In some aspects, the method further comprises culturing the population of CD127- TILs (“population of selected TIL”) without the population of de-selected CD 127+ TILs in a medium comprising IL-2.

[0208] In some aspects, the method further comprises culturing the population of selected TILs in a first expansion medium comprising a cell-support matrix, a cytokine, and a T cell activator (a "first REP step"). In some aspects, the method further comprises culturing the population of selected TILs after the first REP step in a second expansion medium comprising a cell-support matrix, cytokine, and a T cell activator (a "second REP step").

[0209] Some aspects of the present disclosure are directed to methods of culturing tumor infiltrating lymphocytes (TILs) ex vivo or in vitro for a therapy for a subject in need thereof comprising: (i) dissociating a tumor sample obtained from the subject comprising TILs to generate a single cell suspension comprising a heterogeneous population of TILs; (ii) removing or deselecting for TILs that express CD127 ("population of de-selected TILs") from the heterogeneous population of TILs; (iii) culturing the population of TILs without the population of de-selected TILs in a first expansion medium, wherein the first expansion medium comprises a T cell activator and a cell support matrix at a first ratio of cell support matrix to TIL (a "first REP step"); and (iv) culturing the TILs from the first REP step in a second expansion medium, wherein the second expansion medium comprises a T cell activator and a cell support matrix at a second ratio of cell support matrix to TIL (a "second REP step").

[0210] Some aspects of the present disclosure are directed to methods of culturing tumor infiltrating lymphocytes (TILs) ex vivo or in vitro for a therapy for a subject in need thereof comprising: (i) dissociating a tumor sample obtained from the subject comprising TILs to generate a single cell suspension comprising a heterogeneous population of TILs; (ii) culturing the heterogeneous population of TILs in an initial expansion medium, wherein the initial expansion medium comprises IL-2; (iii) removing or de-selecting for TILs that express CD127 ("population of de-selected TILs") from the heterogeneous population of TILs; (iv) culturing the population of TILs without the population of de-selected TILs (“population of selected TILs”) in a first expansion medium, wherein the first expansion medium comprises a T cell activator and a cell support matrix at a first ratio of cell support matrix to TIL (a "first REP step"); and (v) culturing the TILs from the first REP step in a second expansion medium, wherein the second expansion medium comprises a T cell activator and a cell support matrix at a second ratio of cell support matrix to TIL (a "second REP step").

[0211] Some aspects of the present disclosure are directed to methods of culturing tumor infiltrating lymphocytes (TILs) ex vivo or in vitro for a therapy for a subject in need thereof comprising: (i) dissociating a tumor sample obtained from the subject comprising TILs to generate a single cell suspension comprising a heterogeneous population of TILs; (ii) culturing the heterogeneous population of TILs in an initial expansion medium, wherein the initial expansion medium comprises IL-2; (iii) removing or de-selecting for TILs that express CD127 ("population of de-selected TILs") from the heterogeneous population of TILs; (iv) culturing the population of selected TILs without the population of de-selected TILs (“population of selected TILs”) in a first expansion medium, wherein the first expansion medium comprises a T cell activator and a cellsupport matrix at a first ratio of cell support matrix to TIL (a "first REP step"); and (v) culturing the TILs from the first REP step in a second expansion medium, wherein the second expansion medium comprises a T cell activator and a cell support matrix at a second ratio of cell support matrix to TIL (a "second REP step").

[0212] 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.

[0213] 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 CD 19 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.

[0214] The present disclosure provides novel methods of culturing immune cells, e.g., TILs, that generate an expanded population of TILs suitable for an immunotherapy in less time than conventional methods. In some aspects, an expanded population of TILs suitable for an immunotherapy is obtained within less than about 22 days. In some aspects, an expanded population of TILs suitable for an immunotherapy is obtained within less than about 21 days. In some aspects, an expanded population of TILs suitable for an immunotherapy is obtained within less than about 20 days. In some aspects, an expanded population of TILs suitable for an immunotherapy is obtained within less than about 19 days. In some aspects, an expanded population of TILs suitable for an immunotherapy is obtained within less than about 18 days. In some aspects, an expanded population of TILs suitable for an immunotherapy is obtained within less than about 17 days. In some aspects, an expanded population of TILs suitable for an immunotherapy is obtained within less than about 16 days.

[0215] In some aspects, the expanded population of TILs suitable for an immunotherapy comprises 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 IO10, at least about 2 x IO10, at least about 3 x IO10, at least about 4 x IO10, at least about 5 x IO10, at least about 6 x IO10, at least about 7 x IO10, at least about 8 x IO10, or at least about 9 x IO10TILs following the methods disclosed herein, e.g., in less than 22 days. In some aspects, the expanded population of TILs suitable for an immunotherapy comprises at least about 1 x 109TILs following the methods disclosed herein, e.g., in less than 22 days.

[0216] TIL therapy has also shown a potential in mediating clinical responses in patients with advanced cancer. Emerging evidence has demonstrated that TILs are a heterogeneous population composed of both tumor-reactive and non-specific bystander cells. This heterogeneous 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 heterogeneous 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.

[0217] In some aspects, the methods and / or compositions disclosed herein increase the clonal diversity of TILs in culture, as compared to TILs cultured using conventional methods. 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. Immunolog. Mtd. 327:182-95 (2007), which is incorporated by reference herein in its entirety. In some aspects, clonal diversity is assessed using IMMUNOSEQ® (ADAPTIVE BIOTECHNOLOGIES®). In some aspects, clonal diversity is assessed using TCR deep sequencing. In certain aspects, the clonal diversity is assessed by sequencing TCRB CDR3 sequences 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 clonality.

[0218] 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 about100%, 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 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 75% 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 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 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 50% 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 40% to about 100% of the clonal diversity of TILs in a tumor sample.

[0219] In some aspects, clonal diversity is assessed using Simpsons clonality ( ^pi2where, 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. Immunol. Meth. 327: 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.

[0220] 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 thanabout 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 TILs 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 TILs 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 themethods 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.ILA. TIL Isolation, Optional Selection, Expansion, and Harvest

[0221] In some aspects, the present disclosure includes a method of culturing tumor infiltrating lymphocytes (TILs) ex vivo or in vitro for a therapy to a subject in need thereof comprising:(i) dissociating a tumor sample obtained from the subject comprising TILs to generate a single cell suspension;(ii) culturing the single cell suspension in a first expansion medium, wherein the first expansion medium comprises a CD3 agonist and APCs at a first ratio (a "first REP step"); and(iii) culturing the TILs from the first REP step in a second expansion medium, wherein the second expansion medium comprises a CD3 agonist and APCs at a second ratio (a "second REP step").

[0222] In some aspects, the present disclosure includes a method of culturing TILs ex vivo or in vitro for a therapy in a subject in need thereof comprising:(i) culturing a dissociated tumor sample comprising TILs in a first expansion medium, wherein the first expansion medium comprises a CD3 agonist and APCs at a first ratio (a "first REP step"); and(ii) culturing the TILs from the first REP step in a second expansion medium, wherein the second expansion medium comprises a CD3 agonist, and APCs at a second ratio (a "second REP step").

[0223] In some aspects, the dissociated tumor sample is a single cell suspension. Any means of generating a single cell suspension can be used in the methods disclosed herein. Solely as an example, in some aspects, a single cell suspension is prepared using the a MACS system. In some aspects,

[0224] 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.

[0225] In some aspects, a method disclosed herein further comprises selecting for tumor- reactive TILs. In some aspects, the selection of tumor-reactive TILs comprises contacting a heterogeneous population of TILs with an antibody or antigen-binding portion thereof that binds a specific target expressed by tumor-reactive TILs. In some aspects, the antibody or antigen-binding portion thereof that binds an HLA polypeptide.

[0226] Selection of tumor-reactive TILs according to the methods disclosed herein, e.g., by contacting a heterogeneous population of TILs with an anti -HLA antibody, can occur during or between any step of isolation and / or expansion. In some aspects, selection of TILs that express an HLA polypeptide comprises contacting the heterogeneous population of TILs with one or more antibody or antigen-binding portion thereof that specifically binds the HLA polypeptide ("anti- HLA antibody"). In some aspects, the selecting comprises contacting the heterogeneous population of TILs with an anti-HLA antibody and sorting the TILs by FACS. In some aspects, the selecting comprises contacting the heterogeneous population of TILs with one or more antibody or antigenbinding portion thereof that specifically binds HLA-DR ("anti-HLA-DR antibody"). In some aspects, the selecting comprises contacting the heterogeneous population of TILs with an anti- HLA-DR antibody and sorting the TILs by FACS. In some aspects, the selecting comprises contacting the heterogeneous population of TILs with one or more antibody or antigen-bindingportion thereof that specifically binds HLA-DP ("anti-HLA-DP antibody"). In some aspects, the selecting comprises contacting the heterogeneous population of TILs with an anti-HLA-DP antibody and sorting the TILs by FACS. In some aspects, the selecting comprises contacting the heterogeneous population of TILs with one or more antibody or antigen-binding portion thereof that specifically binds HLA-DQ ("anti-HLA-DQ antibody"). In some aspects, the selecting comprises contacting the heterogeneous population of TILs with an anti-HLA-DQ antibody and sorting the TILs by FACS. In some aspects, the selecting comprises contacting the heterogeneous population of TILs with one or more antibody or antigen-binding portion thereof that specifically binds HLA-DM ("anti-HLA-DM antibody"). In some aspects, the selecting comprises contacting the heterogeneous population of TILs with an anti-HLA-DM antibody and sorting the TILs by FACS.

[0227] In some aspects, the selection of tumor-reactive TILs comprises contacting heterogeneous population of TILs with an antibody or antigen-binding portion thereof that binds a target that is not expressed by tumor-reactive TILs. In some aspects, the antibody or antigenbinding portion thereof that binds CD127. Selection of tumor-reactive TILs according to the methods disclosed herein, e.g., by contacting a heterogeneous population of TILs with an anti- CD127 antibody, can occur during or between any step of isolation and / or expansion. In some aspects, removal or de-selection of TILs that express CD127 comprises contacting the heterogeneous population of TILs with one or more antibody or antigen-binding portion thereof that specifically binds CD127 ("anti-CD127 antibody"). In some aspects, the removing or deselecting comprises contacting the heterogeneous population of TILs with an anti-CD127 antibody and sorting the TILs by FACS.

[0228] 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.ILA.l. Tumor Sample

[0229] 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 a 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 MRM 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. In some aspects, the tumor sample is cryopreserved after selecting for TILs that express an HLA polypeptide or that do not express CD127. In some aspects the tumor sample is fresh, e.g., not cryopreserved. In some aspects, the tumor sample is placed directly into MRM media.

[0230] In some aspects, the donor patient (e.g., the subject from which the tumor is obtained) is treatment naive (ie., 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. In some aspects, the one or more prior therapy comprises a BRAF inhibitor. In some aspects, the one or more prior therapy comprises an MEK inhibitor.

[0231] 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.

[0232] 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. WO2019217753, 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, aminopyrimidine-based ITK inhibitors, 3-aminopyride-2-ones-based ITK inhibitors, indolylndazole-based ITK inhibitors, pyrazolyl-indole-based inhibitors, thienopyrazole inhibitors, and ITK inhibitors targeting cysteine- 442 in the ATP pocket. In 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.

[0233] 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 (z.e., disaggregating) 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 someaspects, the mechanical and / or enzymatic dissociation is performed in an MRM medium disclosed herein.

[0234] 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 6 N / 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.

[0235] In some aspects, the tumor sample is dissociated to prepare a single cell suspension. Any methods of dissociation can be used in the methods disclosed herein. In some aspects, the tumor sample is cut into small fragments. In some aspects, the small fragments are then collected in one or more gentleMACS tube and subjected to dissociation according to the gentleMACS system.

[0236] In some aspects, the single cell suspension is taken directly into a first expansion step, e.g., a first REP step disclosed herein. In some aspects, the singe cell suspension is cryopreserved prior to advancing to a first expansion step, e.g., a first REP step.

[0237] In some aspects, the single cell suspension is taken directly into an initial expansion step, e.g., wherein the heterogeneous population of TILs is cultured in an initial expansion medium comprising IL-2.

[0238] In some aspects, the single cell suspension is contacted with an anti-HLA antibody to select for TILs that express an HLA polypeptide. In some aspects, the population of selected TILs is then taken into an initial expansion step, e.g., wherein the heterogeneous population of TILs is cultured in an initial expansion medium comprising IL-2.

[0239] In some aspects, the single cell suspension is contacted with an anti-CD127 antibody to select for and remove TILs that express CD127. In some aspects, the population of selected TILs without the population of de-selected TILs is then taken into an initial expansion step, e.g., wherein the heterogeneous population of TILs is cultured in an initial expansion medium comprising IL-2.

[0240] In some aspects, the method comprises culturing a population of TILs obtained from a tumor sample of a subject in an expansion medium comprising a CD3 agonist and antigen- presenting cells (APCs); wherein, prior to or after culturing in the expansion medium, the TILs are not cultured in a medium that does not comprising APCs. In some aspects, prior to culturing in the expansion medium, the population of TILs is cultured in a medium comprising APCs. In some aspects, after culturing in the expansion medium, the population of TILs is cultured in a medium comprising APCs.

[0241] In some aspects, the TILs are cultured in the expansion medium for less than 14 days. In some aspects, the TILs are cultured in the expansion medium for less than 13 days. In some aspects, the TILs are cultured in the expansion medium for less than 12 days. In some aspects, the TILs are cultured in the expansion medium for less than 11 days. In some aspects, the TILs are cultured in the expansion medium for less than 10 days. In some aspects, the TILs are cultured in the expansion medium for less than 9 days. In some aspects, the TILs are cultured in the expansion medium for less than 8 days.

[0242] In some aspects, prior to culturing in the expansion medium, the population of TILs is cultured for less than 14 days in the medium comprising APCs. In some aspects, prior to culturing in the expansion medium, the population of TILs is cultured for less than 13 days in the medium comprising APCs. In some aspects, prior to culturing in the expansion medium, the population of TILs is cultured for less than 12 days in the medium comprising APCs. In some aspects, prior to culturing in the expansion medium, the population of TILs is cultured for less than 11 days in the medium comprising APCs. In some aspects, prior to culture in the expansionmedium, the population of TILs is cultured for less than 10 days in the medium comprising APCs. In some aspects, prior to culture in the expansion medium, the population of TILs is cultured for less than 9 days in the medium comprising APCs. In some aspects, prior to culture in the expansion medium, the population of TILs is cultured for less than 8 days in the medium comprising APCs.II.A.2. Optional Initial Expansion

[0243] In some aspects, the tumor sample (e.g., the dissociated tumor sample, e.g., the single cell suspension) is placed into an initial expansion medium comprising IL-2. In some aspects, the initial expansion medium comprises from about 1000 lU / ml to at least about 5000 lU / ml IL-2. In some aspects, the initial expansion medium comprises at least about 1000 lU / ml, at least about 1100 lU / ml, at least about 1200 lU / ml, at least about 1300 lU / ml, at least about 1400 lU / ml, at least about 1500 lU / ml, at least about 1600 lU / ml, at least about 1700 lU / ml, at least about 1800 lU / ml, at least about 1900 lU / ml, at least about 2000 lU / ml, at least about 2100 lU / ml, at least about 2200 lU / ml, at least about 2300 lU / ml, at least about 2400 lU / ml, at least about 2500 lU / ml, at least about 2600 lU / ml, at least about 2700 lU / ml, at least about 2800 lU / ml, at least about 2900 lU / ml, at least about 3000 lU / ml, at least about 3100 lU / ml, at least about 3200 lU / ml, at least about 3300 lU / ml, at least about 3400 lU / ml, at least about 3500 lU / ml, at least about 3600 lU / ml, at least about 3700 lU / ml, at least about 3800 lU / ml, at least about 3900 lU / ml, at least about 4000 lU / ml, at least about 4100 lU / ml, at least about 4200 lU / ml, at least about 4300 lU / ml, at least about 4400 lU / ml, at least about 4500 lU / ml, at least about 4600 lU / ml, at least about 4700 lU / ml, at least about 4800 lU / ml, at least about 4900 lU / ml, at least about 5000 lU / ml, at least about 5100 lU / ml, at least about 5200 lU / ml, at least about 5300 lU / ml, at least about 5400 lU / ml, at least about 5500 lU / ml, at least about 5600 lU / ml, at least about 5700 lU / ml, at least about 5800 lU / ml, at least about 5900 lU / ml, at least about 6000 lU / ml, at least about 6100 lU / ml, at least about 6200 lU / ml, at least about 6300 lU / ml, at least about 6400 lU / ml, at least about 6500 lU / ml, at least about 6600 lU / ml, at least about 6700 lU / ml, at least about 6800 lU / ml, at least about 6900 lU / ml, at least about 7000 lU / ml, at least about 7500 lU / ml, at least about 8000 lU / ml, at least about 9000 lU / ml, at least about 10,000 lU / ml IL-2. In some aspects, the initial expansion medium comprises about 6000 lU / ml IL-2.

[0244] In some aspects, the initial expansion medium comprises IL-21. In some aspects, the initial expansion medium comprises from about 1 ng / ml to at least about 100 ng / ml IL-21. In some aspects, the initial expansion medium comprises 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 leastabout 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 20 ng / ml, at least about 25 ng / ml, at least about 30 ng / ml, at least about 35 ng / ml, at least about 40 ng / ml, at least about 45 ng / ml, 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, or at least about 100 ng / ml IL-21. In some aspects, the initial expansion medium comprises about 10 ng / ml IL-21.

[0245] In some aspects, the step of selecting for TILs that express an HLA polypeptide occurs before the selected TILs are contacted with the initial expansion medium. For example, in some aspects, the method comprises selecting for TILs that express an HLA polypeptide from a heterogeneous population of TILs obtained from a tumor, then culturing the population of selected TILs in the initial expansion medium.

[0246] In some aspects, the heterogeneous population of TILs is cultured in the initial expansion medium, and then TILs that express an HLA polypeptide are selected for from the expanded heterogeneous population of TILs from the initial expansion.

[0247] In some aspects, the step of removing or de-selecting TILs that express CD 127 occurs before the selected TILs are contacted with the initial expansion medium. For example, in some aspects, the method comprises removing or de-selecting TILs that express CD127 from a heterogeneous population of TILs obtained from a tumor, then culturing the population of selected TILs without the population of de-selected TILs in the initial expansion medium.

[0248] In some aspects, the heterogeneous population of TILs is cultured in the initial expansion medium, and then TILs that express CD 127 are selected for and removed from the expanded heterogeneous population of TILs from the initial expansion.

[0249] In some aspects, the initial expansion does not comprise a T cell activator (e.g., a CD3 agonist). In some aspects, the initial expansion does not comprise a cell-support matrix (e.g., a population of APCs).

[0250] In some aspects, the TILs are cultured in the initial expansion step for less than about 7 days, e.g., about 1 to 7 days. In some aspects, the TILs are cultured in the initial expansion step for less than about 6 days, e.g., about 1 to 6 days. In some aspects, the TILs are cultured in the initial expansion step for less than about 5 days, e.g., about 1 to 5 days. In some aspects, the TILs are cultured in the initial expansion step for less than about 4 days, e.g., about 1 to 4 days. In some aspects, the TILs are cultured in the initial expansion step for less than about 3 days, e.g., about 1 to 3 days. In some aspects, the TILs are cultured in the initial expansion step for less than about 2days, e.g., about 1 to 2 days. In some aspects, the TILs are cultured in the initial expansion step for about 4 days.II.A.3. First REP Step

[0251] In some aspects, the tumor sample e.g., the dissociated tumor sample, e.g., the single cell suspension) is placed into a first expansion medium, wherein the first expansion medium comprises a CD3 agonist and APCs at a first ratio (a "first REP step"). In some aspects, the TILs are cultured in the first REP step for less than about 14 days. In some aspects, the TILs are cultured in the first REP step for less than about 13 days. In some aspects, the TILs are cultured in the first REP step for less than about 12 days. In some aspects, the TILs are cultured in the first REP step for less than about 11 days. In some aspects, the TILs are cultured in the first REP step for less than about 10 days. In some aspects, the TILs are cultured in the first REP step for less than about 9 days. In some aspects, the TILs are cultured in the first REP step for less than about 8 days. In some aspects, the TILs are cultured in the first REP step for about 8 days.

[0252] In some aspects, the TILs are cultured in the first REP step for a period of time until a certain number of cells is reached. In some aspects, the number of TILs present in the dissociated tumor sample is increased by 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 following culture in the first REP step. In some aspects, the number of TILs present in the dissociated tumor sample is increased by at least about 10-fold following culture in the first REP step. In some aspects, the number of TILs present in the dissociated tumor sample is increased by at least about 15-fold following culture in the first REP step. In some aspects, the number of TILs present in the dissociated tumor sample is increased by at least about 20-fold following culture in the first REP step. In some aspects, the number of TILs present in the dissociated tumor sample is increased by at least about 25-fold following culture in the first REP step. In some aspects, the number of TILs present in the dissociated tumor sample is increased by at least about 30-fold following culture in the first REP step. In some aspects, the number of TILs preset in the dissociated tumor sample is increased by at least about 35-fold following culture in the first REP step. In some aspects, the number of TILs present in the dissociated tumor sample is increased by at least about 40-fold following culture in the first REP step. In some aspects, the number of TILs present in the dissociated tumor sample is increased by at least about 45-fold following culture in the first REP step. In some aspects, the number of TILspresent in the dissociated tumor sample is increased by at least about 50-fold following culture in the first REP step.

[0253] In some aspects, the number of TILs following culture in the first REP step is at least about 5 x 107, at least about 1 x 108, at least about 2 x 108, at least about 3 x 108, at least about 4 x 108, at least about 5 x 108, at least about 6 x 108, at least about 7 x 108, at least about 8 x 108, at least about 9 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 IO10, or at least about 2 x IO10TILs. In some aspects, the number of TILs following culture in the first REP step is at least about 1 x 109. In some aspects, the number of TILs following culture in the first REP step is at least about 2 x 109. In some aspects, the number of TILs following culture in the first REP step is at least about 3 x 109. In some aspects, the number of TILs following culture in the first REP step is at least about 4 x 109. In some aspects, the number of TILs following culture in the first REP step is at least about 5 x 109. In some aspects, the number of TILs following culture in the first REP step is at least about 6 x 109. In some aspects, the number of TILs following culture in the first REP step is at least about 7 x 109. In some aspects, the number of TILs following culture in the first REP step is at least about 8 x 109. In some aspects, the number of TILs following culture in the first REP step is at least about 9 x 109. In some aspects, the number of TILs following culture in the first REP step is at least about 10 x 109.

[0254] In some aspects, the first REP step continues until at least about 1 x 109TILs are obtained. In some aspects, the first REP step continues until at least about 2 x 109TILs are obtained. In some aspects, the first REP step continues until at least about 3 x 109TILs are obtained. In some aspects, the first REP step continues until at least about 4 x 109TILs are obtained. In some aspects, the first REP step continues until at least about 5 x 109TILs are obtained. In some aspects, the first REP step continues until at least about 6 x 109TILs are obtained. In some aspects, the first REP step continues until at least about 7 x 109TILs are obtained. In some aspects, the first REP step continues until at least about 8 x 109TILs are obtained. In some aspects, the first REP step continues until at least about 9 x 109TILs are obtained. In some aspects, the first REP step continues until at least about 10 x 109TILs are obtained.

[0255] In some aspects, the first REP step lasts for less than 8 days, wherein at least about 1 x 109TILs are obtained. In some aspects, the first REP step lasts for less than 8 days, wherein at least about 2 x 109TILs are obtained. In some aspects, the first REP step lasts for less than 8 days, wherein at least about 3 x 109TILs are obtained. In some aspects, the first REP step lasts for less than 8 days, wherein at least about 4 x 109TILs are obtained. In some aspects, the first REP steplasts for less than 8 days, wherein at least about 5 x 109TILs are obtained. In some aspects, the first REP step lasts for less than 8 days, wherein at least about 6 x 109TILs are obtained. In some aspects, the first REP step lasts for less than 8 days, wherein at least about 7 x 109TILs are obtained. In some aspects, the first REP step lasts for less than 8 days, wherein at least about 8 x 109TILs are obtained. In some aspects, the first REP step lasts for less than 8 days, wherein at least about 9 x 109TILs are obtained. In some aspects, the first REP step lasts for less than 8 days, wherein at least about 10 x 109TILs are obtained.

[0256] In some aspects, the first REP step lasts for less than 9 days, wherein at least about 1 x 109TILs are obtained. In some aspects, the first REP step lasts for less than 9 days, wherein at least about 2 x 109TILs are obtained. In some aspects, the first REP step lasts for less than 9 days, wherein at least about 3 x 109TILs are obtained. In some aspects, the first REP step lasts for less than 9 days, wherein at least about 4 x 109TILs are obtained. In some aspects, the first REP step lasts for less than 9 days, wherein at least about 5 x 109TILs are obtained. In some aspects, the first REP step lasts for less than 9 days, wherein at least about 6 x 109TILs are obtained. In some aspects, the first REP step lasts for less than 9 days, wherein at least about 7 x 109TILs are obtained. In some aspects, the first REP step lasts for less than 9 days, wherein at least about 8 x 109TILs are obtained. In some aspects, the first REP step lasts for less than 9 days, wherein at least about 9 x 109TILs are obtained. In some aspects, the first REP step lasts for less than 9 days, wherein at least about 10 x 109TILs are obtained.

[0257] In some aspects, the first REP step lasts for less than 10 days, wherein at least about 1 x 109TILs are obtained. In some aspects, the first REP step lasts for less than 10 days, wherein at least about 2 x 109TILs are obtained. In some aspects, the first REP step lasts for less than 10 days, wherein at least about 3 x 109TILs are obtained. In some aspects, the first REP step lasts for less than 10 days, wherein at least about 4 x 109TILs are obtained. In some aspects, the first REP step lasts for less than 10 days, wherein at least about 5 x 109TILs are obtained. In some aspects, the first REP step lasts for less than 10 days, wherein at least about 6 x 109TILs are obtained. In some aspects, the first REP step lasts for less than 10 days, wherein at least about 7 x 109TILs are obtained. In some aspects, the first REP step lasts for less than 10 days, wherein at least about 8 x 109TILs are obtained. In some aspects, the first REP step lasts for less than 10 days, wherein at least about 9 x 109TILs are obtained. In some aspects, the first REP step lasts for less than 10 days, wherein at least about 10 x 109TILs are obtained.

[0258] In some aspects, the first REP step lasts for less than 11 days, wherein at least about 1 x 109TILs are obtained. In some aspects, the first REP step lasts for less than 11 days, whereinat least about 2 x 109TILs are obtained. In some aspects, the first REP step lasts for less than 11 days, wherein at least about 3 x 109TILs are obtained. In some aspects, the first REP step lasts for less than 11 days, wherein at least about 4 x 109TILs are obtained. In some aspects, the first REP step lasts for less than 11 days, wherein at least about 5 x 109TILs are obtained. In some aspects, the first REP step lasts for less than 11 days, wherein at least about 6 x 109TILs are obtained. In some aspects, the first REP step lasts for less than 11 days, wherein at least about 7 x 109TILs are obtained. In some aspects, the first REP step lasts for less than 11 days, wherein at least about 8 x 109TILs are obtained. In some aspects, the first REP step lasts for less than 11 days, wherein at least about 9 x 109TILs are obtained. In some aspects, the first REP step lasts for less than 11 days, wherein at least about 10 x 109TILs are obtained.

[0259] In some aspects, the first expansion medium comprises a dissociated tumor cell to APC ratio of about 1 :5, about 1 :6, about 1 :7, about 1 :8, about 1 :9, about 1 : 10, about 1 : 11, about 1 : 12, about 1 :13, about 1 : 14, about 1 : 15, about 1 : 16, about 1 : 17, about 1 : 18, about 1 : 19, about 1 :20, about 1 :25, about 1 :30, about 1 :35, about 1 :40, about 1 :45, or about 1 :50. In some aspects, the first expansion medium comprises a dissociated tumor cell to APC ratio of about 1 :5. In some aspects, the first expansion medium comprises a dissociated tumor cell to APC ratio of about 1 :6. In some aspects, the first expansion medium comprises a dissociated tumor cell to APC ratio of about 1 :7. In some aspects, the first expansion medium comprises a dissociated tumor cell to APC ratio of about 1 :8. In some aspects, the first expansion medium comprises a dissociated tumor cell to APC ratio of about 1 :9. In some aspects, the first expansion medium comprises a dissociated tumor cell to APC ratio of about 1 : 10. In some aspects, the first expansion medium comprises a dissociated tumor cell to APC ratio of about 1 : 11. In some aspects, the first expansion medium comprises a dissociated tumor cell to APC ratio of about 1 : 12. In some aspects, the first expansion medium comprises a dissociated tumor cell to APC ratio of about 1 : 13. In some aspects, the first expansion medium comprises a dissociated tumor cell to APC ratio of about 1 : 14. In some aspects, the first expansion medium comprises a dissociated tumor cell to APC ratio of about 1 : 15. In some aspects, the first expansion medium comprises a dissociated tumor cell to APC ratio of about 1 : 16. In some aspects, the first expansion medium comprises a dissociated tumor cell to APC ratio of about 1 : 17. In some aspects, the first expansion medium comprises a dissociated tumor cell to APC ratio of about 1 : 18. In some aspects, the first expansion medium comprises a dissociated tumor cell to APC ratio of about 1 :20.

[0260] In some aspects, the first expansion medium comprises a CD3 agonist. In some aspects, the CD3 agonist comprises OKT3. In some aspects, the first expansion medium comprisesfrom about 1 to about 100 ng / mL OKT3. In some aspects, the concentration of OKT3 is at least about 1 ng / ml, at least about 5 ng / ml, at least about 10 ng / ml, at least about 15 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, at least about 40 ng / ml, at least about 45 ng / ml, 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, or at least about 100 ng / ml. In some aspects, the first expansion medium comprises about 10 ng / ml OKT3. In some aspects, the first expansion medium comprises about 15 ng / ml OKT3. In some aspects, the first expansion medium comprises about 20 ng / ml OKT3. In some aspects, the first expansion medium comprises about 25 ng / ml OKT3. In some aspects, the first expansion medium comprises about 30 ng / ml OKT3. In some aspects, the first expansion medium comprises about 35 ng / ml OKT3. In some aspects, the first expansion medium comprises about 40 ng / ml OKT3. In some aspects, the first expansion medium comprises about 45 ng / ml OKT3. In some aspects, the first expansion medium comprises about 50 ng / ml OKT3.

[0261] In some aspects, the first expansion medium comprises (i) a dissociated tumor cell to APC ratio of about 1 : 10 and (ii) about 10 ng / ml OKT3. In some aspects, the first expansion medium comprises (i) a dissociated tumor cell to APC ratio of about 1 : 10 and (ii) about 15 ng / ml OKT3. In some aspects, the first expansion medium comprises (i) a dissociated tumor cell to APC ratio of about 1 : 10 and (ii) about 20 ng / ml OKT3. In some aspects, the first expansion medium comprises (i) a dissociated tumor cell to APC ratio of about 1 : 10 and (ii) about 25 ng / ml OKT3. In some aspects, the first expansion medium comprises (i) a dissociated tumor cell to APC ratio of about 1 : 10 and (ii) about 30 ng / ml OKT3. In some aspects, the first expansion medium comprises(i) a dissociated tumor cell to APC ratio of about 1 : 10 and (ii) about 35 ng / ml OKT3. In some aspects, the first expansion medium comprises (i) a dissociated tumor cell to APC ratio of about 1 : 10 and (ii) about 40 ng / ml OKT3. In some aspects, the first expansion medium comprises (i) a dissociated tumor cell to APC ratio of about 1 : 10 and (ii) about 45 ng / ml OKT3. In some aspects, the first expansion medium comprises (i) a dissociated tumor cell to APC ratio of about 1 : 10 and(ii) about 50 ng / ml OKT3.

[0262] 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 pm, an at least about 15 pm, an at least about 20 pm, an at least about 25 pm, an at least about 30 pm, an at least about 35 pm, an at least about 40 pm, an at least about 45 pm, an at least about 50 pm strainer following the first REP step. In some aspects, the cells (e.g., TILs) are passed through an about 40 pm strainer following the first REP step.

[0263] In some aspects, the first REP step is carried out in one or more gas permeable flasks (e.g., GREX flasks). In some aspects, the first REP step is carried out in static GREX. In some aspects, the first REP step is carried out in a stirred tank. In some aspects, the first REP step is carried out in a bioreactor. In some aspects, the first REP step is carried out in a closed system (e.g., using a GREX closed system).

[0264] In some aspects, the first REP step further comprises contacting the tumor samples or fragments / cells thereof with 4-1BB ligand. 4-1BBL (4-1BB ligand, CD137L) is found on APCs (antigen presenting cells) and binds to 4- IBB (also known as CD 137), a type 2 transmembrane glycoprotein receptor belonging to the TNF superfamily, which is expressed on activated T Lymphocytes. 4- IBB ligand can be used to activate T cells in vitro. In some aspects, the first REP step comprises contacting the tumor samples or fragments thereof with 4- IBB ligand on about day 3, about day 4, about day 5, about day 6, or about day 7 of the first REP step; wherein first REP step is performed less than about 14 days. In some aspects, the first REP step comprises contacting the tumor samples or fragments thereof with 4-1BB ligand on about day 3, about day 4, about day 5, about day 6, or about day 7 of the first REP step; wherein first REP step is performed less than about 13 days. In some aspects, the first REP step comprises contacting the tumor samples or fragments thereof with 4-1BB ligand on about day 3, about day 4, about day 5, about day 6, or about day 7 of the first REP step; wherein first REP step is performed less than about 12 days. In some aspects, the first REP step comprises contacting the tumor samples or fragments thereof with 4-1BB ligand on about day 3, about day 4, about day 5, about day 6, or about day 7 of the first REP step; wherein first REP step is performed less than about 11 days. In some aspects, the first REP step comprises contacting the tumor samples or fragments thereof with 4- IBB ligand on about day 3, about day 4, about day 5, about day 6, or about day 7 of the first REP step; wherein first REP step is performed less than about 10 days. In some aspects, the first REP step comprises contacting the tumor samples or fragments thereof with 4-1BB ligand on about day 3, about day 4, about day 5, about day 6, or about day 7 of the first REP step; wherein first REP step is performed less than about 9 days. In some aspects, the first REP step comprises contacting the tumor samples or fragments thereof with 4-1BB ligand on about day 3, about day 4, about day 5, about day 6, or about day 7 of the first REP step; wherein first REP step is performed less than about 8 days.

[0265] In some aspects, the TILs are cryopreserved following the first REP step. In some aspects, the TILs from the first REP step are inoculated directly into a secondary REP step, e.g. , as described herein, following the first REP step.

[0266] In some aspects, the method comprises selecting for TILs that express an HL A polypeptide from the heterogeneous population of TILs obtained from the tumor sample (e.g., the dissociated tumor sample, e.g., the single cell suspension), and then culturing the population of selected TILs in a first expansion medium, wherein the first expansion medium comprises a T cell activator and a cell support matrix at a first ratio of cell support matrix to TIL (a "first REP step").

[0267] In some aspects, the method comprises (i) dissociating a tumor sample obtained from the subject comprising TILs to generate a single cell suspension comprising a heterogeneous population of TILs; (ii) selecting for TILs that express an HLA polypeptide ("population of selected TILs") from the heterogeneous population of TILs; and (iii) culturing the population of selected TILs in a first expansion medium, wherein the first expansion medium comprises a T cell activator and a cell support matrix at a first ratio of cell support matrix to TIL (a "first REP step").

[0268] In some aspects, the method comprises (i) dissociating a tumor sample obtained from the subject comprising TILs to generate a single cell suspension comprising a heterogeneous population of TILs; (ii) culturing the heterogeneous population of TILs in an initial expansion medium disclosed herein; (iii) selecting for TILs that express an HLA polypeptide ("population of selected TILs") from the heterogeneous population of TILs; and (iv) culturing the population of selected TILs in a first expansion medium, wherein the first expansion medium comprises a T cell activator and a cell support matrix at a first ratio of cell support matrix to TIL (a "first REP step").

[0269] In some aspects, the method comprises (i) dissociating a tumor sample obtained from the subject comprising TILs to generate a single cell suspension comprising a heterogeneous population of TILs; (ii) selecting for TILs that express an HLA polypeptide ("population of selected TILs") from the heterogeneous population of TILs; (iii) culturing the population of selected TILs in an initial expansion medium disclosed herein; and (iv) culturing the population of selected TILs in a first expansion medium, wherein the first expansion medium comprises a T cell activator and a cell support matrix at a first ratio of cell support matrix to TIL (a "first REP step").

[0270] In some aspects, the tumor sample (e.g., the dissociated tumor sample, e.g., the single cell suspension comprising the heterogeneous population of TILs) is placed into a first expansion medium, wherein the first expansion medium comprises a T cell activator and a cell support matrix at a first ratio of cell support matrix to TIL (a "first REP step"). In some aspects, the method comprises subjecting the heterogeneous population of TILs to a first REP step, and then removing or de-selecting TILs that express CD 127 from the expanded heterogeneous population of TILs arising from the first REP step.

[0271] In some aspects, the method comprises removing or de-selecting TILs that express CD 127 from the heterogeneous population of TILs obtained from the tumor sample (e.g., the dissociated tumor sample, e.g., the single cell suspension), and then culturing the population of TILs without the population of de-selected TILs in a first expansion medium, wherein the first expansion medium comprises a T cell activator and a cell support matrix at a first ratio of cell support matrix to TIL (a "first REP step").

[0272] In some aspects, the method comprises (i) dissociating a tumor sample obtained from the subject comprising TILs to generate a single cell suspension comprising a heterogeneous population of TILs; (ii) removing or de-selecting TILs that express CD127 ("population of deselected TILs") from the heterogeneous population of TILs; and (iii) culturing the population of TILs without the population of de-selected TILs in a first expansion medium, wherein the first expansion medium comprises a T cell activator and a cell support matrix at a first ratio of cell support matrix to TIL (a "first REP step").

[0273] In some aspects, the method comprises (i) dissociating a tumor sample obtained from the subject comprising TILs to generate a single cell suspension comprising a heterogeneous population of TILs; (ii) culturing the heterogeneous population of TILs in an initial expansion medium disclosed herein; (iii) removing or de-selecting TILs that express CD127 ("population of de-selected TILs") from the heterogeneous population of TILs; and (iv) culturing the population of TILs without the population of de-selected TILs in a first expansion medium, wherein the first expansion medium comprises a T cell activator and a cell support matrix at a first ratio of cell support matrix to TIL (a "first REP step").

[0274] In some aspects, the method comprises (i) dissociating a tumor sample obtained from the subject comprising TILs to generate a single cell suspension comprising a heterogeneous population of TILs; (ii) removing or de-selecting TILs that express CD127 ("population of deselected TILs") from the heterogeneous population of TILs; (iii) culturing the population of selected TILs in an initial expansion medium disclosed herein; and (iv) culturing the population of selected TILs in a first expansion medium, wherein the first expansion medium comprises a T cell activator and a cell support matrix at a first ratio of cell support matrix to TIL (a "first REP step").ILA.3. i. Cytokines

[0275] In some aspects, the first expansion medium comprises IL-2. In some aspects, the first expansion medium 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 leastabout 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 first expansion medium comprises at least about 50 ng / mL IL-2. In some aspects, the first expansion medium comprises at least about 60 ng / mL IL-2. In some aspects, the first expansion medium comprises at least about 70 ng / mL IL-2. In some aspects, the first expansion medium comprises at least about 73.6 ng / mL IL-2. In some aspects, the first expansion medium comprises at least about 75 ng / mL IL-2. In some aspects, the first expansion medium comprises at least about 80 ng / mL IL-2. In some aspects, the first expansion medium comprises at least about 90 ng / mL IL-2. In some aspects, the first expansion medium comprises at least about 100 ng / mL IL-2. In some aspects, the first expansion medium comprises at least about 200 ng / mL IL-2. In some aspects, the first expansion medium comprises at least about 300 ng / mL IL-2. In some aspects, the first expansion medium comprises at least about 400 ng / mL IL-2. In some aspects, the first expansion medium comprises at least about 500 ng / mL IL-2. In some aspects, the first expansion medium comprises at least about 600 ng / mL IL- 2.

[0276] In some aspects, the first expansion medium comprises at least about 1500 lU / mL IL-2. In some aspects, the first expansion medium comprises from about 1500 lU / mL to about 12,000 lU / mL IL-2. In some aspects, the first expansion medium comprises at least about 1500 lU / mL, at least about 1600 lU / mL, at least about 1700 lU / mL, at least about 1800 lU / mL, at least about 1900 lU / mL, at least about 2000 lU / mL, at least about 2100 lU / mL, at least about 2200 lU / mL, at least about 2300 lU / mL, at least about 2400 lU / mL, at least about 2500 lU / mL, at least about 2600 lU / mL, at least about 2700 lU / mL, at least about 2800 lU / mL, at least about 2900lU / mL, at least about 3000 lU / mL, at least about 3100 lU / mL, at least about 3200 lU / mL, at least about 3300 lU / mL, at least about 3400 lU / mL, at least about 3500 lU / mL, at least about 3600 lU / mL, at least about 3700 lU / mL, at least about 3800 lU / mL, at least about 3900 lU / mL, at least about 4000 lU / mL, at least about 4100 lU / mL, at least about 4200 lU / mL, at least about 4300 lU / mL, at least about 4400 lU / mL, at least about 4500 lU / mL, at least about 4600 lU / mL, at least about 4700 lU / mL, at least about 4800 lU / mL, at least about 4900 lU / mL, at least about 5000 lU / mL, at least about 5100 lU / mL, at least about 5200 lU / mL, at least about 5300 lU / mL, at least about 5400 lU / mL, at least about 5500 lU / mL, at least about 5600 lU / mL, at least about 5700 lU / mL, at least about 5800 lU / mL, at least about 5900 lU / mL, at least about 6000 lU / mL, at least about 6100 lU / mL, at least about 6200 lU / mL, at least about 6300 lU / mL, at least about 6400 lU / mL, at least about 6500 lU / mL, at least about 6600 lU / mL, at least about 6700 lU / mL, at least about 6800 lU / mL, at least about 6900 lU / mL, at least about 7000 lU / mL IL-2, at least about 7100 lU / mL, at least about 7200 lU / mL, at least about 7300 lU / mL, at least about 7400 lU / mL, at least about 7500 lU / mL, at least about 7600 lU / mL, at least about 7700 lU / mL, at least about 7800 lU / mL, at least about 7900 lU / mL, or at least about 8000 lU / mL IL-2. In some aspects, the first expansion medium comprises at least about 3000 lU / mL IL-2. In some aspects, the first expansion medium comprises at least about 3100 lU / mL IL-2. In some aspects, the first expansion medium comprises at least about 3200 lU / mL IL-2. In some aspects, the first expansion medium comprises at least about 3300 lU / mL IL-2. In some aspects, the first expansion medium comprises at least about 3400 lU / mL IL-2. In some aspects, the first expansion medium comprises at least about 3500 lU / mL IL-2.

[0277] In some aspects, the first expansion medium comprises at least about 0.1 ng / mL IL- 21. In some aspects, the first expansion medium comprises from about 0.1 ng / mL to about 50 ng / mL, about 1 ng / mL to about 50 ng / mL, about 1 ng / mL to about 40 ng / mL, about 1 ng / mL to about 35 ng / mL, about 10 ng / mL to about 40 ng / mL, about 10 ng / mL to about 35 ng / mL, about 10 ng / mL to about 30 ng / mL, about 15 ng / mL to about 40 ng / mL, about 15 ng / mL to about 35 ng / mL, about 15 ng / mL to about 30 ng / mL, about 20 ng / mL to about 40 ng / mL, about 20 ng / mL to about 35 ng / mL, about 20 ng / mL to about 30 ng / mL, about 25 ng / mL to about 35 ng / mL, about 25 ng / mL to about 30 ng / mL, or about 30 ng / mL to about 35 ng / mL IL-21.

[0278] In some aspects, the first expansion medium 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 leastabout 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 first expansion medium comprises at least about 1.0 ng / mL IL-21. In some aspects, the first expansion medium comprises at least about 2.0 ng / mL IL-21. In some aspects, the first expansion medium comprises at least about 3.0 ng / mL IL-21. In some aspects, the first expansion medium comprises at least about 4.0 ng / mL IL-21. In some aspects, the first expansion medium comprises at least about 5.0 ng / mL IL-21. In some aspects, the first expansion medium comprises at least about 6.0 ng / mL IL-21. In some aspects, the first expansion medium comprises at least about 7.0 ng / mL IL-21. In some aspects, the first expansion medium comprises at least about 8.0 ng / mL IL-21. In some aspects, the first expansion medium comprises at least about 9.0 ng / mL IL-21. In some aspects, the first expansion medium comprises at least about 10 ng / mL IL-21. In some aspects, the first expansion medium comprises at least about 15 ng / mL IL-21. In some aspects, the first expansion medium comprises at least about 20 ng / mL IL-21. In some aspects, the first expansion medium comprises at least about 25 ng / mL IL- 21. In some aspects, the first expansion medium comprises at least about 30 ng / mL IL-21. In some aspects, the first expansion medium comprises at least about 35 ng / mL IL-21.

[0279] In some aspects, the first expansion medium comprises between about 50 lU / mL to about 500 lU / mL of IL-21. In some aspects, the first expansion medium comprises about 50 lU / mL, about 60 lU / mL, about 70 lU / mL, about 80 lU / mL, about 90 lU / mL, about 100 lU / mL, about 125 lU / mL, about 150 lU / mL, about 175 lU / mL, about 200 lU / mL, about 225 lU / mL, about 250 lU / mL, about 275 lU / mL, about 300 lU / mL, about 350 lU / mL, about 400 lU / mL, about 450 lU / mL, or about 500 lU / mL of IL-21.

[0280] In some aspects, the first expansion medium does not comprise IL-7. In some aspects, the first expansion medium comprises IL-7. In some aspects, the first expansion medium comprises at least about 0.1 ng / mL IL-7. In some aspects, the first expansion medium 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 toabout 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.

[0281] In some aspects, the first expansion medium 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 first expansion medium comprises at least about 2.0 ng / mL IL-7. In some aspects, the first expansion medium comprises at least about 2.3 ng / mL IL-7. In some aspects, the first expansion medium comprises at least about 2.5 ng / mL IL-7. In some aspects, the first expansion medium comprises at least about 2.7 ng / mL IL-7. In some aspects, the first expansion medium comprises at least about 3.0 ng / mL IL-7. In some aspects, the first expansion medium comprises at least about 3.3 ng / mL IL-7. In some aspects, the first expansion medium comprises at least about 3.5 ng / mL IL-7. In some aspects, the first expansion medium comprises at least about 3.7 ng / mL IL-7.

[0282] In some aspects, the first expansion medium comprises between about 500 lU / mL to about 1,500 lU / mL of IL-7. In some aspects, the first expansion medium comprises about 500 lU / mL, about 550 lU / mL, about 600 lU / mL, about 650 lU / mL, about 700 lU / mL, about 750 lU / mL, about 800 lU / mL, about 850 lU / mL, about 900 lU / mL, about 950 lU / mL, about 1,000 lU / mL, about 1,050 lU / mL, about 1,100 lU / mL, about 1,150 lU / mL, about 1,200 lU / mL, about 1,250 lU / mL, about 1,300 lU / mL, about 1,350 lU / mL, about 1,400 lU / mL, about 1,450 lU / mL, or about 1,500 lU / mL of IL-7.

[0283] In some aspects, the first expansion medium does not comprise IL-15. In some aspects, the first expansion medium comprises IL-15. In some aspects, the first expansion medium comprises at least about 0.1 ng / mL IL-15. In some aspects, the first expansion medium 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 1ng / 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.

[0284] In some aspects, the first expansion medium 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 first expansion medium comprises at least about 0.1 ng / mL IL-15. In some aspects, the first expansion medium comprises at least about 0.2 ng / mL IL-15. In some aspects, the first expansion medium comprises at least about 0.3 ng / mL IL-15. In some aspects, the first expansion medium comprises at least about 0.4 ng / mL IL-15. In some aspects, the first expansion medium comprises at least about 0.5 ng / mL IL-15. In some aspects, the first expansion medium comprises at least about 0.6 ng / mL IL-15. In some aspects, the first expansion medium comprises at least about 0.7 ng / mL IL-15. In some aspects, the first expansion medium comprises at least about 0.8 ng / mL IL-15. In some aspects, the first expansion medium comprises at least about 0.9 ng / mL IL-15. In some aspects, the first expansion medium comprises at least about 1.0 ng / mL IL-15. In some aspects, the first expansion medium comprises at least about 2.0 ng / mL IL-15. In some aspects, the first expansion medium comprises at least about 3.0 ng / mL IL- 15. In some aspects, the first expansion medium comprises at least about 4.0 ng / mL IL-15. In some aspects, the first expansion medium comprises at least about 5.0 ng / mL IL-15. In some aspects, the first expansion medium comprises at least about 6.0 ng / mL IL-15. In some aspects, the first expansion medium comprises at least about 7.0 ng / mL IL-15. In some aspects, the first expansion medium comprises at least about 8.0 ng / mL IL-15. In some aspects, the first expansion medium comprises at least about 9.0 ng / mL IL-15. In some aspects, the first expansion medium comprises at least about 10 ng / mL IL-15.

[0285] In some aspects, the first expansion medium comprises between about 50 lU / mL to about 500 lU / mL of IL-15. In some aspects, the first expansion medium comprises about 50 lU / mL, about 60 lU / mL, about 70 lU / mL, about 80 lU / mL, about 90 lU / mL, about 100 lU / mL, about 125 lU / mL, about 150 lU / mL, about 175 lU / mL, about 200 lU / mL, about 225 lU / mL, about 250 lU / mL, about 275 lU / mL, about 300 lU / mL, about 350 lU / mL, about 400 lU / mL, about 450 lU / mL, or about 500 lU / mL of IL-15.

[0286] In some aspects, the first expansion medium comprises about 3000 lU / mL IL-2 and about 30 ng / M IL-21. In some aspects, the first expansion medium comprises about 3000 lU / mL IL-2 and about 30 ng / M IL-21, wherein the medium does not comprise IL-7, IL-15, or both IL-7 and IL-15.II.A.4. Second REP Step

[0287] In some aspects, the TILs are subjected to a second REP step, z.e., a secondary expansion step. 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 second REP step without opening the closed system. In some aspects, the secondary expansion is carried out in a bioreactor. In some aspects, the secondary expansion comprises two phases, wherein the first phase is carried out in a first container, and the second phase is carried out in a second container, wherein the first container is not a bioreactor, and wherein the second container is a bioreactor. In some aspects, the TILs from the first REP step are screened for tumor-specific cytolytic activity prior to advancing the TILs to the second REP step. In some aspects, the TILs are screened for expression of one or more biomarkers prior to advancing to the second REP step. In some aspects, the biomarker comprises expression of one or more gene typically expressed by more naive TILs, e.g., CD8+, CD27+, CD3+, CD95+, CD45RA+, CCR7+, CD62L+, TCF7+, or any combination thereof. In some aspects, the TILs are screened for expression of PD-1 prior to advancing to the second REP step. In some aspects, the TILs from the first REP step are not screened prior to advancing the TILs to the second REP step. In some aspects, all TILs obtained in the first REP step are subjected to the second REP step. In some aspects, the TILs from the first REP step are pooled prior to advancement to the second REP step.

[0288] In some aspects, the TILs from the first REP step are placed into a second expansion medium, wherein the second expansion medium comprises a CD3 agonist and APCs at a second ratio (a "second REP step"). In some aspects, the TILs are cultured in the second REP step for less than about 14 days. In some aspects, the TILs are cultured in the second REP step for less thanabout 13 days. In some aspects, the TILs are cultured in the second REP step for less than about 12 days. In some aspects, the TILs are cultured in the second REP step for less than about 11 days. In some aspects, the TILs are cultured in the second REP step for less than about 10 days. In some aspects, the TILs are cultured in the second REP step for less than about 9 days. In some aspects, the TILs are cultured in the second REP step for less than about 8 days. In some aspects, the TILs are cultured in the second REP step for about 12 days. In some aspects, the TILs are cultured in the second REP step for about 11 days. In some aspects, the TILs are cultured in the second REP step for about 10 days. In some aspects, the TILs are cultured in the second REP step for about 9 days. In some aspects, the TILs are cultured in the second REP step for about 8 days. In some aspects, the TILs are cultured in the second REP step for about 7 days.

[0289] In some aspects, the TILs are cultured in the second REP step for a period of time until a certain number of cells is reached. In some aspects, the number of TILs present following the first REP step is increased by 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 following culture in the second REP step. In some aspects, the number of TILs present in the dissociated tumor sample is increased by at least about 10-fold following culture in the second REP step. In some aspects, the number of TILs present in the dissociated tumor sample is increased by at least about 15-fold following culture in the second REP step. In some aspects, the number of TILs present in the dissociated tumor sample is increased by at least about 20-fold following culture in the second REP step. In some aspects, the number of TILs present in the dissociated tumor sample is increased by at least about 25-fold following culture in the second REP step. In some aspects, the number of TILs present in the dissociated tumor sample is increased by at least about 30-fold following culture in the second REP step. In some aspects, the number of TILs preset in the dissociated tumor sample is increased by at least about 35-fold following culture in the second REP step. In some aspects, the number of TILs present in the dissociated tumor sample is increased by at least about 40-fold following culture in the second REP step. In some aspects, the number of TILs present in the dissociated tumor sample is increased by at least about 45-fold following culture in the second REP step. In some aspects, the number of TILs present in the dissociated tumor sample is increased by at least about 50-fold following culture in the second REP step.

[0290] In some aspects, the number of TILs following culture in the second REP step is 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 IO10, at least about 2 x IO10, at least about 3 x IO10, at least about 4 x IO10, at least about 5 x IO10, at least about 6 x IO10, at least about 7 x IO10, at least about 8 x IO10, or at least about 9 x IO10TILs. In some aspects, the number of TILs following culture in the second REP step is at least about 5 x 109. In some aspects, the number of TILs following culture in the second REP step is at least about 1 x IO10. In some aspects, the number of TILs following culture in the second REP step is at least about 2 x IO10. In some aspects, the number of TILs following culture in the second REP step is at least about 3 x IO10. In some aspects, the number of TILs following culture in the second REP step is at least about 4 x IO10. In some aspects, the number of TILs following culture in the second REP step is at least about 5 x IO10. In some aspects, the number of TILs following culture in the second REP step is at least about 6 x IO10. In some aspects, the number of TILs following culture in the second REP step is at least about 7 x IO10. In some aspects, the number of TILs following culture in the second REP step is at least about 8 x IO10. In some aspects, the number of TILs following culture in the second REP step is at least about 9 x IO10. In some aspects, the number of TILs following culture in the second REP step is at least about 10 x IO10.

[0291] In some aspects, the second REP step continues until at least about 5 x 109TILs are obtained. In some aspects, the second REP step continues until at least about 1 x IO10TILs are obtained. In some aspects, the second REP step continues until at least about 2 x IO10TILs are obtained. In some aspects, the second REP step continues until at least about 3 x IO10TILs are obtained. In some aspects, the second REP step continues until at least about 4 x IO10TILs are obtained. In some aspects, the second REP step continues until at least about 5 x IO10TILs are obtained. In some aspects, the second REP step continues until at least about 6 x IO10TILs are obtained. In some aspects, the second REP step continues until at least about 7 x IO10TILs are obtained. In some aspects, the second REP step continues until at least about 8 x IO10TILs are obtained. In some aspects, the second REP step continues until at least about 9 x IO10TILs are obtained. In some aspects, the second REP step continues until at least about 10 x IO10TILs are obtained.

[0292] In some aspects, the second REP step lasts for less than 8 days, wherein at least about 1 x IO10TILs are obtained. In some aspects, the second REP step lasts for less than 8 days, wherein at least about 2 x IO10TILs are obtained. In some aspects, the second REP step lasts for less than 108 days, wherein at least about 3 x 10° TILs are obtained. In some aspects, the second REP step lasts for less than 8 days, wherein at least about 4 x 1010TILs are obtained. In some aspects, the second REP step lasts for less than 8 days, wherein at least about 5 x 1010TILs are obtained. In some aspects, the second REP step lasts for less than 8 days, wherein at least about 6x IO10TILs are obtained. In some aspects, the second REP step lasts for less than 8 days, wherein at least about 7 x IO10TILs are obtained. In some aspects, the second REP step lasts for less than8 days, wherein at least about 8 x 1010TILs are obtained. In some aspects, the second REP step lasts for less than 8 days, wherein at least about 9 x IO10TILs are obtained. In some aspects, the second REP step lasts for less than 8 days, wherein at least about 10 x IO10TILs are obtained.

[0293] In some aspects, the second REP step lasts for less than 9 days, wherein at least about 1 x IO10TILs are obtained. In some aspects, the second REP step lasts for less than 9 days, wherein at least about 2 x IO10TILs are obtained. In some aspects, the second REP step lasts for less than 9 days, wherein at least about 3 x IO10TILs are obtained. In some aspects, the second REP step lasts for less than 9 days, wherein at least about 4 x IO10TILs are obtained. In some aspects, the second REP step lasts for less than 9 days, wherein at least about 5 x 1010TILs are obtained. In some aspects, the second REP step lasts for less than 9 days, wherein at least about 6 x IO10TILs are obtained. In some aspects, the second REP step lasts for less than 9 days, wherein at least about 7 x IO10TILs are obtained. In some aspects, the second REP step lasts for less than9 days, wherein at least about 8 x 1010TILs are obtained. In some aspects, the second REP step lasts for less than 9 days, wherein at least about 9 x 1010TILs are obtained. In some aspects, the second REP step lasts for less than 9 days, wherein at least about 10 x 1010TILs are obtained.

[0294] In some aspects, the second REP step lasts for less than 10 days, wherein at least about 1 x 1010TILs are obtained. In some aspects, the second REP step lasts for less than 10 days, wherein at least about 2 x 1010TILs are obtained. In some aspects, the second REP step lasts for less than 10 days, wherein at least about 3 x 1010TILs are obtained. In some aspects, the second REP step lasts for less than 10 days, wherein at least about 4 x 1010TILs are obtained. In some aspects, the second REP step lasts for less than 10 days, wherein at least about 5 x 1010TILs are obtained. In some aspects, the second REP step lasts for less than 10 days, wherein at least about 6 x 1010TILs are obtained. In some aspects, the second REP step lasts for less than 10 days, wherein at least about 7 x 1010TILs are obtained. In some aspects, the second REP step lasts for less than10 days, wherein at least about 8 x 1010TILs are obtained. In some aspects, the second REP step lasts for less than 10 days, wherein at least about 9 x 1010TILs are obtained. In some aspects, the second REP step lasts for less than 10 days, wherein at least about 10 x 1010TILs are obtained.

[0295] In some aspects, the second REP step lasts for less than 11 days, wherein at least about 1 x 1010TILs are obtained. In some aspects, the second REP step lasts for less than 11 days, wherein at least about 2 x 1010TILs are obtained. In some aspects, the second REP step lasts for less than 11 days, wherein at least about 3 x 1010TILs are obtained. In some aspects, the secondREP step lasts for less than 11 days, wherein at least about 4 x IO10TILs are obtained. In some aspects, the second REP step lasts for less than 11 days, wherein at least about 5 x IO10TILs are obtained. In some aspects, the second REP step lasts for less than 11 days, wherein at least about6 x IO10TILs are obtained. In some aspects, the second REP step lasts for less than 11 days, wherein at least about 7 x IO10TILs are obtained. In some aspects, the second REP step lasts for less than 11 days, wherein at least about 8 x IO10TILs are obtained. In some aspects, the second REP step lasts for less than 11 days, wherein at least about 9 x IO10TILs are obtained. In some aspects, the second REP step lasts for less than 11 days, wherein at least about 10 x IO10TILs are obtained.

[0296] In some aspects, the TILs are cultured in the first REP step for 7 to 10 days, and the TILs are cultured in the second REP step for 7 to 10 days, wherein the total number of TILs following culture in the second REP step is at least about 1 x IO10TILs. In some aspects, the TILs are cultured in the first REP step for 7 days, and the TILs are cultured in the second REP step for7 days, wherein the total number of TILs following culture in the second REP step is at least about 1 x IO10TILs. In some aspects, the TILs are cultured in the first REP step for 10 days or less, and the TILs are cultured in the second REP step for 10 days or less, wherein the total number of TILs following culture in the second REP step is at least about 1 x 1010TILs.

[0297] In some aspects, the second expansion medium comprises a dissociated tumor cell to APC ratio of about 1 : 1, about 1 : 1.5, about 1 :2, about 1 :2.5, about 1 :3, about 1 :3.5, about 1 :4, about 1 :4.5, about 1 :5, about 1 :5.5, about 1 :6, about 1:6.5, about 1 :7, about 1:7.5, about 1 :8, about 1 :8.5, about 1 :9, about 1 :9.5, about 1 : 10, about 1 : 11, about 1 : 12, about 1 : 13, about 1 : 14, about 1 : 15, or about 1 :20. In some aspects, the second expansion medium comprises a dissociated tumor cell to APC ratio of about 1 : 1. In some aspects, the second expansion medium comprises a dissociated tumor cell to APC ratio of about 1 :2. In some aspects, the second expansion medium comprises a dissociated tumor cell to APC ratio of about 1 :2.5. In some aspects, the second expansion medium comprises a dissociated tumor cell to APC ratio of about 1 :3. In some aspects, the second expansion medium comprises a dissociated tumor cell to APC ratio of about 1 :4. In some aspects, the second expansion medium comprises a dissociated tumor cell to APC ratio of about 1 :5. In some aspects, the second expansion medium comprises a dissociated tumor cell to APC ratio of about 1 :6. In some aspects, the second expansion medium comprises a dissociated tumor cell to APC ratio of about 1 :7. In some aspects, the second expansion medium comprises a dissociated tumor cell to APC ratio of about 1 :7.5. In some aspects, the second expansion medium comprises a dissociated tumor cell to APC ratio of about 1 :8. In some aspects, the second expansionmedium comprises a dissociated tumor cell to APC ratio of about 1 :9. In some aspects, the second expansion medium comprises a dissociated tumor cell to APC ratio of about 1 : 10.

[0298] In some aspects, the first expansion medium comprises a dissociated tumor cell to APC ratio of about 1 : 10, and the second expansion medium comprises a first REP expanded TIL to APC ratio of about 1 : 1. In some aspects, the first expansion medium comprises a dissociated tumor cell to APC ratio of about 1 : 10, and the second expansion medium comprises a first REP expanded TIL to APC ratio of about 1 :2. In some aspects, the first expansion medium comprises a dissociated tumor cell to APC ratio of about 1 : 10, and the second expansion medium comprises a first REP expanded TIL to APC ratio of about 1 :2.5. In some aspects, the first expansion medium comprises a dissociated tumor cell to APC ratio of about 1 : 10, and the second expansion medium comprises a first REP expanded TIL to APC ratio of about 1 :3. In some aspects, the first expansion medium comprises a dissociated tumor cell to APC ratio of about 1 :10, and the second expansion medium comprises a first REP expanded TIL to APC ratio of about 1 :4. In some aspects, the first expansion medium comprises a dissociated tumor cell to APC ratio of about 1 : 10, and the second expansion medium comprises a first REP expanded TIL to APC ratio of about 1 :5. In some aspects, the first expansion medium comprises a dissociated tumor cell to APC ratio of about 1 : 10, and the second expansion medium comprises a first REP expanded TIL to APC ratio of about 1 :6. In some aspects, the first expansion medium comprises a dissociated tumor cell to APC ratio of about 1 : 10, and the second expansion medium comprises a first REP expanded TIL to APC ratio of about 1 :7. In some aspects, the first expansion medium comprises a dissociated tumor cell to APC ratio of about 1 : 10, and the second expansion medium comprises a first REP expanded TIL to APC ratio of about 1 :7.5. In some aspects, the first expansion medium comprises a dissociated tumor cell to APC ratio of about 1 : 10, and the second expansion medium comprises a first REP expanded TIL to APC ratio of about 1 :8. In some aspects, the first expansion medium comprises a dissociated tumor cell to APC ratio of about 1 : 10, and the second expansion medium comprises a first REP expanded TIL to APC ratio of about 1 :9. In some aspects, the first expansion medium comprises a dissociated tumor cell to APC ratio of about 1 : 10, and the second expansion medium comprises a first REP expanded TIL to APC ratio of about 1 : 10.

[0299] In some aspects, the second expansion medium comprises a CD3 agonist. In some aspects, the CD3 agonist comprises OKT3. In some aspects, the second expansion medium comprises from about 1 to about 100 ng / mL OKT3. In some aspects, the concentration of OKT3 is at least about 1 ng / ml, at least about 5 ng / ml, at least about 10 ng / ml, at least about 15 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, atleast about 40 ng / ml, at least about 45 ng / ml, at least about 50 ng / ml, at least about 55 ng / ml, at least about 60 ng / ml, at least about 65 ng / ml, at least about 70 ng / ml, at least about 75 ng / ml, at least about 80 ng / ml, at least about 90 ng / ml, or at least about 100 ng / ml. In some aspects, the second expansion medium comprises about 10 ng / ml OKT3. In some aspects, the second expansion medium comprises about 15 ng / ml OKT3. In some aspects, the second expansion medium comprises about 20 ng / ml OKT3. In some aspects, the second expansion medium comprises about 25 ng / ml OKT3. In some aspects, the second expansion medium comprises about 30 ng / ml OKT3. In some aspects, the second expansion medium comprises about 35 ng / ml OKT3. In some aspects, the second expansion medium comprises about 40 ng / ml OKT3. In some aspects, the second expansion medium comprises about 45 ng / ml OKT3. In some aspects, the second expansion medium comprises about 50 ng / ml OKT3. In some aspects, the second expansion medium comprises about 55 ng / ml OKT3. In some aspects, the second expansion medium comprises about 60 ng / ml OKT3. In some aspects, the second expansion medium comprises about 65 ng / ml OKT3. In some aspects, the second expansion medium comprises about 70 ng / ml OKT3. In some aspects, the second expansion medium comprises about 75 ng / ml OKT3.

[0300] In some aspects, the second expansion medium comprises (i) a dissociated tumor cell to APC ratio of about 1 :3 and (ii) about 30 ng / ml OKT3. In some aspects, the second expansion medium comprises (i) a dissociated tumor cell to APC ratio of about 1 :3 and (ii) about 35 ng / ml OKT3. In some aspects, the second expansion medium comprises (i) a dissociated tumor cell to APC ratio of about 1 :3 and (ii) about 40 ng / ml OKT3. In some aspects, the second expansion medium comprises (i) a dissociated tumor cell to APC ratio of about 1 :3 and (ii) about 45 ng / ml OKT3. In some aspects, the second expansion medium comprises (i) a dissociated tumor cell to APC ratio of about 1 :3 and (ii) about 50 ng / ml OKT3. In some aspects, the second expansion medium comprises (i) a dissociated tumor cell to APC ratio of about 1 :3 and (ii) about 55 ng / ml OKT3. In some aspects, the second expansion medium comprises (i) a dissociated tumor cell to APC ratio of about 1 :3 and (ii) about 60 ng / ml OKT3. In some aspects, the second expansion medium comprises (i) a dissociated tumor cell to APC ratio of about 1 :3 and (ii) about 65 ng / ml OKT3. In some aspects, the second expansion medium comprises (i) a dissociated tumor cell to APC ratio of about 1 :3 and (ii) about 70 ng / ml OKT3.

[0301] In some aspects, the second expansion medium comprises (i) a dissociated tumor cell to APC ratio of about 1 :4 and (ii) about 30 ng / ml OKT3. In some aspects, the second expansion medium comprises (i) a dissociated tumor cell to APC ratio of about 1 :4 and (ii) about 35 ng / ml OKT3. In some aspects, the second expansion medium comprises (i) a dissociated tumor cell toAPC ratio of about 1:4 and (ii) about 40 ng / ml OKT3. In some aspects, the second expansion medium comprises (i) a dissociated tumor cell to APC ratio of about 1 :4 and (ii) about 45 ng / ml OKT3. In some aspects, the second expansion medium comprises (i) a dissociated tumor cell to APC ratio of about 1 :4 and (ii) about 50 ng / ml OKT3. In some aspects, the second expansion medium comprises (i) a dissociated tumor cell to APC ratio of about 1 :4 and (ii) about 55 ng / ml OKT3. In some aspects, the second expansion medium comprises (i) a dissociated tumor cell to APC ratio of about 1 :4 and (ii) about 60 ng / ml OKT3. In some aspects, the second expansion medium comprises (i) a dissociated tumor cell to APC ratio of about 1 :4 and (ii) about 65 ng / ml OKT3. In some aspects, the second expansion medium comprises (i) a dissociated tumor cell to APC ratio of about 1 :4 and (ii) about 70 ng / ml OKT3.

[0302] In some aspects, the second expansion medium comprises (i) a dissociated tumor cell to APC ratio of about 1 :5 and (ii) about 30 ng / ml OKT3. In some aspects, the second expansion medium comprises (i) a dissociated tumor cell to APC ratio of about 1 :5 and (ii) about 35 ng / ml OKT3. In some aspects, the second expansion medium comprises (i) a dissociated tumor cell to APC ratio of about 1 :5 and (ii) about 40 ng / ml OKT3. In some aspects, the second expansion medium comprises (i) a dissociated tumor cell to APC ratio of about 1 :5 and (ii) about 45 ng / ml OKT3. In some aspects, the second expansion medium comprises (i) a dissociated tumor cell to APC ratio of about 1 :5 and (ii) about 50 ng / ml OKT3. In some aspects, the second expansion medium comprises (i) a dissociated tumor cell to APC ratio of about 1 :5 and (ii) about 55 ng / ml OKT3. In some aspects, the second expansion medium comprises (i) a dissociated tumor cell to APC ratio of about 1 :5 and (ii) about 60 ng / ml OKT3. In some aspects, the second expansion medium comprises (i) a dissociated tumor cell to APC ratio of about 1 :5 and (ii) about 65 ng / ml OKT3. In some aspects, the second expansion medium comprises (i) a dissociated tumor cell to APC ratio of about 1 :5 and (ii) about 70 ng / ml OKT3.

[0303] In some aspects, the second expansion medium comprises (i) a dissociated tumor cell to APC ratio of about 1 :6 and (ii) about 30 ng / ml OKT3. In some aspects, the second expansion medium comprises (i) a dissociated tumor cell to APC ratio of about 1 :6 and (ii) about 35 ng / ml OKT3. In some aspects, the second expansion medium comprises (i) a dissociated tumor cell to APC ratio of about 1 :6 and (ii) about 40 ng / ml OKT3. In some aspects, the second expansion medium comprises (i) a dissociated tumor cell to APC ratio of about 1 :6 and (ii) about 45 ng / ml OKT3. In some aspects, the second expansion medium comprises (i) a dissociated tumor cell to APC ratio of about 1 :6 and (ii) about 50 ng / ml OKT3. In some aspects, the second expansion medium comprises (i) a dissociated tumor cell to APC ratio of about 1 :6 and (ii) about 55 ng / ml0KT3. In some aspects, the second expansion medium comprises (i) a dissociated tumor cell to APC ratio of about 1 :6 and (ii) about 60 ng / ml OKT3. In some aspects, the second expansion medium comprises (i) a dissociated tumor cell to APC ratio of about 1 :6 and (ii) about 65 ng / ml OKT3. In some aspects, the second expansion medium comprises (i) a dissociated tumor cell to APC ratio of about 1 :6 and (ii) about 70 ng / ml OKT3.

[0304] In some aspects, the second expansion medium comprises (i) a dissociated tumor cell to APC ratio of about 1 :7 and (ii) about 30 ng / ml OKT3. In some aspects, the second expansion medium comprises (i) a dissociated tumor cell to APC ratio of about 1 :7 and (ii) about 35 ng / ml OKT3. In some aspects, the second expansion medium comprises (i) a dissociated tumor cell to APC ratio of about 1 :7 and (ii) about 40 ng / ml OKT3. In some aspects, the second expansion medium comprises (i) a dissociated tumor cell to APC ratio of about 1 :7 and (ii) about 45 ng / ml OKT3. In some aspects, the second expansion medium comprises (i) a dissociated tumor cell to APC ratio of about 1 :7 and (ii) about 50 ng / ml OKT3. In some aspects, the second expansion medium comprises (i) a dissociated tumor cell to APC ratio of about 1 :7 and (ii) about 55 ng / ml OKT3. In some aspects, the second expansion medium comprises (i) a dissociated tumor cell to APC ratio of about 1 :7 and (ii) about 60 ng / ml OKT3. In some aspects, the second expansion medium comprises (i) a dissociated tumor cell to APC ratio of about 1 :7 and (ii) about 65 ng / ml OKT3. In some aspects, the second expansion medium comprises (i) a dissociated tumor cell to APC ratio of about 1 :7 and (ii) about 70 ng / ml OKT3.

[0305] In some aspects, the cells (e.g., TILs) are passed through a strainer following the second expansion. In some aspects, the cells (e.g., TILs) are passed through an at least about 10 pm, an at least about 15 pm, an at least about 20 pm, an at least about 25 pm, an at least about 30 pm, an at least about 35 pm, an at least about 40 pm, an at least about 45 pm, an at least about 50 pm strainer following the second REP step. In some aspects, the cells (e.g., TILs) are passed through an about 40 pm strainer following the second REP step.

[0306] In some aspects, the second REP step is carried out in one or more gas permeable flasks (e.g., GREX flasks). In some aspects, the second REP step is carried out in static GREX. In some aspects, the second REP step is carried out in a stirred tank. In some aspects, the second REP step is carried out in a bioreactor. In some aspects, the second REP step is carried out in a closed system (e.g., using a GREX closed system).

[0307] In some aspects, the second REP step comprises two phases. In some aspects the second REP comprises culturing the TILs in a first phase medium followed by culturing the TILs in a second phase medium. In some aspects, the TILs are cultured in the first phase medium forabout 12 hours, about 24 hours, about 36 hours, about 48 hours, about 50 hours, about 62 hours, about 74 hours, about 86 hours, or about 98 hours before the TILs are cultured in the second phase medium. In some aspects, the TILs are cultured in the first phase medium for about 1 day, about 2 days, about 3 days, about 4 days, about 5 days, or about 6 days before the TILs are cultured in the second phase medium. In some aspects, the TILs are cultured in the first phase medium for about 1 day before the TILs are cultured in the second phase medium. In some aspects, the TILs are cultured in the first phase medium for about 2 days before the TILs are cultured in the second phase medium. In some aspects, the TILs are cultured in the first phase medium for about 3 days before the TILs are cultured in the second phase medium. In some aspects, the TILs are cultured in the first phase medium for about 4 days before the TILs are cultured in the second phase medium. In some aspects, the TILs are cultured in the first phase medium for about 5 days before the TILs are cultured in the second phase medium.

[0308] In some aspects, the first phase medium comprises culturing the TILs in a metabolic reprogramming medium (MRM) disclosed herein. In some aspects, the first phase medium comprises about 45 mM potassium ion. In some aspects, the first phase medium comprises about 50 mM potassium ion. In some aspects, the first phase medium comprises about 55 mM potassium ion. In some aspects, the first phase medium comprises about 60 mM potassium ion. In some aspects, the first phase medium comprises about 65 mM potassium ion. In some aspects, the first phase medium comprises about 70 mM potassium ion. In some aspects, the first phase medium comprises about 75 mM potassium ion. In some aspects, the first phase medium comprises about 80 mM potassium ion. In some aspects, the first phase medium comprises about 85 mM potassium ion. In some aspects, the first phase medium comprises about 90 mM potassium ion.

[0309] In some aspects, the contents of the first phase medium culture are transferred to a second phase medium, wherein the first phase medium is diluted by fresh medium to arrive at the second phase medium. In some aspects, the first phase medium is diluted by the fresh medium at a ratio of 1 : 1, respectively, z.e., a 50% solution. In some aspects, the first phase medium is diluted by the fresh medium at a ratio of 1 :2, respectively, ie., a 33% solution. In some aspects, the first phase medium is diluted by the fresh medium at a ratio of 1 :3, respectively, z.e., a 25% solution. In some aspects, the first phase medium is diluted by the fresh medium at a ratio of 3: 1, respectively, z.e., a 75% solution. In some aspects, the fresh medium does not comprise greater than about 5% potassium ion. In some aspects, the MRM of the first phase medium culture is diluted to a final concentration of about 75% MRM in the second phase medium. In some aspects, the MRM of the first phase medium culture is diluted to a final concentration of about 70% MRMin the second phase medium. In some aspects, the MRM of the first phase medium culture is diluted to a final concentration of about 65% MRM in the second phase medium. In some aspects, the MRM of the first phase medium culture is diluted to a final concentration of about 60% MRM in the second phase medium. In some aspects, the MRM of the first phase medium culture is diluted to a final concentration of about 55% MRM in the second phase medium. In some aspects, the MRM of the first phase medium culture is diluted to a final concentration of about 50% MRM in the second phase medium. In some aspects, the MRM of the first phase medium culture is diluted to a final concentration of about 45% MRM in the second phase medium. In some aspects, the MRM of the first phase medium culture is diluted to a final concentration of about 40% MRM in the second phase medium. In some aspects, the MRM of the first phase medium culture is diluted to a final concentration of about 35% MRM in the second phase medium. In some aspects, the MRM of the first phase medium culture is diluted to a final concentration of about 30% MRM in the second phase medium. In some aspects, the MRM of the first phase medium culture is diluted to a final concentration of about 25% MRM in the second phase medium. In some aspects, the MRM of the first phase medium culture is diluted to a final concentration of about 20% MRM in the second phase medium. In some aspects, the MRM of the first phase medium culture is diluted to a final concentration of about 15% MRM in the second phase medium. In some aspects, the MRM of the first phase medium culture is diluted to a final concentration of about 10% MRM in the second phase medium. In some aspects, the MRM of the first phase medium culture is diluted to a final concentration of about 5% MRM in the second phase medium. In some aspects, culturing the TILs in a medium comprising less MRM (z.e., in the second phase medium) allows the TILs to recover prior to administration to a subject.

[0310] In some aspects, the fresh medium does not comprise a CD3 agonist. In some aspects, the fresh medium does not comprise additional antigen-presenting cells (e.g., irradiated PBMCs). As a result, the number of viable antigen-presenting cells present in the first phase medium (z.e., at day 0 of the second REP step) will decrease throughout the second REP step.

[0311] In some aspects, the duration of the first phase of the second REP step is about 1 day. In some aspects, the duration of the first phase of the second REP step is about 2 days. In some aspects, the duration of the first phase of the second REP step is about 3 days. In some aspects, the duration of the first phase of the second REP step is about 4 days. In some aspects, the duration of the first phase of the second REP step is about 5 days.

[0312] In some aspects, the duration of the second phase of the second REP step is about 3 days to about 13 days. In some aspects, the duration of the second phase of the second REP stepis about 4 days to about 13 days. In some aspects, the duration of the second phase of the second REP step is about 5 days to about 13 days. In some aspects, the duration of the second phase of the second REP step is about 6 days to about 13 days. In some aspects, the duration of the second phase of the second REP step is about 7 days to about 13 days. In some aspects, the duration of the second phase of the second REP step is about 9 days to about 13 days. In some aspects, the duration of the second phase of the second REP step is about 5 days. In some aspects, the duration of the second phase of the second REP step is about 6 days. In some aspects, the duration of the second phase of the second REP step is about 7 days. In some aspects, the duration of the second phase of the second REP step is about 8 days. In some aspects, the duration of the second phase of the second REP step is about 9 days. In some aspects, the duration of the second phase of the second REP step is about 10 days. In some aspects, the duration of the second phase of the second REP step is about 11 days. In some aspects, the duration of the second phase of the second REP step is about 12 days. In some aspects, the duration of the second phase of the second REP step is about 13 days.

[0313] In some aspects, the total duration of the first phase of the second REP step and the second phase of the second REP step is about 7 to about 13 days. In some aspects, the total duration of the first phase of the second REP step and the second phase of the second REP step is about 7 to about 12 days. In some aspects, the total duration of the first phase of the second REP step and the second phase of the second REP step is about 7 to about 11 days. In some aspects, the total duration of the first phase of the second REP step and the second phase of the second REP step is about 7 to about 10 days. In some aspects, the total duration of the first phase of the second REP step and the second phase of the second REP step is about 7 to about 9 days. In some aspects, the total duration of the first phase of the second REP step and the second phase of the second REP step is about 8 to about 12 days. In some aspects, the total duration of the first phase of the second REP step and the second phase of the second REP step is about 8 to about 11 days. In some aspects, the total duration of the first phase of the second REP step and the second phase of the second REP step is about 8 to about 10 days. In some aspects, the total duration of the first phase of the second REP step and the second phase of the second REP step is about 9 to about 12 days. In some aspects, the total duration of the first phase of the second REP step and the second phase of the second REP step is about 9 to about 11 days. In some aspects, the total duration of the first phase of the second REP step and the second phase of the second REP step is about 9 to about 10 days. In some aspects, the total duration of the first phase of the second REP step and the second phase of the second REP step is about 10 to about 13 days. In some aspects, the total duration of the first phase of the secondREP step and the second phase of the second REP step is about 10 to about 12 days. In some aspects, the total duration of the first phase of the second REP step and the second phase of the second REP step is about 10 to about 11 days. In some aspects, the total duration of the first phase of the second REP step and the second phase of the second REP step is about 8 days. In some aspects, the total duration of the first phase of the second REP step and the second phase of the second REP step is about 9 days. In some aspects, the total duration of the first phase of the second REP step and the second phase of the second REP step is about 10 days. In some aspects, the total duration of the first phase of the second REP step and the second phase of the second REP step is about 11 days. In some aspects, the total duration of the first phase of the second REP step and the second phase of the second REP step is about 12 days. In some aspects, the total duration of the first phase of the second REP step and the second phase of the second REP step is about 13 days.

[0314] In some aspects, duration of the first phase of the second REP step is about 3 days, and the duration of the second phase of the second REP step is about 7 days. In some aspects, duration of the first phase of the second REP step is about 3 days, and the duration of the second phase of the second REP step is about 8 days. In some aspects, duration of the first phase of the second REP step is about 3 days, and the duration of the second phase of the second REP step is about 9 days. In some aspects, duration of the first phase of the second REP step is about 2 days, and the duration of the second phase of the second REP step is about 8 days. In some aspects, duration of the first phase of the second REP step is about 2 days, and the duration of the second phase of the second REP step is about 9 days. In some aspects, duration of the first phase of the second REP step is about 2 days, and the duration of the second phase of the second REP step is about 10 days. In some aspects, duration of the first phase of the second REP step is about 2 days, and the duration of the second phase of the second REP step is about 11 days. In some aspects, duration of the first phase of the second REP step is about 4 days, and the duration of the second phase of the second REP step is about 6 days. In some aspects, duration of the first phase of the second REP step is about 4 days, and the duration of the second phase of the second REP step is about 7 days. In some aspects, duration of the first phase of the second REP step is about 4 days, and the duration of the second phase of the second REP step is about 8 days. In some aspects, duration of the first phase of the second REP step is about 4 days, and the duration of the second phase of the second REP step is about 9 days. In some aspects, duration of the first phase of the second REP step is about 5 days, and the duration of the second phase of the second REP step is about 5 days. In some aspects, duration of the first phase of the second REP step is about 5 days, and the duration of the second phase of the second REP step is about 6 days. In some aspects,duration of the first phase of the second REP step is about 5 days, and the duration of the second phase of the second REP step is about 7 days. In some aspects, duration of the first phase of the second REP step is about 5 days, and the duration of the second phase of the second REP step is about 8 days. In some aspects, duration of the first phase of the second REP step is about 5 days, and the duration of the second phase of the second REP step is about 9 days. In some aspects, duration of the first phase of the second REP step is about 5 days, and the duration of the second phase of the second REP step is about 10 days. In some aspects, duration of the first phase of the second REP step is about 5 days, and the duration of the second phase of the second REP step is about 11 days. In some aspects, duration of the first phase of the second REP step is about 5 days, and the duration of the second phase of the second REP step is about 12 days. In some aspects, the TILs are cryopreserved following conclusion of the second phase of the second REP step.

[0315] In some aspects, the duration of the first REP plus the duration of the second REP is about 18 days. In some aspects, the duration of the first REP plus the duration of the second REP is about 19 days. In some aspects, the duration of the first REP plus the duration of the second REP is about 20 days. In some aspects, the duration of the first REP plus the duration of the second REP is about 21 days. In some aspects, the duration of the first REP plus the duration of the second REP is about 22 days.

[0316] In some aspects, the TILs are cryopreserved following the second REP step. In some aspects, the TILs from the second REP step are administered directly to a subject.

[0317] In some aspects, the method comprises selecting for TILs that express an HLA polypeptide from the heterogeneous population of TILs obtained from the tumor sample (e.g., the dissociated tumor sample, e.g., the single cell suspension); then culturing the population of selected TILs in a first expansion medium, wherein the first expansion medium comprises a T cell activator and a cell support matrix at a first ratio of cell support matrix to TIL (a "first REP step"); and then culturing the expanded population of selected TILs in a second expansion medium, wherein the second expansion medium comprises a T cell activator and a cell support matrix at a second ratio of cell support matrix to TIL (a "second REP step").

[0318] In some aspects, the method comprises (i) dissociating a tumor sample obtained from the subject comprising TILs to generate a single cell suspension comprising a heterogeneous population of TILs; (ii) selecting for TILs that express an HLA polypeptide ("population of selected TILs") from the heterogeneous population of TILs; (iii) culturing the population of selected TILs in a first expansion medium, wherein the first expansion medium comprises a T cell activator and a cell support matrix at a first ratio (a "first REP step"); and (iv) culturing theexpanded population of selected TILs in a second expansion medium, wherein the second expansion medium comprises a T cell activator and a cell support matrix at a second ratio of cell support matrix to TIL (a "second REP step").

[0319] In some aspects, the method comprises (i) dissociating a tumor sample obtained from the subject comprising TILs to generate a single cell suspension comprising a heterogeneous population of TILs; (ii) culturing the heterogeneous population of TILs in an initial expansion medium disclosed herein; (iii) selecting for TILs that express an HLA polypeptide ("population of selected TILs") from the heterogeneous population of TILs; (iv) culturing the population of selected TILs in a first expansion medium, wherein the first expansion medium comprises a T cell activator and a cell support matrix at a first ratio of cell support matrix to TIL (a "first REP step"); and (v) culturing the expanded population of selected TILs in a second expansion medium, wherein the second expansion medium comprises a T cell activator and a cell support matrix at a second ratio of cell support matrix to TIL (a "second REP step").

[0320] In some aspects, the method comprises (i) dissociating a tumor sample obtained from the subject comprising TILs to generate a single cell suspension comprising a heterogeneous population of TILs; (ii) selecting for TILs that express an HLA polypeptide ("population of selected TILs") from the heterogeneous population of TILs; (iii) culturing the population of selected TILs in an initial expansion medium disclosed herein; (iv) culturing the population of selected TILs in a first expansion medium, wherein the first expansion medium comprises a T cell activator and a cell support matrix at a first ratio of cell support matrix to TIL (a "first REP step"); and (v) culturing the expanded population of selected TILs in a second expansion medium, wherein the second expansion medium comprises a T cell activator and a cell support matrix at a second ratio of cell support matrix to TIL (a "second REP step").

[0321] In some aspects, the TILs from the first REP step are de-selected for TILs that express CD127 after the first REP step and before the second REP step. In some aspects, the population of selected TILs without the population of de-selected TILs from the first REP step are subjected to the second REP step.

[0322] In some aspects, the method comprises removing or de-selecting TILs that express CD 127 from the heterogeneous population of TILs obtained from the tumor sample (e.g., the dissociated tumor sample, e.g., the single cell suspension); then culturing the population of selected TILs without the population of de-selected TILs in a first expansion medium, wherein the first expansion medium comprises a T cell activator and a cell support matrix at a first ratio of cell support matrix to TIL (a "first REP step"); and then culturing the expanded population of selectedTILs without the population of de-selected TILs in a second expansion medium, wherein the second expansion medium comprises a T cell activator and a cell support matrix at a second ratio of cell support matrix to TIL (a "second REP step").

[0323] In some aspects, the method comprises (i) dissociating a tumor sample obtained from the subject comprising TILs to generate a single cell suspension comprising a heterogeneous population of TILs; (ii) removing or de-selecting TILs that express CD127 ("population of deselected TILs") from the heterogeneous population of TILs; (iii) culturing the population of TILs without the population of de-selected TILs in a first expansion medium, wherein the first expansion medium comprises a T cell activator and a cell support matrix at a first ratio of cell support matrix to TIL (a "first REP step"); and (iv) culturing the expanded population of selected TILs without the population of de-selected TILs in a second expansion medium, wherein the second expansion medium comprises a T cell activator and a cell support matrix at a second ratio of cell support matrix to TIL (a "second REP step").

[0324] In some aspects, the method comprises (i) dissociating a tumor sample obtained from the subject comprising TILs to generate a single cell suspension comprising a heterogeneous population of TILs; (ii) culturing the heterogeneous population of TILs in an initial expansion medium disclosed herein; (iii) removing or de-selecting TILs that express CD127 ("population of de-selected TILs") from the heterogeneous population of TILs; (iv) culturing the population of TILs without the population of de-selected TILs in a first expansion medium, wherein the first expansion medium comprises a T cell activator and a cell support matrix at a first ratio of cell support matrix to TIL (a "first REP step"); and (v) culturing the expanded population of TILs without the population of de-selected TILs in a second expansion medium, wherein the second expansion medium comprises a T cell activator and a cell support matrix at a second ratio of cell support matrix to TIL (a "second REP step").

[0325] In some aspects, the method comprises (i) dissociating a tumor sample obtained from the subject comprising TILs to generate a single cell suspension comprising a heterogeneous population of TILs; (ii) removing or de-selecting TILs that express CD127 ("population of deselected TILs") from the heterogeneous population of TILs; (iii) culturing the population of selected TILs in an initial expansion medium disclosed herein; (iv) culturing the population of selected TILs in a first expansion medium, wherein the first expansion medium comprises a T cell activator and a cell support matrix at a first ratio of cell support matrix to TIL (a "first REP step"); and (v) culturing the expanded population of selected TILs in a second expansion medium, whereinthe second expansion medium comprises a T cell activator and a cell support matrix at a second ratio of cell support matrix to TIL (a "second REP step").ILA.4.L Cytokines

[0326] In some aspects, the second expansion medium comprises IL-2. In some aspects, the second expansion medium 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 second expansion medium comprises at least about 50 ng / mL IL-2. In some aspects, the second expansion medium comprises at least about 60 ng / mL IL-2. In some aspects, the second expansion medium comprises at least about 70 ng / mL IL-2. In some aspects, the second expansion medium comprises at least about 73.6 ng / mLIL-2. In some aspects, the second expansion medium comprises at least about 75 ng / mL IL-2. In some aspects, the second expansion medium comprises at least about 80 ng / mL IL-2. In some aspects, the second expansion medium comprises at least about 90 ng / mL IL-2. In some aspects, the second expansion medium comprises at least about 100 ng / mL IL-2. In some aspects, the second expansion medium comprises at least about 200 ng / mL IL-2. In some aspects, the second expansion medium comprises at least about 300 ng / mL IL-2, In some aspects, the second expansion medium comprises at least about 400 ng / mL IL-2, In some aspects, the second expansion medium comprises at least about 500 ng / mL IL-2. In some aspects, the second expansion medium comprises at least about 600 ng / mL IL-2.

[0327] In some aspects, the second expansion medium comprises at least about 500 lU / mL IL-2. In some aspects, the second expansion medium comprises from about 500 lU / mL to about 12,000 lU / mL IL-2. In some aspects, the second expansion medium comprises at least about 500 lU / mL, at least about 600 lU / mL, at least about 700 lU / mL, at least about 800 lU / mL, at least about 900 lU / mL, at least about 1000 lU / mL, at least about 21100 lU / mL, at least about 1200 lU / mL, at least about 1300 lU / mL, at least about 1400 lU / mL, at least about 1500 lU / mL, at least about 1600 lU / mL, at least about 1700 lU / mL, at least about 1800 lU / mL, at least about 1900 lU / mL, at least about 2000 lU / mL, at least about 2100 lU / mL, at least about 2200 lU / mL, at least about 2300 lU / mL, at least about 2400 lU / mL, at least about 2500 lU / mL, at least about 2600 lU / mL, at least about 2700 lU / mL, at least about 2800 lU / mL, at least about 2900 lU / mL, at least about 3000 lU / mL, at least about 3100 lU / mL, at least about 3200 lU / mL, at least about 3300 lU / mL, at least about 3400 lU / mL, at least about 3500 lU / mL, at least about 3600 lU / mL, at least about 3700 lU / mL, at least about 3800 lU / mL, at least about 3900 lU / mL, or at least about 1000 lU / mL. In some aspects, the second expansion medium comprises at least about 1100 lU / mL IL-2. In some aspects, the second expansion medium comprises at least about 1200 lU / mL IL-2. In some aspects, the second expansion medium comprises at least about 1300 lU / mL IL-2. In some aspects, the second expansion medium comprises at least about 1400 lU / mL IL-2. In some aspects, the second expansion medium comprises at least about 1500 lU / mL IL-2. In some aspects, the second expansion medium comprises at least about 1600 lU / mL IL-2. In some aspects, the second expansion medium comprises at least about 1700 lU / mL IL-2. In some aspects, the second expansion medium comprises at least about 1800 lU / mL IL-2. In some aspects, the second expansion medium comprises at least about 1900 lU / mL IL-2. In some aspects, the second expansion medium comprises at least about 2000 lU / mL IL-2.

[0328] In some aspects, the second expansion medium comprises at least about 0.1 ng / mLIL-21. In some aspects, the second expansion medium comprises from about 0.1 ng / mL to about 50 ng / mL, about 1 ng / mL to about 50 ng / mL, about 1 ng / mL to about 40 ng / mL, about 1 ng / mL to about 35 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 20 ng / mL, about 5 ng / mL to about 15 ng / mL IL-21, about 5 ng / mL to about 10 ng / mL, about 10 ng / mL to about 20 ng / mL, or about 10 ng / mL to about 15 ng / mL.

[0329] In some aspects, the second expansion medium 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 leastabout 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-21. In some aspects, the second expansion medium comprises at least about 1.0 ng / mL IL-21. In some aspects, the second expansion medium comprises at least about 2.0 ng / mL IL-21. In some aspects, the second expansion medium comprises at least about 3.0 ng / mL IL-21. In some aspects, the second expansion medium comprises at least about 4.0 ng / mLIL-21. In some aspects, the second expansion medium comprises at least about 5.0 ng / mL IL-21. In some aspects, the second expansion medium comprises at least about 6.0 ng / mL IL-21. In some aspects, the second expansion medium comprises at least about 7.0 ng / mL IL-21. In some aspects, the second expansion medium comprises at least about 8.0 ng / mL IL-21. In some aspects, the second expansion medium comprises at least about 9.0 ng / mL IL-21. In some aspects, the second expansion medium comprises at least about 10 ng / mL IL-21, In some aspects, the second expansion medium comprises at least about 11 ng / mL IL-21, In some aspects, the second expansion medium comprises at least about 12 ng / mL IL-21. In some aspects, the second expansion medium comprises at least about 13 ng / mL IL-21. In some aspects, the second expansion medium comprises at least about 14 ng / mL IL-21. In some aspects, the second expansion medium comprises at least about 15 ng / mL IL-21.

[0330] In some aspects, the second expansion medium comprises between about 10 lU / mL to about 500 lU / mL of IL-21. In some aspects, the second expansion medium comprises about 10 lU / mL, about 20 lU / mL, about 30 lU / mL, about 40 lU / mL, about 50 HJ / mL, about 60 lU / mL, about 70 lU / mL, about 80 lU / mL, about 90 lU / mL, about 100 lU / mL, about 125 lU / mL, about 150 lU / mL, about 175 lU / mL, about 200 lU / mL, about 225 lU / mL, about 250 lU / mL, about 275 lU / mL, about 300 lU / mL, about 350 lU / mL, about 400 lU / mL, about 450 lU / mL, or about 500 lU / mL of IL-21.

[0331] In some aspects, the second expansion medium does not comprise IL-15. In some aspects, the second expansion medium comprises IL-15. In some aspects, the second expansion medium comprises at least about 0.1 ng / mL IL-15. In some aspects, the second expansion medium comprises from about 0.1 ng / mL to about 20 ng / mL, about 0.1 ng / mL to about 10 ng / mL, about 0.1 ng / mL to about 5 ng / mL, or about 0.1 ng / mL to about 1 ng / mL IL-15.

[0332] In some aspects, the second expansion medium 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 about0.9 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, or at least about 10 ng / mL IL-15. In some aspects, the second expansion medium comprises at least about 0.1 ng / mL IL-15. In some aspects, the second expansion medium comprises at least about 0.2 ng / mL IL-15. In some aspects, the second expansion medium comprises at least about 0.3 ng / mL IL-15. In some aspects, the second expansion medium comprises at least about 0.4 ng / mL IL-15. In some aspects, the second expansion medium comprises at least about 0.5 ng / mL IL-15. In some aspects, the second expansion medium comprises at least about 0.6 ng / mL IL-15. In some aspects, the second expansion medium comprises at least about 0.7 ng / mL IL-15. In some aspects, the second expansion medium comprises at least about 0.8 ng / mL IL-15. In some aspects, the second expansion medium comprises at least about 0.9 ng / mL IL-15. In some aspects, the second expansion medium comprises at least about 1.0 ng / mL IL-15.

[0333] In some aspects, the second expansion medium comprises between about 50 lU / mL to about 500 lU / mL of IL-15. In some aspects, the second expansion medium comprises about 50 lU / mL, about 60 lU / mL, about 70 HJ / mL, about 80 lU / mL, about 90 HJ / mL, about 100 lU / mL, about 125 lU / mL, about 150 lU / mL, about 175 lU / mL, about 200 lU / mL, about 225 lU / mL, about 250 lU / mL, about 275 lU / mL, about 300 lU / mL, about 350 lU / mL, about 400 lU / mL, about 450 lU / mL, or about 500 lU / mL of IL-15.

[0334] In some aspects, the second expansion medium does not comprise IL-7. In some aspects, the second expansion medium comprises IL-7. In some aspects, the second expansion medium comprises at least about 0.1 ng / mL IL-7. In some aspects, the second expansion medium 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.

[0335] In some aspects, the second expansion medium 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 second expansion medium comprises at least about 2.0 ng / mL IL-7. In some aspects, the second expansion medium comprises at least about 2.3 ng / mL IL-7. In some aspects, the second expansion medium comprises at least about 2.5 ng / mL IL-7. In some aspects, the second expansion medium comprises at least about 2.7 ng / mL IL-7. In some aspects, the second expansion medium comprises at least about 3.0 ng / mL IL-7. In some aspects, the second expansion medium comprises at least about 3.3 ng / mL IL-7. In some aspects, the second expansion medium comprises at least about 3.5 ng / mL IL-7. In some aspects, the second expansion medium comprises at least about 3.7 ng / mL IL-7.

[0336] In some aspects, the second expansion medium comprises between about 500 lU / mL to about 1,500 lU / mL of IL-7. In some aspects, the second expansion medium comprises about 500 lU / mL, about 550 lU / mL, about 600 lU / mL, about 650 lU / mL, about 700 lU / mL, about 750 lU / mL, about 800 lU / mL, about 850 lU / mL, about 900 lU / mL, about 950 lU / mL, about 1,000 lU / mL, about 1,050 lU / mL, about 1,100 lU / mL, about 1,150 lU / mL, about 1,200 lU / mL, about 1,250 lU / mL, about 1,300 lU / mL, about 1,350 lU / mL, about 1,400 lU / mL, about 1,450 lU / mL, or about 1,500 lU / mL of IL-7.

[0337] In some aspects, the second expansion medium comprises about 1500 lU / mL IL-2 and about 10 ng / M IL-21. In some aspects, the second expansion medium comprises about 1500 lU / mL IL-2 and about 0.4 ng / M IL-15. In some aspects, the second expansion medium comprises about 1500 lU / mL IL-2, about 10 ng / M IL-21, and about 0.4 ng / mL IL-15.

[0338] In some aspects, the TILs are subjected to a second REP step using a Rapid Expansion Protocol (REP). See, e.g., Dudley, et al., Science 295:850-54 (2002); Dudley, et al., J. Clin. Oncol. 23:2346-57 (2005); Dudley, et al., J. Clin. Oncol. 26:5233-39 (2008); Riddell, et al., Science 257:238-41 (1992); and Dudley, et al., J. 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.

[0339] 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 10 ng / ml, about 11 ng / ml, about 12 ng / ml, about 13 ng / ml, about 14 ng / ml, about 15 ng / ml, about16 ng / ml, about 17 ng / ml, about 18 ng / ml, about 19 ng / ml, about 20 ng / ml, about 21 ng / ml, about22 ng / ml, about 23 ng / ml, about 24 ng / ml, about 25 ng / ml, about 26 ng / ml, about 27 ng / ml, about28 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 IL-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.

[0340] 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.

[0341] 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 gpl00:209-217 (210M)), in the presence of a T-cell growth factor, such as around 200-400 lU / ml of a T-cell growth factor, such as 300 lU / ml IL-2 or IL-15. In some aspects, TILs are expanded by stimulation using TRANSACT™. In some aspects, the in vztro-induced TILs are rapidly expanded by stimulationwith the same antigen(s) of the cancer pulsed onto HLA-A2-expressing antigen-presenting cells. In some aspects, the TILs can be stimulated with irradiated, autologous lymphocytes or with irradiated HLA-A2+ allogeneic lymphocytes and IL-2.

[0342] In some aspects, the TILs are stimulated during the second expansion by culturing the cells in a medium comprising TRANSACT™ 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 TRANSACT™, 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 TRANSACT™, at least about 1 pg / ml 4-1BBL, and at least about 5 pg / ml CD27L.

[0343] In some aspects, one or more TILs are genetically modified before, during, or after TIL expansion. Genetic modification of the TILs 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 OX40L. 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-PDl antibody.

[0344] 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.

[0345] In some aspects, the TILs are expanded in a culture medium that further comprises a 4- IBB agonist. Any 4- IBB agonist can be used in the methods disclosed herein. In some aspects,the 4- IBB agonist comprises a 4- IBB antibody. Non-limiting examples of 4- IBB 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- IBB agonist is added during the second or final expansion.

[0346] In some aspects, the TILs are stimulated during the second expansion by culturing the cells in a medium comprising TRANSACT™ 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 TRANSACT™, 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 TRANSACT™, at least about 1 pg / ml 4-1BBL, and at least about 5 pg / ml CD27L.

[0347] 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 7MMG-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.

[0348] 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. Nonlimiting 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.

[0349] 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.

[0350] 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, OC40L, 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.

[0351] In some aspects, the TILs are cultured in a secondary TIL media until cell yield in the secondary expansion reaches at least about IxlO7to at least about 50xl07, at least about 2xl07to at least about 40x107, at least about 3xl07to at least about 30xl07, at least about 4xl07to at least about 25xl07, at least about 5xl07to at least about 20xl07, at least about IxlO7to at least about 20x107, at least about 2xl07to at least about 20xl07, at least about 3xl07to at least about 20x107, or at least about 4xl07to at least about 20xl07cells. In some aspects, the TILs are cultured in a secondary TIL media until cell yield in the secondary expansion reaches at least about 5xl07to at least about 20xl07cells. In some aspects, the TILs are cultured in a secondary TIL media until cell yield in the secondary expansion reaches at least about IxlO7, at least about 2xl07, at least about 3xl07, at least about 4xl07, at least about 5xl07, at least about 6xl07, at least about 7xl07, at least about 8xl07, at least about 9xl07, at least about 10xl07, at least about 1 IxlO7, at least about 12xl07, at least about 13xl07, at least about 14xl07, at least about 15xl07, at least about 16xl07, at least about 17xl07, at least about 18xl07, at least about 19xl07, or at least about 20xl07cells. In some aspects, the TILs are cultured in a secondary TIL media until cell yield in the secondary expansion reaches at least about 5xl07cells. In some aspects, the TILs are cultured in a secondary TIL media until cell yield in the secondary expansion reaches at least about 6xl07cells. In some aspects, the TILs are cultured in a secondary TIL media until cell yield in the secondary expansion reaches at least about 7xl07cells. In some aspects, the TILs are cultured in a secondary expansion until cellyield in the secondary TIL media reaches at least about 8xl07cells. In some aspects, the TILs are cultured in a secondary expansion until cell yield in the secondary TIL media reaches at least about 9xl07cells. In some aspects, the TILs are cultured in a secondary TIL media until cell yield in the secondary expansion reaches at least about 10xl07cells. In some aspects, the TILs are cultured in a secondary TIL media until cell yield in the secondary expansion reaches at least about 15xl07cells. In some aspects, the TILs are cultured in a secondary TIL media until cell yield in the secondary expansion reaches at least about 20xl07cells.

[0352] In some aspects, TILs 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 corresp...

Claims

WHAT IS CLAIMED IS:

1. A method of culturing tumor infiltrating lymphocytes (TILs) ex vivo or in vitro for a therapy to a subject in need thereof comprising:(i) dissociating a tumor sample obtained from the subject comprising TILs to generate a single cell suspension;(ii) culturing the single cell suspension in a first expansion medium, wherein the first expansion medium comprises a CD3 agonist and APCs at a first ratio (a "first REP step"); and(iii) culturing the TILs from the first REP step in a second expansion medium, wherein the second expansion medium comprises a CD3 agonist and APCs at a second ratio (a "second REP step").

2. A method of culturing TILs ex vivo or in vitro for a therapy in a subject in need thereof comprising:(i) culturing a dissociated tumor sample comprising TILs in a first expansion medium, wherein the first expansion medium comprises a CD3 agonist and APCs at a first ratio (a "first REP step"); and(ii) culturing the TILs from the first REP step in a second expansion medium, wherein the second expansion medium comprises a CD3 agonist, and APCs at a second ratio (a "second REP step").

3. The method of claim 2, wherein the dissociated tumor sample is a single cell suspension.

4. A method of culturing TILs ex vivo or in vitro for a therapy in a subject in need thereof comprising culturing a population of TILs obtained from a tumor sample of the subject in an expansion medium comprising a CD3 agonist and antigen-presenting cells (APCs); wherein, prior to or after culturing in the expansion medium, the TILs are not cultured in a medium that does not comprising APCs.

5. The method of claim 4, wherein prior to culturing in the expansion medium, the population of TILs is cultured in a medium comprising APCs.

6. The method of claim 4, wherein after culturing in the expansion medium, the population ofTILs is cultured in a medium comprising APCs.

7. The method of any one of claims 4 to 6, wherein the TILs are cultured in the expansion medium for less than 11 days.

8. The method of any one of claims 4 to 6, wherein the TILs are cultured in the expansion medium for less than 10 days.

9. The method of any one of claims 4 to 6, wherein the TILs are cultured in the expansion medium for less than 9 days.

10. The method of any one of claims 4 to 9, wherein prior to culturing in the expansion medium, the population of TILs is cultured for less than 11 days in the medium comprising APCs.

11. The method of any one of claims 4 to 9, wherein prior to culture in the expansion medium, the population of TILs is cultured for less than 10 days in the medium comprising APCs.

12. The method of any one of claims 4 to 9, wherein prior to culture in the expansion medium, the population of TILs is cultured for less than 9 days in the medium comprising APCs.

13. The method of any one of claims 4 to 9, wherein prior to culture in the expansion medium, the population of TILs is cultured for less than 8 days in the medium comprising APCs.

14. The method of any one of claims 4 to 13, wherein the population of TILs is obtained from a dissociated tumor sample.

15. The method of any one of claims 1 to 3, wherein the TILs are cultured in the first REP step for less than 11 days.

16. The method of any one of claims 1 to 3, wherein the TILs are cultured in the first REP step for less than 10 days.

17. The method of any one of claims 1 to 3, wherein the TILs are cultured in the first REP step for less than 9 days.

18. The method of any one of claims 1 to 3, wherein the TILs are cultured in the first REP step for less than 8 days.

19. The method of any one of claims 1 to 3 and 15 to 18, wherein the number of TILs present in the dissociated tumor sample is increased by 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 following culture in the first REP step.

20. The method of any one of claims 1 to 3 and 15 to 19, wherein the number of TILs present in the dissociated tumor sample is increased by at least about 20-fold following culture in the first REP step.

21. The method of any one of claims 1 to 3 and 15 to 20, wherein the number of TILs following culture in the first REP step is at least about 5 x 107, at least about 1 x 108, at least about 2 x 108, at least about 3 x 108, at least about 4 x 108, at least about 5 x 108, at least about 6 x108, at least about 7 x 108, at least about 8 x 108, at least about 9 x 108, at least about 1 x109, at least about 2 x 109, at least about 3 x 109, at least about 4 x 109, at least about 5 x109, at least about 6 x 109, at least about 7 x 109, at least about 8 x 109, at least about 9 x109, at least about 1 x IO10, or at least about 2 x IO10TILs.

22. The method of any one of claims 1 to 3 and 15 to 21, wherein the TILs are cultured in the second REP step for less than 11 days.

23. The method of any one of claims 1 to 3 and 15 to 21, wherein the TILs are cultured in the second REP step for less than 10 days.

24. The method of any one of claims 1 to 3 and 15 to 21, wherein the TILs are cultured in the second REP step for less than 9 days.

25. The method of any one of claims 1 to 3 and 15 to 21, wherein the TILs are cultured in the second REP step for less than 8 days.

26. The method of any one of claims 1 to 3 and 15 to 25, wherein the number of TILs present following the first REP step is increased by 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 following culture in the second REP step.

27. The method of any one of claims 1 to 3 and 15 to 25, wherein the number of TILs present following the first REP step is increased by at least about 20-fold following culture in the second REP step.

28. The method of any one of claims 1 to 3 and 15 to 27, wherein the number of TILs following culture in the second REP step is 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 IO10, at least about 2 x IO10, at least about 3 x IO10, at least about 4 x IO10, at least about 5 x IO10, at least about 6 x IO10, at least about 7 x IO10, at least about 8 x IO10, at least about 9 x IO10, or at least about 1 x 1011TILs.

29. The method of any one of claims 1 to 3 and 15 to 28, wherein the TILs are cultured in the first REP step for 7 to 10 days, and the TILs are cultured in the second REP step for 7 to 10 days, wherein the total number of TILs following culture in the second REP step is at least about 1 x 109TILs.

30. The method of any one of claims 1 to 3 and 15 to 28, wherein the TILs are cultured in the first REP step for 7 days, and the TILs are cultured in the second REP step for 7 days, wherein the total number of TILs following culture in the second REP step is at least about 1 x 109TILs.

31. The method of any one of claims 1 to 3 and 15 to 28, wherein the TILs are cultured in the first REP step for 10 days or less, and the TILs are cultured in the second REP step for 10 days or less, wherein the total number of TILs following culture in the second REP step is at least about 1 x 109TILs.

32. The method of any one of claims 1 to 3 and 15 to 31, wherein the first expansion medium comprises a dissociated tumor cell to APC ratio of about 1 :5, about 1 :6, about 1 :7, about 1 :8, about 1 :9, about 1 : 10, about 1 :11, about 1 : 12, about 1 : 13, about 1 : 14, about 1 :15, about 1 : 16, about 1 :17, about 1 : 18, about 1 : 19, about 1 :20, about 1 :25, about 1 :30, about 1 :35, about 1 :40, about 1 :45, or about 1 :50.

33. The method of any one of claims 1 to 3 and 15 to 32, wherein the first expansion medium comprises a dissociated tumor cell to APC ratio of about 1 : 10.

34. The method of any one of claims 1 to 3 and 15 to 33, wherein the second expansion medium comprises a dissociated tumor cell to APC ratio of about 1:5, about 1:6, about 1:7, about 1:8, about 1:9, about 1:10, about 1:11, about 1:12, about 1:13, about 1:14, about 1:15, about 1:16, about 1:17, about 1:18, about 1:19, about 1:20, about 1:25, about 1:30, about 1:35, about 1:40, about 1:45, or about 1:50.

35. The method of any one of claims 1 to 3 and 15 to 33, wherein the second expansion medium comprises a first REP expanded TIL to APC ratio of about 1:1, about 1 :2, about 1 :3, about 1:4, about 1:5, about 1:6, about 1:7, about 1:8, about 1:9, or about 1:10.

36. The method of any one of claims 1 to 3 and 15 to 33, wherein the second expansion medium comprises a first REP expanded TIL to APC ratio of about 1 :3.

37. The method of any one of claims 1 to 3 and 15 to 33, wherein the second expansion medium comprises a first REP expanded TIL to APC ratio of about 1 :4.

38. The method of any one of claims 1 to 3 and 15 to 33, wherein the second expansion medium comprises a first REP expanded TIL to APC ratio of about 1:5.

39. The method of any one of claims 1 to 3 and 15 to 33, wherein the first expansion medium comprises a dissociated tumor cell to APC ratio of about 1:10, and wherein the second expansion medium comprises a first REP expanded TIL to APC ratio of about 1 :3.

40. The method of any one of claims 1 to 3 and 15 to 33, wherein the first expansion medium comprises a dissociated tumor cell to APC ratio of about 1:10, and wherein the second expansion medium comprises a first REP expanded TIL to APC ratio of about 1 :4.

41. The method of any one of claims 1 to 3 and 15 to 33, wherein the first expansion medium comprises a dissociated tumor cell to APC ratio of about 1:10, and wherein the second expansion medium comprises a first REP expanded TIL to APC ratio of about 1:5.

42. The method of any one of claims 1 to 41, wherein the subject is previously received one or more therapy for treating the tumor.

43. The method of claim 42, wherein the subject is relapsed or refractory to one or more prior therapy for treating the tumor.

44. The method of claim 42 or 43, wherein the one or more therapy for treating the tumor is selected from a chemotherapy, an immunotherapy, a radiotherapy, a surgery, or any combination thereof.

45. The method of any one of claims 42 to 44, wherein the tumor is refractory to a checkpoint inhibitor.

46. The method of claim 45, wherein 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.

47. The method of claim 45, wherein the checkpoint inhibitor comprises an anti-PDl 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.

48. The method of any one of claims 1 to 47, wherein the tumor is metastatic.

49. The method of any one of claims 1 to 48, wherein the tumor comprises a solid tumor.

50. The method of claim 49, wherein the solid tumor is 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.

51. The method of any one of claims 1 to 3 and 15 to 50, wherein the first expansion medium, the second expansion medium, or both comprises potassium ion.

52. The method of claim 51 wherein the first expansion medium, the second expansion medium, or both comprises potassium ion higher than 4 mM.

53. The method of any one of claims 1 to 3 and 15 to 52, wherein the first expansion medium, the second expansion medium, or both further comprises sodium ion, calcium ion, glucose, or any combination thereof.

54. The method of any one of claims 1 to 3 and 15 to 53, wherein the first expansion medium, the second expansion medium, or both further comprises a cell expansion agent.

55. The method of claim 54, wherein the cell expansion agent comprises a GSK3B inhibitor, an ACLY inhibitor, a PI3K inhibitor, an AKT inhibitor, or any combination thereof.

56. The method of claim 55, wherein the PI3K inhibitor comprises LY294002, pictilisib, CAL101, IC87114, or any combination thereof.

57. The method of claim 56, wherein the AKT inhibitor comprises MK2206, A443654, AKTi- VIII, or any combination thereof.

58. The method of any one of claims 51 to 57, 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.

59. The method of any one of claims 51 to 58, 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 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.

60. The method of any one of claims 51 to 59, wherein the concentration of potassium ion is about 40 mM to about 90 mM.

61. The method of any one of claims 51 to 60, wherein the concentration of potassium ion is about 50 mM to about 90 mM.

62. The method of any one of claims 51 to 61, wherein the concentration of potassium ion is about 50 mM to about 80 mM.

63. The method of any one of claims 1 to 3 and 15 to 62, wherein the first expansion medium, the second expansion medium, or both further comprises sodium ion.

64. The method of claim 63, wherein the concentration of the sodium ion is from about 25 mM to about 100 mM.

65. The method of claim 63 or 64, 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 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.

66. The method of any one of claims 63 to 65, 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.

67. The method of any one of claims 63 to 66, wherein the concentration of the sodium ion is about 55 mM.

68. The method of any one of claims 63 to 66, wherein the concentration of the sodium ion is about 60 mM.

69. The method of any one of claims 63 to 66, wherein the concentration of the sodium ion is about 65 mM.

70. The method of any one of claims 1 to 3 and 15 to 69, wherein the first expansion medium, the second expansion medium, or both further comprises glucose.

71. The method of claim 70, wherein the concentration of glucose is more than about 10 mM.

72. The method of claim 70 or 71, 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 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.

73. The method of any one of claims 70 to 72, wherein 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.

74. The method of any one of claims 1 to 3 and 15 to 73, wherein the first expansion medium, the second expansion medium, or both further comprises calcium ion.

75. The method of claim 74, wherein the concentration of calcium ion is more than about 0.4 mM.

76. The method of claim 74 or 75, 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 about1.6 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 about1.7 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.

77. The method of any one of claims 74 to 76, 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.

78. The method of any one of claims 1 to 3 and 15 to 77, wherein the first expansion medium, the second expansion medium, or both comprise 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 25 mM glucose;(iii) about 0.5 mM to about 2.8 mM calcium ion; or(iv) any combination of (i)-(iii).

79. The method of any one of claims 1 to 3 and 15 to 78, wherein the first expansion medium, the second expansion medium, or both further comprises IL2.

80. The method of claim 79, wherein the concentration of IL2 is from about 1000 lU / ml to at least about 2000 lU / ml.

81. The method of claim 79 or 80, wherein the concentration of IL2 is at least about 1000 lU / ml, at least about 1100 lU / ml, at least about 1200 lU / ml, at least about 1300 lU / ml, at least about 1400 lU / ml, at least about 1500 lU / ml, at least about 1600 lU / ml, at least about 1700 lU / ml, at least about 1800 lU / ml, at least about 1900 lU / ml, or at least about 2000 lU / ml.

82. The method of any one of claims 79 to 81, wherein the concentration of IL2 is about 1500 lU / ml.

83. The method of any one of claims 1 to 3 or 15 to 82, wherein the first expansion medium, the second expansion medium, or both further comprises IL21.

84. The method of claim 83, wherein the concentration of IL21 is from about 1 ng / ml to at least about 100 ng / ml.

85. The method of claim 83 or 84, wherein the concentration of IL21 is 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 20 ng / ml, at least about 25 ng / ml, at least about 30 ng / ml, at least about 35 ng / ml, at least about 40 ng / ml, at least about 45 ng / ml, 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, or at least about 100 ng / ml.

86. The method of any one of claims 83 to 85, wherein the concentration of IL21 is about 10 ng / ml.

87. The method of any one of claims 1 to 3 or 15 to 86, wherein the first expansion medium, the second expansion medium, or both further comprises IL15.

88. The method of claim 87, wherein the concentration of IL15 is from about 0.1 ng / ml to at least about 10 ng / ml.

89. The method of claim 87 or 88, wherein the concentration of IL15 is 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 1.1 ng / ml, at least about 1.2 ng / ml, at least about 1.3 ng / ml, at least about 1.4 ng / ml, at least about 1.5 ng / ml, at least about 2.0 ng / ml, at least about 2.5 ng / ml, at least about 3.0 ng / ml, at least about 3.5 ng / ml, at least about 4.0 ng / ml, at least about 4.5 ng / ml, at least about 5.0 ng / ml, at least about 6.0 ng / ml, at least about 7.0 ng / ml, at least about 8.0 ng / ml, at least about 9.0 ng / ml, or at least about 10.0 ng / ml.

90. The method of any one of claims 87 to 89, wherein the concentration of IL15 is about 0.4 ng / ml.

91. The method of any one of claims 1 to 3 or 15 to 90, wherein first expansion medium, the second expansion medium, or both further comprises IL7.

92. The method of any one of claims 1 to 91, wherein the CD3 agonist comprises OKT3.

93. The method of claim 92, wherein the concentration of OKT3 is from about 1 to about 100 ng / ml.

94. The method of claim 92 or 93, wherein the concentration of OKT3 is at least about 1 ng / ml, at least about 5 ng / ml, at least about 10 ng / ml, at least about 15 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, at least about 40 ng / ml, at least about 45 ng / ml, 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, or at least about 100 ng / ml.

95. The method of any one of claims 92 to 94, wherein the concentration of OKT3 is about 20 ng / ml.

96. A population of TILs expanded according to the method of any one of claims 1 to 95.

97. A method of treating a cancer in a subject in need thereof, comprising administering the population of expanded TILs of claim 96.

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 LAG3 antagonist, or any combination thereof.

100. The method of claim 98, wherein the checkpoint inhibitor comprises an anti-PDl 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 1 to 3 and 15 to 95, wherein the second REP step comprises a first phase followed by a second phase, wherein the first phase of the second REP step comprises a first phase medium and the second phase of the second REP step comprises a second phase medium, and wherein the first phase medium and the second phase medium are not the same.

104. The method of claim 103, wherein the first phase medium comprises about 45 mM potassium ion to about 90 mM potassium ion.

105. The method of claim 103 or 104, wherein the first phase medium comprises 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 potassium ion.

106. The method of any one of claims 103 to 104, wherein fresh medium is added to the TILs and the first phase medium to generate the second phase medium.

107. The method of claim 105, wherein the first phase medium is diluted by the fresh medium at a ratio of 1 : 1, 1 :2, 1 :3, 1 :4, 1 :5, 2: 1, 3: 1, or 4: 1 (first phase mediunrfresh medium).

108. The method of claim 105 or 106, wherein the first phase medium is diluted by the fresh medium at a ratio of 1 : 1 (first phase mediunrfresh medium).

109. The method of claim 105 or 106, wherein the first phase medium is diluted by the fresh medium at a ratio of 1 :2 (first phase mediunrfresh medium).

110. The method of claim 105 or 106, wherein the first phase medium is diluted by the fresh medium at a ratio of 1 :3 (first phase mediunrfresh medium).

111. The method of claim 105, wherein the first phase medium is diluted by the fresh medium to a final concentration of about 75%, about 70%, about 65%, about 60%, about 55%, about 50%, about 45%, about 40%, about 35%, about 30%, about 25%, about 20%, about 15%, about 10%, or about 5% first phase medium.

112. The method of claim 105, wherein the first phase medium is diluted by the fresh medium to a final concentration of about 50% first phase medium.

113. The method of claim 105, wherein the first phase medium is diluted by the fresh medium to a final concentration of about 40% first phase medium.

114. The method of claim 105, wherein the first phase medium is diluted by the fresh medium to a final concentration of about 30% first phase medium.

115. The method of claim 105, wherein the first phase medium is diluted by the fresh medium to a final concentration of about 25% first phase medium.

116. The method of claim 105, wherein the first phase medium is diluted by the fresh medium to a final concentration of about 20% first phase medium.

117. The method of claim 105, wherein the first phase medium is diluted by the fresh medium to a final concentration of about 15% first phase medium.

118. The method of claim 105, wherein the first phase medium is diluted by the fresh medium to a final concentration of about 10% first phase medium.

119. The method of claim 105, wherein the first phase medium is diluted by the fresh medium to a final concentration of about 5% first phase medium.

120. The method of claim 105, wherein the first phase medium is diluted by the fresh medium to a final concentration of less than about 5% first phase medium.

121. The method of any one of claims 105 to 119, wherein the concentration of potassium ion in the second phase medium is about 75%, about 70%, about 65%, about 60%, about 55%,about 50%, about 45%, about 40%, about 35%, about 30%, about 25%, about 20%, about 15%, about 10%, about 5%, or less than about 5% the concentration of potassium in the first phase medium.

122. The method of claim 120, wherein the concentration of potassium ion in the second phase medium is about 50% the concentration of potassium in the first phase medium.

123. The method of claim 120, wherein the concentration of potassium ion in the second phase medium is about 40% the concentration of potassium in the first phase medium.

124. The method of claim 120, wherein the concentration of potassium ion in the second phase medium is about 30% the concentration of potassium in the first phase medium.

125. The method of claim 120, wherein the concentration of potassium ion in the second phase medium is about 25% the concentration of potassium in the first phase medium.

126. The method of claim 120, wherein the concentration of potassium ion in the second phase medium is about 20% the concentration of potassium in the first phase medium.

127. The method of claim 120, wherein the concentration of potassium ion in the second phase medium is about 15% the concentration of potassium in the first phase medium.

128. The method of claim 120, wherein the concentration of potassium ion in the second phase medium is about 10% the concentration of potassium in the first phase medium.

129. The method of claim 120, wherein the concentration of potassium ion in the second phase medium is about 5% the concentration of potassium in the first phase medium.

130. The method of claim 120, wherein the concentration of potassium ion in the second phase medium is about 5% the concentration of potassium in the first phase medium.

131. The method of any one of claims 103 to 129, wherein the first phase of the second REP comprises culturing the TILs in a first container, and the second phase of the second REP comprises culturing the TILs in a second container, wherein the second container comprises a bioreactor.

132. The method of claims 130, wherein the first container does not comprise a bioreactor.

133. The method of any one of claims 103 to 131, wherein the duration of the first phase of the second REP step is about 1 day to about 5 days.

134. The method of any one of claims 103 to 132, wherein the duration of the first phase of the second REP step is about 1 day, about 2 days, about 3 days, about 4 days, or about 5 days.

135. The method of any one of claims 103 to 133, wherein the duration of the second phase of the second REP step is about 3 days to about 13 days.

136. The method of any one of claims 103 to 134, wherein the duration of the second phase of the second REP step is about 3 days, about 4 days, about 5 days, about 6 days, about 7 days, about 8 days, about 9 days, about 10 days, about 11 days, about 12 days, or about 13 days.

137. The method of any one of claims 103 to 135, wherein the duration of the first phase of the second REP step is about 3 days, and the duration of the second phase of the second REP step is about 7 days.

138. The method of any one of claims 103 to 135, wherein the duration of the first phase of the second REP step is about 3 days, and the duration of the second phase of the second REP step is about 8 days.

139. The method of any one of claims 103 to 135, wherein the duration of the first phase of the second REP step is about 3 days, and the duration of the second phase of the second REP step is about 9 days.

140. The method of any one of claims 103 to 135, wherein the duration of the first phase of the second REP step is about 4 days, and the duration of the second phase of the second REP step is about 6 days.

141. The method of any one of claims 103 to 135, wherein the duration of the first phase of the second REP step is about 4 days, and the duration of the second phase of the second REP step is about 7 days.

142. The method of any one of claims 103 to 135, wherein the duration of the first phase of the second REP step is about 4 days, and the duration of the second phase of the second REP step is about 8 days.

143. The method of any one of claims 103 to 135, wherein the duration of the first phase of the second REP step is about 4 days, and the duration of the second phase of the second REP step is about 9 days.

144. The method of any one of claims 1 to 3 and 15 to 95, wherein the second REP step comprises a static-REP step followed by dynamic-REP step, wherein the static-REP step comprises culturing the TILs in a medium comprising greater than 4 mM potassium ion, a CD3 agonist, and antigen-presenting cells; and wherein the dynamic-REP step comprises adding the TILs from the static-REP step to fresh medium, wherein the fresh medium does not comprise a CD3 agonist or antigen-presenting cells, and wherein agitation is applied to the culture.

145. The method of claim 143, wherein the dynamic-REP step comprises inoculating the contents of the static-REP into a rocking motion bioreactor, wherein the rocking motion bioreactor comprises fresh medium.

146. The method of claim 143 or 144, wherein the medium of the static-REP comprises about 45 mM potassium ion to about 90 mM potassium ion.

147. The method of claim 143 to 145, wherein the medium of the static-REP comprises 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 potassium ion.

148. The method of any one of claims 143 to 146, wherein the medium of the static-REP is diluted by the fresh medium of the dynamic-REP step at a ratio of 1 : 1, 1 :2, 1 :3, 1 :4, 1 :5, 2: 1, 3: 1, or 4: 1 (static-REP: fresh medium).

149. The method of any one of claims 143 to 146, wherein the medium of the static-REP is diluted by the fresh medium of the dynamic-REP step at a ratio of 1 : 1 (static-REP:fresh medium).

150. The method of any one of claims 143 to 146, wherein the medium of the static-REP is diluted by the fresh medium of the dynamic-REP step at a ratio of 1 :2 (static-REP:fresh medium).

151. The method of any one of claims 143 to 146, wherein the medium of the static-REP is diluted by the fresh medium of the dynamic-REP step at a ratio of 1 :3 (static-REP:fresh medium).

152. The method of any one of claims 143 to 146, wherein the medium of the static-REP is diluted by the fresh medium to a final concentration of about 75%, about 70%, about 65%,about 60%, about 55%, about 50%, about 45%, about 40%, about 35%, about 30%, about25%, about 20%, about 15%, about 10%, or about 5% static-REP medium.

153. The method of claim 151, wherein the medium of the static-REP is diluted by the fresh medium to a final concentration of about 50% first phase medium.

154. The method of claim 151, wherein the medium of the static-REP is diluted by the fresh medium to a final concentration of about 40% static-REP medium.

155. The method of claim 151, wherein the medium of the static-REP is diluted by the fresh medium to a final concentration of about 30% static-REP medium.

156. The method of claim 151, wherein the medium of the static-REP is diluted by the fresh medium to a final concentration of about 25% static-REP medium.

157. The method of claim 151, wherein the medium of the static-REP is diluted by the fresh medium to a final concentration of about 20% static-REP medium.

158. The method of claim 151, wherein the medium of the static-REP is diluted by the fresh medium to a final concentration of about 15% static-REP medium.

159. The method of claim 151, wherein the medium of the static-REP is diluted by the fresh medium to a final concentration of about 10% static-REP medium.

160. The method of claim 151, wherein the medium of the static-REP is diluted by the fresh medium to a final concentration of about 5% static-REP medium.

161. The method of claim 151, wherein the medium of the static-REP is diluted by the fresh medium to a final concentration of less than about 5% static-REP medium.

162. The method of any one of claims 151 to 160, wherein the concentration of potassium ion in the medium of the dynamic-REP is about 75%, about 70%, about 65%, about 60%, about 55%, about 50%, about 45%, about 40%, about 35%, about 30%, about 25%, about 20%, about 15%, about 10%, about 5%, or less than about 5% the concentration of potassium in the medium of the static-REP.

163. The method of claim 161, wherein the concentration of potassium ion in the medium of the dynamic-REP is about 50% the concentration of potassium in the medium of the static- REP.

164. The method of claim 161, wherein the concentration of potassium ion in the medium of the dynamic-REP is about 40% the concentration of potassium in the medium of the static- REP.

165. The method of claim 161, wherein the concentration of potassium ion in the medium of the dynamic-REP is about 30% the concentration of potassium in the medium of the static- REP.

166. The method of claim 161, wherein the concentration of potassium ion in the medium of the dynamic-REP is about 25% the concentration of potassium in the medium of the static- REP.

167. The method of claim 161, wherein the concentration of potassium ion in the medium of the dynamic-REP is about 20% the concentration of potassium in the medium of the static- REP.

168. The method of claim 161, wherein the concentration of potassium ion in the medium of the dynamic-REP is about 15% the concentration of potassium in the medium of the static- REP.

169. The method of claim 161, wherein the concentration of potassium ion in the medium of the dynamic-REP is about 10% the concentration of potassium in the medium of the static- REP.

170. The method of claim 161, wherein the concentration of potassium ion in the medium of the dynamic-REP is about 5% the concentration of potassium in the medium of the static-REP.

171. The method of claim 161, wherein the concentration of potassium ion in the medium of the dynamic-REP is less than about 5% the concentration of potassium in the medium of the static-REP.

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