Methods for producing tumor-infiltrating lymphocytes and their use in immunotherapy

Abstract

Methods for producing tumor-infiltrating lymphocytes and their use in immunotherapy are provided. The present invention provides improved and / or shortened methods for expanding TILs and producing therapeutic TIL populations, including novel methods for expanding TIL populations in a closed system that result in improved efficacy, improved phenotype, and increased metabolic health of TILs in a shorter period of time, while allowing for reduced microbial contamination and cost savings. Such TILs find use in therapeutic treatment regimens.

Description

[Technical Field]

[0001] CROSS-REFERENCE TO RELATED APPLICATIONS This application is a continuation of U.S. Provisional Patent Application No. 62 / 478,506, filed March 29, 2017, U.S. Provisional Patent Application No. 62 / 539,410, filed July 31, 2017, U.S. Provisional Patent Application No. 62 / 548,306, filed August 21, 2017, U.S. Provisional Patent Application No. 62 / 554,538, filed September 5, 2017, U.S. Provisional Patent Application No. 62 / 559,374, filed September 15, 2017, U.S. Provisional Patent Application No. 62 / 559,374, filed October 1, 2017, U.S. Provisional Patent Application No. 62 / 559,374, filed October 1, 2017, U.S. Provisional Patent Application No. 62 / 559,374, filed October 1, 2017, U.S. Provisional Patent Application No. 62 / 559,374, filed December ... This application claims priority to U.S. Provisional Patent Application No. 62 / 567,121, filed on November 2, 2017, U.S. Provisional Patent Application No. 62 / 577,655, filed on October 26, 2017, U.S. Provisional Patent Application No. 62 / 582,874, filed on November 7, 2017, and U.S. Provisional Patent Application No. 62 / 596,374, filed on December 8, 2017, which are hereby incorporated by reference in their entireties.

[0002] Sequence Listing

[0002] This application contains a Sequence Listing that has been submitted electronically in ASCII format and is hereby incorporated by reference in its entirety. Said ASCII copy was created on January 4, 2018, under the name 116983-5017_ST25.txt, and is 14 kilobytes in size. [Background technology]

[0003] Background of the Invention

[0003] The treatment of large, refractory cancers using adoptive transfer of tumor-infiltrating lymphocytes (TILs) represents a powerful therapeutic approach for patients with poor prognosis. Gattinoni, et al., Nat. Rev. Immunol. 2006, 6, 383-393. Successful immunotherapy requires large amounts of TILs, necessitating a robust and reliable method for commercialization. This has been challenging due to the technical, logistical, and regulatory challenges associated with cell expansion. IL-2-based TIL expansion followed by the "Rapid Expansion Process" (REP) is a promising approach. Due to its speed and efficiency, it is becoming the preferred method for TIL expansion. Dudley, et al., Science 2002, 298, 850-54; Dudley, et al., J. Clin. Oncol. 2005, 23, 2346-57; Dudley, et al., J. Clin. Oncol. 2008, 26, 5233-39; Riddell, et al., Science 1992, 257, 238-41; Dudley, et al., J. Immunother. 2003, 26, 332-42. REP can result in a 1,000-fold expansion of TILs in a 14-day period, but it requires a large excess (e.g., 200-fold) of irradiated allogeneic peripheral blood mononuclear cells (PBMCs, also known as mononuclear cells (MNCs)) as feeder cells, often from multiple donors, as well as anti-CD3 antibodies (OKT3) and high doses of IL-2. (Dudley, et al., J. Immunother.) 2003, 26, 332-42. Summary of the Invention [Problem to be solved by the invention]

[0004]

[0004] Current TIL manufacturing processes are limited by length, expense, sterility concerns, and other factors described herein, which significantly limits the potential for commercializing such processes, and for these and other reasons, no commercial processes are currently available. There is an urgent need to provide TIL manufacturing processes and therapies based on such processes that are suitable for commercial-scale manufacturing and regulatory approval for use in human patients in multiple clinical centers. [Means for solving the problem]

[0005]

[0005] The present invention provides improved and / or shortened methods for expanding TILs and producing therapeutic TIL populations.

[0006] The present invention also provides a method for expanding tumor-infiltrating lymphocytes (TILs) into a therapeutic TIL population, the method comprising: (a) obtaining a first population of TILs from a tumor excised from a patient by processing a tumor sample obtained from the patient into multiple tumor fragments; (b) adding tumor fragments to a closed system; (c) performing a first expansion culture by culturing the first TIL population in a cell culture medium containing IL-2 to generate a second TIL population, wherein the first expansion culture is performed in a closed vessel providing a first gas permeable surface area, the first expansion culture is performed for about 3 to 14 days to obtain a second TIL population, wherein the second TIL population is at least 50-fold more numerous than the first TIL population, and the transition from step (b) to step (c) occurs without opening the system; (d) performing a second expansion culture by adding additional IL-2, OKT-3, and antigen-presenting cells (APCs) to the cell culture medium of the second TIL population to generate a third TIL population, wherein the second expansion culture is performed for about 7 to 14 days to obtain the third TIL population, the third TIL population being a therapeutic TIL population, wherein the second expansion culture is performed in a closed vessel providing a second gas-permeable surface area, and wherein the transition from step (c) to step (d) occurs without opening the system; (e) recovering the therapeutic TIL population obtained from step (d), wherein the transition from step (d) to step (e) occurs without releasing the system; and (f) transferring the TIL population collected in step (e) into an infusion bag, wherein the transition from step (e) to (f) occurs without opening the system. Includes.

[0007]

[0007] In some embodiments, the method further comprises the step of cryopreserving the infusion bag containing the TIL population collected in step (f) using a cryopreservation process.

[0008]

[0008] In some embodiments, the cryopreservation process is performed using a 1:1 ratio of recovered TIL population to cryopreservation medium.

[0009] In some embodiments, the antigen-presenting cells are peripheral blood mononuclear cells (PBMCs). In some embodiments, the PBMCs are irradiated and allogeneic. In some embodiments, the PBMCs are added to the cell culture in step (d) on any of days 9 to 14. In some embodiments, the antigen-presenting cells are artificial antigen-presenting cells.

[0010] In some embodiments, the harvesting of step (e) is carried out using a membrane-based cell processing system.

[0011] In some embodiments, the harvesting of step (e) is carried out using a LOVO cell processing system.

[0012] In some embodiments, the plurality of fragments comprises about 4 to about 50 fragments, each fragment being about 27 mm 3 It has a volume of

[0013] In some embodiments, the plurality of segments is about 1300 mm 3 ~about 1500mm 3It contains about 30 to about 60 fragments with a total volume of .

[0014] In some embodiments, the plurality of segments is about 1350 mm 3 It contains approximately 50 fragments with a total volume of 1000 s.p.m.

[0015] In some embodiments, the plurality of fragments comprises about 50 fragments having a total mass of about 1 g to about 1.5 g.

[0016]

[0016] In some embodiments, the cell culture medium is provided in a container selected from the group consisting of a G container and a Xuri cell culture bag.

[0017] In some embodiments, the cell culture medium in step (d) further comprises IL-15 and / or IL-21.

[0018] In some embodiments, the IL-2 concentration is about 10,000 IU / mL to about 5,000 IU / mL.

[0019] In some embodiments, the IL-15 concentration is about 500 IU / mL to about 100 IU / mL.

[0020] In some embodiments, the IL-21 concentration is from about 20 IU / mL to about 0.5 IU / mL.

[0021] In some embodiments, the infusion bag of step (f) contains HypoThermosol. It is a transfusion bag.

[0022]

[0022] In some embodiments, the cryopreservation medium comprises dimethyl sulfoxide (DMSO). In some embodiments, the cryopreservation medium comprises 7% to 10% dimethyl sulfoxide (DMSO).

[0023] In some embodiments, the first period of step (c) and the second period of step (e) are each independently performed within a period of 10 days, 11 days, or 12 days.

[0024] In some embodiments, the first period of step (c) and the second period of step (e) are each performed separately within a period of 11 days.

[0025] In some embodiments, steps (a) through (f) are carried out within a period of about 10 days to about 22 days.

[0026] In some embodiments, steps (a) through (f) are carried out within a period of about 20 days to about 22 days.

[0027] In some embodiments, steps (a) through (f) are carried out within a period of about 15 days to about 20 days.

[0028] In some embodiments, steps (a) through (f) are carried out within a period of about 10 days to about 20 days.

[0029] In some embodiments, steps (a) through (f) are carried out within a period of about 10 days to about 15 days.

[0030] In some embodiments, steps (a) through (f) are performed within 22 days.

[0031] In some embodiments, steps (a) through (f) are performed within 20 days.

[0032] In some embodiments, steps (a) through (f) are performed within 15 days.

[0033] In some embodiments, steps (a) through (f) are performed within 10 days.

[0034] In some embodiments, steps (a)-(f) and cryopreservation are performed within 22 days.

[0035]

[0035] In some embodiments, the therapeutic TIL population collected in step (e) comprises sufficient TILs to provide a therapeutically effective dosage of TILs.

[0036] In some embodiments, the number of TILs sufficient to provide a therapeutically effective dosage is about 2.3×10 10 to about 13.7×10 10 .

[0037]

[0037] In some embodiments, steps (b) to (e) are performed in a single container, and performing steps (b) to (e) in a single container results in an increased TIL yield per resected tumor compared to performing steps (b) to (e) in two or more containers.

[0038] In some embodiments, the antigen-presenting cells are added to the TILs during the second period of step (d) without releasing the system.

[0039]

[0039] In some embodiments, the third TIL population of step (d) provides increased efficacy, increased interferon gamma production, increased polyclonality, increased mean IP-10, and / or increased mean MCP-1 when administered to a subject.

[0040] In some embodiments, the third population of TILs in step (d) provides at least a 5-fold or greater increase in interferon gamma production when administered to a subject.

[0041]

[0041] In some embodiments, the third TIL population of step (d) is a therapeutic TIL population comprising an increased subpopulation of effector T cells and / or central memory T cells compared to the second TIL population, and the effector T cells and / or central memory T cells in the therapeutic TIL population exhibit one or more characteristics selected from the group consisting of CD27+ expression, CD28+ expression, longer telomeres, increased CD57 expression, and decreased CD56 expression compared to the effector T cells and / or central memory T cells obtained from the second cell population.

[0042]

[0042] In some embodiments, effector T cells and / or central memory T cells obtained from the third TIL population exhibit increased CD57 expression and decreased CD56 expression compared to effector T cells and / or central memory T cells obtained from the second cell population.

[0043] In some embodiments, the risk of microbial contamination is reduced compared to open systems.

[0044] In some embodiments, the TILs from step (g) are infused into the patient.

[0045] In some embodiments, the plurality of fragments comprises about 4 fragments.

[0046] The present invention also provides a method of treating a subject with cancer comprising administering expanded tumor-infiltrating lymphocytes (TILs), the method comprising: (a) obtaining a first population of TILs from a tumor resected from a patient by processing a tumor sample obtained from the subject into multiple tumor fragments; (b) adding tumor fragments to a closed system; (c) performing a first expansion culture by culturing the first TIL population in a cell culture medium containing IL-2 to generate a second TIL population, wherein the first expansion culture is performed in a closed vessel providing a first gas permeable surface area, the first expansion culture is performed for about 3 to 14 days to obtain a second TIL population, wherein the second TIL population is at least 50-fold more numerous than the first TIL population, and the transition from step (b) to step (c) occurs without opening the system; (d) performing a second expansion culture by adding additional IL-2, OKT-3, and antigen-presenting cells (APCs) to the cell culture medium of the second TIL population to generate a third TIL population, wherein the second expansion culture is performed for about 7 to 14 days to obtain the third TIL population, the third TIL population being a therapeutic TIL population, wherein the second expansion culture is performed in a closed vessel providing a second gas-permeable surface area, and wherein the transition from step (c) to step (d) occurs without opening the system; (e) recovering the therapeutic TIL population obtained from step (d), wherein the transition from step (d) to step (e) occurs without releasing the system; and (f) transferring the TIL population collected in step (e) to an infusion bag, wherein the transition from step (e) to (f) occurs without opening the system; (g) optionally cryopreserving the infusion bag containing the TIL population collected in step (f) using a cryopreservation process; and (h) administering a therapeutically effective dose of the third population of TILs from the infusion bag in step (g) to the patient. Includes.

[0047]

[0047] In some embodiments, the therapeutic TIL population collected in step (e) comprises sufficient TILs to administer a therapeutically effective dosage of TILs in step (h).

[0048] In some embodiments, the number of TILs sufficient for administration of a therapeutically effective dosage in step (h) is from about 2.3×10 10 to about 13.7×10 10 .

[0049] In some embodiments, the antigen presenting cells (APCs) are PBMCs.

[0050] In some embodiments, the PBMCs are added to the cell culture in step (d) on any of days 9-14.

[0051]

[0051] In some embodiments, the patient has been administered a non-myeloablative lymphodepletion regimen prior to administering the therapeutically effective dose of TIL cells in step (h).

[0052]

[0052] In some embodiments, the non-myeloablative lymphodepletion regimen includes administering cyclophosphamide at a dose of 60 mg / m2 / day for two days, followed by administering fludarabine at a dose of 25 mg / m2 / day for five days.

[0053]

[0053] In some embodiments, the method further comprises treating the patient with a high-dose IL-2 regimen beginning the day after administering the TIL cells to the patient in step (h).

[0054] In some embodiments, the high-dose IL-2 regimen comprises 600,000 or 720,000 IU / kg administered as a 15-minute bolus intravenous infusion every 8 hours to a tolerated dose.

[0055]

[0055] In some embodiments, the third TIL population of step (d) is a therapeutic TIL population comprising an increased subpopulation of effector T cells and / or central memory T cells compared to the second TIL population, and the effector T cells and / or central memory T cells in the therapeutic TIL population exhibit one or more characteristics selected from the group consisting of CD27+ expression, CD28+ expression, longer telomeres, increased CD57 expression, and decreased CD56 expression compared to the effector T cells and / or central memory T cells obtained from the second cell population.

[0056]

[0056] In some embodiments, the effector T cells and / or central memory T cells of the therapeutic TIL population exhibit increased CD57 expression and decreased CD56 expression compared to the effector T cells and / or central memory T cells obtained from the second cell population.

[0057]

[0057] In some embodiments, the cancer is selected from the group consisting of melanoma, ovarian cancer, cervical cancer, non-small cell lung cancer (NSCLC), lung cancer, bladder cancer, breast cancer, cancer caused by human papillomavirus, head and neck cancer (including head and neck squamous cell carcinoma (HNSCC)), renal cancer, and renal cell carcinoma.

[0058] In some embodiments, the cancer is selected from the group consisting of melanoma, HNSCC, cervical cancer, and NSCLC.

[0059] In some embodiments, the cancer is melanoma.

[0060] In some embodiments, the cancer is HNSCC.

[0061] In some embodiments, the cancer is cervical cancer.

[0062] In some embodiments, the cancer is NSCLC.

[0063] The present invention also provides a method for expanding tumor infiltrating lymphocytes (TILs) into a therapeutic TIL population, the method comprising: (a) Addition of processed tumor fragments from tumors resected from patients to a closed system to obtain a first TIL population; (b) performing a first expansion culture by culturing the first TIL population in a cell culture medium containing IL-2 to generate a second TIL population, wherein the first expansion culture is performed in a closed vessel providing a first gas permeable surface area, and wherein the first expansion culture is performed for about 3 to 14 days to obtain a second TIL population, wherein the second TIL population is at least 50-fold more numerous than the first TIL population, and wherein the transition from step (a) to step (b) occurs without opening the system; (c) performing a second expansion culture by adding additional IL-2, OKT-3, and antigen-presenting cells (APCs) to the cell culture medium of the second TIL population to generate a third TIL population, wherein the second expansion culture is performed for about 7 to 14 days to obtain the third TIL population, the third TIL population being a therapeutic TIL population, wherein the second expansion culture is performed in a closed vessel providing a second gas-permeable surface area, and wherein the transition from step (b) to step (c) occurs without opening the system; (d) recovering the therapeutic TIL population obtained from step (c), wherein the transition from step (c) to step (d) occurs without releasing the system; and (e) transferring the TIL population collected in step (d) into an infusion bag, wherein the transition from step (d) to (e) occurs without opening the system. Includes.

[0064]

[0064] In some embodiments, the therapeutic TIL population collected in step (d) comprises sufficient TILs to provide a therapeutically effective dosage of TILs.

[0065] In some embodiments, the number of TILs sufficient to provide a therapeutically effective dosage is about 2.3×10 10 to about 13.7×10 10 .

[0066]

[0066] In some embodiments, the method further comprises the step of cryopreserving the infusion bag containing the collected TIL population using a cryopreservation process.

[0067]

[0067] In some embodiments, the cryopreservation process is performed using a 1:1 ratio of recovered TIL population to cryopreservation medium.

[0068] In some embodiments, the antigen-presenting cells are peripheral blood mononuclear cells (PBMCs).

[0069] In some embodiments, the PBMCs are irradiated and allogeneic.

[0070]

[0070] The method described in claim 68, wherein the PBMCs are added to the cell culture in step (c) on any of days 9 to 14.

[0071]

[0071] In some embodiments, the antigen-presenting cell is an artificial antigen-presenting cell.

[0072] In some embodiments, the harvesting of step (d) is carried out using a LOVO cell processing system.

[0073] In some embodiments, the plurality of fragments comprises about 4 to about 50 fragments, each fragment being about 27 mm 3 It has a volume of

[0074] In some embodiments, the plurality of segments is about 1300 mm 3 ~about 1500mm 3 It contains about 30 to about 60 fragments with a total volume of .

[0075] In some embodiments, the plurality of segments is about 1350 mm 3 It contains approximately 50 fragments with a total volume of 1000 s.p.m.

[0076] In some embodiments, the plurality of fragments comprises about 50 fragments having a total mass of about 1 g to about 1.5 g.

[0077] In some embodiments, the plurality of fragments comprises about 4 fragments.

[0078]

[0078] In some embodiments, the second cell culture medium is provided in a container selected from the group consisting of a G container and a Xuri cell culture bag.

[0079] In some embodiments, the infusion bag of step (e) contains HypoThermosol. It is a transfusion bag.

[0080] In some embodiments, the first period of step (b) and the second period of step (c) are each independently performed within a period of 10 days, 11 days, or 12 days.

[0081] In some embodiments, the first period of step (b) and the second period of step (c) are each performed separately within a period of 11 days.

[0082] In some embodiments, steps (a) through (e) are carried out within a period of about 10 days to about 22 days.

[0083] In some embodiments, steps (a) through (e) are carried out within a period of about 10 days to about 20 days.

[0084] In some embodiments, steps (a) through (e) are carried out within a period of about 10 days to about 15 days.

[0085] In some embodiments, steps (a) through (e) are performed within 22 days.

[0086] In some embodiments, steps (a)-(e) and cryopreservation are performed within 22 days.

[0087]

[0087] In some embodiments, steps (b) to (e) are performed in a single container, and performing steps (b) to (e) in a single container results in an increased TIL yield per resected tumor compared to performing steps (b) to (e) in two or more containers.

[0088] In some embodiments, the antigen-presenting cells are added to the TILs during the second period of step (c) without releasing the system.

[0089]

[0089] In some embodiments, the third TIL population of step (d) is a therapeutic TIL population comprising an increased subpopulation of effector T cells and / or central memory T cells compared to the second TIL population, and the effector T cells and / or central memory T cells obtained in the therapeutic TIL population exhibit one or more characteristics selected from the group consisting of CD27+ expression, CD28+ expression, longer telomeres, increased CD57 expression, and decreased CD56 expression compared to the effector T cells and / or central memory T cells obtained from the second cell population.

[0090]

[0090] In some embodiments, the effector T cells and / or central memory T cells obtained from the therapeutic TIL population exhibit increased CD57 expression and decreased CD56 expression compared to the effector T cells and / or central memory T cells obtained from the second cell population.

[0091]

[0091] In some embodiments, the risk of microbial contamination is reduced compared to open systems.

[0092] In some embodiments, the TILs from step (e) are infused into the patient.

[0093]

[0093] In some embodiments, the closed vessel comprises a single bioreactor.

[0094] In some embodiments, the closed container comprises G-REX-10.

[0095]

[0095] In some embodiments, the closed container comprises G-REX-100.

[0096] In some embodiments, in step (d), antigen presenting cells (APCs) are added to the cell culture of the second TIL population at an APC:TIL ratio of 25:1 to 100:1.

[0097] In some embodiments, the cell culture comprises 2.5×10 9 APC vs 100x10 6 With the ratio of TILs.

[0098] In some embodiments, in step (c), antigen presenting cells (APCs) are added to the cell culture of the second TIL population at an APC:TIL ratio of 25:1 to 100:1.

[0099] In some embodiments, the cell culture comprises 2.5×10 9 APC vs 100x10 6 With the ratio of TILs.

[0100] The present invention also provides a collection of expanded TILs for use in treating a subject with cancer. Providing a group and expanding a population of cultured TILs (a) obtaining a first population of TILs from a tumor resected from a patient by processing a tumor sample obtained from the subject into multiple tumor fragments; (b) adding tumor fragments to a closed system; (c) performing a first expansion culture by culturing the first TIL population in a cell culture medium containing IL-2 to generate a second TIL population, wherein the first expansion culture is performed in a closed vessel providing a first gas permeable surface area, the first expansion culture is performed for about 3 to 14 days to obtain a second TIL population, wherein the second TIL population is at least 50-fold more numerous than the first TIL population, and the transition from step (b) to step (c) occurs without opening the system; (d) performing a second expansion culture by adding additional IL-2, OKT-3, and antigen-presenting cells (APCs) to the cell culture medium of the second TIL population to generate a third TIL population, wherein the second expansion culture is performed for about 7 to 14 days to obtain the third TIL population, the third TIL population being a therapeutic TIL population, wherein the second expansion culture is performed in a closed vessel providing a second gas-permeable surface area, and wherein the transition from step (c) to step (d) occurs without opening the system; (e) recovering the therapeutic TIL population obtained from step (d), wherein the transition from step (d) to step (e) occurs without releasing the system; and (f) transferring the TIL population collected in step (e) to an infusion bag, wherein the transition from step (e) to (f) occurs without opening the system; and (g) optionally cryopreserving the infusion bag containing the TIL population collected in step (f) using a cryopreservation process; and a third TIL population obtainable by a method comprising:

[0101] In some embodiments, the population of TILs is selected from the group consisting of those described above and herein. and the method further comprises one or more of the features listed above and herein. [Brief explanation of the drawings]

[0102] BRIEF DESCRIPTION OF THE DRAWINGS [Figure 1]

[0102] Process 2A shows a schematic diagram of an embodiment of a 22-day process for TIL fabrication. [Figure 2]

[0103] 1 shows a comparison of embodiments of the 1C and 2A processes for TIL fabrication. [Figure 3]

[0104] Shows the timeline of the 1C process. [Figure 4]

[0105] 1 shows a process for an embodiment of TIL therapy using Process 2A for TIL production, including administration and co-therapy steps for higher cell numbers. [Figure 5]

[0106] 1 shows a process for an embodiment of TIL therapy using Process 2A for TIL production, including administration and co-therapy steps, for lower cell numbers. [Figure 6]

[0107] 2A shows a detailed schematic diagram of an embodiment of the 2A process. [Figure 7]

[0108] The characteristics of TILs prepared using an embodiment of the 2A process are shown by comparing the expression of interferon gamma (IFN-γ) between fresh and thawed TILs. [Figure 8]

[0109] Characterization of TILs prepared using an embodiment of the 2A process is shown by examining CD3 of fresh and thawed TILs. [Figure 9]

[0110] The recovery between fresh and thawed TILs is examined to demonstrate the properties of TILs prepared using an embodiment of the 2A process. [Figure 10]

[0111] The viability of fresh and thawed TILs is examined to characterize TILs prepared using an embodiment of the 2A process. [Figure 11a]

[0112] 1 shows the main steps of an embodiment of the 2A process, including a cryopreservation step. [Figure 11b]

[0112] The main steps of an embodiment of the 2A process, including a cryopreservation step, are shown. [Figure 11c]

[0112] The main steps of an embodiment of the 2A process, including a cryopreservation step, are shown. [Figure 12]

[0113] 1 shows the cell numbers obtained from process 1C and process 2A embodiments. [Figure 13]

[0114] 1 shows the cell viability obtained from embodiments of the 1C and 2A processes. [Figure 14]

[0115] 1 shows the percentage of CD45 and CD3 cells (i.e., T cells) measured by flow cytometry for TILs obtained in embodiments of process 1C and process 2A. [Figure 15]

[0116] Figures 80 and 98 show the IFN-γ release obtained with embodiments of the 1C and 2A processes, measured by an assay different from the assay used to generate the data. [Figure 16]

[0117] Figures 80 and 98 show the IFN-γ release obtained with embodiments of the 1C and 2A processes, measured by an assay different from the assay used to generate the data. [Figure 17]

[0118] 1 shows the percentage of TCRa / b and NK cells obtained from embodiments of the 1C and 2A processes. [Figure 18]

[0119] The percentages of CD8+ and CD4+ cells measured by flow cytometry of TILs obtained by embodiments of the 1C and 2A processes, as well as the ratios between each subset, are shown. [Figure 19]

[0120] 1 shows the percentage of memory subsets measured by flow cytometry of TILs obtained from embodiments of the 1C and 2A processes. [Figure 20]

[0121] 1 shows the percentage of PD-1, LAG-3, and TIM-3 expression by flow cytometry of TILs obtained from embodiments of process 1C and process 2A. [Figure 21]

[0122] 1 shows the percentage of 4-1BB, CD69, and KLRG1 expression by flow cytometry of TILs obtained from embodiments of process 1C and process 2A. [Figure 22]

[0123] 1 shows the percentage of TIGIT expression by flow cytometry of TILs obtained from embodiments of process 1C and process 2A. [Figure 23]

[0124] 1 shows the percentage of CD27 and CD28 expression by flow cytometry of TILs obtained from embodiments of process 1C and process 2A. [Figure 24]

[0125] 1 shows the results of flow FISH telomere length analysis. [Figure 25]

[0126] 1 shows the results of flow FISH telomere length analysis (after removal of outlier data points). [Figure 26]

[0127] 1 shows a clinical trial design including cohorts treated with embodiments of Process 1C and Process 2A. [Figure 27]

[0128] 2A is an exemplary process chart providing an overview of steps A-F. [Figure 28a]

[0129] Process flow chart for Process 2A. [Figure 28b]

[0129] Process flow chart for Process 2A. [Figure 28c]

[0129] Process flow chart for Process 2A. [Figure 29]

[0130] Process 2A Data Collection Plan Process Flowchart [Figure 30]

[0131] Viability of fresh and thawed TILs [Figure 31]

[0132] Expansion of fresh and thawed TILs in re-REP culture [Figure 32]

[0133] Normal laboratory values ​​of blood metabolites. [Figure 33a]

[0134] Metabolite analysis of process 2A pre-REP TILs. [Figure 33b]

[0134] Metabolite analysis of pre-REP TILs from Process 2A. [Figure 34]

[0135] Quantification of IL-2 in pre-REP TIL cell cultures in Process 2A. [Figure 35]

[0136] Release of the cytotoxic cytokine IFN-γ by stimulation of TILs with anti-CD3, anti-CD28, and anti-4-1BB. [Figure 36]

[0137] Granzyme B release following anti-CD3, anti-CD28, and anti-4-1BB stimulation of TILs. [Figure 37]

[0138] TCR αβ+ TILs. Most human CD3+ T cells express receptors formed by α and β chains that recognize antigens in an MHC-restricted manner. A) With the exception of M1061, fresh and thawed TIL products contained greater than 80% TCR αβ+ expressing TILs. Equivalent expression of TCR αβ was observed in both fresh and thawed TILs (P value -0.9582). Although a decrease in TCR αβ+ expressing TILs was observed after Re-REP, this decrease was not significant within Re-REP TILs (p=0.24). B) There was a 9.2% and 15.7% decrease in TCR αβ expression in fresh RE-REP and thawed RE-REP TILs compared to fresh and thawed TILs, respectively. [Figure 38]

[0139] TCR αβ-CD56+. Tumor-infiltrating natural killer (NK) and NKT cells also have the ability to lyse cells lacking MHC expression as well as CD1-presented lipid antigens and provide immunoregulatory cytokines. However, robust NK cell infiltration is associated with advanced disease and may promote cancer development. Figure A shows that in all cases except M1063, there was a slight, though not significant, reduction in the NK population in thawed TILs compared to fresh TILs (p=0.27). No significant differences were observed between re-REP TIL populations (p=0.88). Fresh TILs, fresh re-REP TILs, and thawed re-REP TILs demonstrate similar expression of CD56, as shown in Figure B. Thawed TIL products contained fewer NK-expressing cells (1.9±1.3) than fresh TILs (3.0±2.2), likely as a result of the cryopreservation procedure. [Figure 39]

[0140] CD4+ cells. No substantial differences in CD4 populations were observed between the individual conditions. Panel A shows the mean CD4 population for each condition. The table in Panel B shows SD and SEM values. There was a slight decrease in the CD4 population in the fresh re-REP population, which is primarily due to a decrease in CD4 in the fresh re-REP population of EP11001T. [Figure 40]

[0141] CD8+ cells. A) In all but EP11001T, both fresh and thawed TILs showed comparable CD8+ populations (P = 0.10, not significant difference). In most experiments, there was a slight decrease in CD8+ expressing TILs in the fresh re-REP TIL product (M1061T and M1065T were exceptions). The CD8+ population of thawed re-REP TILs was reduced by approximately 10-30%. Comparison of re-REP TILs from both fresh and thawed TILs showed a significant difference (p = 0.03, Student's t-test). Panel B shows the mean CD8+ expressing TILs in all conditions. Both fresh and thawed TILs showed similar results. However, there was a 10.8% decrease in the CD8+ population in the thawed re-REP TIL product compared to fresh re-REP TILs. [Figure 41]

[0142] CD4+CD154+ cells. CD154, also known as CD40L, is a marker for activated T cells. Figure A: No substantial differences in the CD4+CD154+ populations were observed across the different conditions, except for a 34.1% decrease in freshly re-REP CD4+ TILs from EP11001T. CD154 expression was not measured in M1061T and M1062T because these experiments were performed before the expanded phenotyping panel was deployed.

[0142] CD4+CD154+ cells. CD154, also known as CD40L, is a marker for activated T cells. Figure B: The slight decrease in the thawed TIL status may be due to CD154 not being measured in M1061T and M1062T. All conditions showed very similar CD154 expression in the CD4 population, suggesting activated CD4+ T cells. [Figure 42]

[0143] CD8+CD154+ cells. The activation marker CD154 expressed on CD8+ TILs was also analyzed. A) Overall, CD154 expression was low in the CD8+ population of both fresh and thawed TIL products. This is not surprising, as CD154 is primarily expressed on activated CD4+ T cells. When CD154 expression was measured in both fresh and thawed TIL products, either no difference or an increase in CD154 expression was observed in the thawed TIL products. Student's t-test showed no significant differences between the two conditions. Increased CD154 expression in thawed re-REP compared to fresh re-REP was demonstrated in all experiments (p=0.02). B) Increased CD154 expression was observed in both thawed and thawed re-REP TIL products compared to the corresponding products. Thawed re-REP TILs showed a 29.1% increase in CD154 expression compared to fresh re-REP TILs. [Figure 43]

[0144] CD4+CD69+ cells. CD69 is an early activation marker of T cells after stimulation or activation. A) For all TILs except EP11001T, both fresh and thawed re-REPs showed a slight increase in CD69 expression, likely due to the re-REP length (7 days instead of 11 days). No difference was observed between fresh and thawed TILs (p=0.89). No difference was observed between fresh and thawed re-REPs (p=0.82). B) A slight increase in CD69 expression is observed in the re-REP TIL product. (Note: CD69 staining was not performed on either the M1061T or M1062T thawed TIL products. CD69 expression in the M1061T fresh TIL product was 33.9%). [Figure 44]

[0145] CD8+CD69+ cells. As observed in the CD4+ population, Figure A shows increased CD69 expression in CD8+ re-REP TILs. CD69 expression was not significantly different between fresh and thawed TILs (p=0.68) or between fresh and thawed re-REP TILs (p=0.76). Figure B confirms the observation that there is a slight increase in CD69 expression in the re-REP TIL product. [Figure 45]

[0146] CD4+CD137+ cells. CD137 (4-11313) is a T cell costimulatory receptor induced by TCR activation. It is activated on CD4+ and CD8+ T cells. A) CD137 expression showed a significant increase in the re-REP TIL population after 7 days of stimulation. However, no difference was observed between fresh and thawed TILs or between fresh and thawed re-REP TILs (p<0.05 in both cases in Figure B, confirming this observation). Thawed TILs also showed a slight decrease in CD137 expression. The increase in CD137 expression in re-REP TILs is likely due to the second 7-day re-REP stimulation. [Figure 46]

[0147] CD8+CD137+ cells. A) The CD8+ population showed an overall increase in the re-REP product. B) The fresh re-REP product showed a 33.4% increase in CD8+CD137+ expression compared to the fresh TIL product. The thawed re-REP product also showed a 33.15% increase in CD137 expression in the CD8+ population compared to the thawed TIL. No significant differences were observed between fresh and thawed re-REP TILs. Similar observations were seen when comparing fresh TILs to the thawed TIL product. This increase in CD137 expression may be due to the second activation of re-REP. (Note that CD137 expression was not measured in three experiments, so only six TILs were used for analysis.) [Figure 47]

[0148] CD4+ CM cells. The central memory (CM) population is defined by CD45RA- (negative) and CCR7+ (positive) expression. A) An increase in the CM population was observed in the re-REP condition. M1063T and M1064T showed decreased CM expression in the CD4+ population obtained from thawed TILs compared to fresh TIL products. Neither fresh nor thawed TIL products (p=0.1658) nor fresh or thawed re-REP TILs (p=0.5535) showed significant differences in the CM population. B) A 14.4% and 15.4% increase in the CM population was observed in fresh and thawed re-REP TILs compared to fresh and thawed TILs, respectively. [Figure 48]

[0149] CD8+ CM cells. A) In the CD8+ population, a dramatic increase in CM expression was observed in the fresh TIL product, but not in the TIL product. This increase did not reach significance (p=0.3086), suggesting no difference between fresh and thawed TILs. A similar trend was observed in the re-REP TIL product. Figure 48B) An overall increase in the CM population was observed in fresh TILs compared to thawed TILs. The figures show that the difference between fresh TILs and fresh re-REP TILs was only approximately 2%. Fresh TILs showed a very high standard deviation due to M1064T. Excluding M1064T CM expression resulted in very similar CM expression between fresh and thawed TIL products (not shown). [Figure 49]

[0150] CD4+ EM cells. Effector memory (EM) populations are defined by the lack of CCR7 and CD45RA expression. A) As expected, the CD4+ populations of fresh and thawed TILs possessed high levels of the effector memory phenotype. A dramatic decrease in effector memory expression was found in the M1056T re-REP TIL population. Five other experiments also demonstrated a decrease in the effector memory phenotype in both fresh and thawed re-REP TILs. B) Both fresh and thawed TILs exhibited similar expression of the effector memory phenotype. A comparison of fresh and fresh Re-REP TILs showed a 16% decrease in the latter. A similar decrease was observed in thawed Re-REP TILs (9%) when compared to thawed TILs. [Figure 50]

[0151] CD8+ EM cells. A) A similar pattern of increased effector memory in fresh TILs was also observed in the CD8+ population. The exception was M1064T, where only 20% of fresh TILs had an effector memory profile. This is due to the CM phenotype of 73% of these TILs, as noted in A and B. All samples showing a decrease in the effector memory population of CD4+ TILs from the re-REP product followed the same trend as CD8+ TILs. B) Unlike the CD4+ TIL population, CD8+ TILs showed a similar effector memory phenotype in fresh, thawed, and re-REP products. (Note the high standard deviations for fresh and thawed TILs. This is due to the low effector memory population in M1064T fresh TILs and the absence of expression in the M1061T thawed TIL samples.) [Figure 51]

[0152] CD4+CD28+ cells. CD28 expression correlates with the decline in immature TILs with age. A) Despite an increase in the CM population observed in re-REP TILs, the decrease in CD28 expression was seen as a trend, suggesting that CM status alone does not determine TIL dynamics. Decreased CD28 expression was observed in the re-REP product, except for M1061T CD4+ TILs. CD28 expression correlates with the decline in immature TILs with age. B) A decrease of 8.89% and 5.71% was observed in fresh and thawed TILs compared to fresh and thawed TIL products, respectively. [Figure 52]

[0153] CD8+CD28+ cells. A) CD28 expression in the CD8+ TIL population was higher in fresh and thawed TILs than in the re-REP product. In most cases, thawed re-REP TILs showed a dramatic decrease when compared with thawed and fresh re-REP TILs. However, Student's t-test showed no significant differences between fresh and thawed TILs (p=0.3668) or between fresh and thawed re-REP products (p=0.7940). B) As seen in the CD4+ TIL population, there was a decrease in the CD8+CD28+ population in fresh re-REP (21.5%) and thawed re-REP (18.2%) when compared with those not restimulated. [Figure 53]

[0154] CD4+PD-1+ cells. PD-1 expression on TILs correlates with antigen-reactive and exhausted T cells. Thus, it is not surprising that an exhausted phenotype was observed in TILs that underwent REP for 11 days. A) This exhausted phenotype was maintained or increased in thawed TIL products (specifically, EP11001T and M1056T). No significant differences were observed between fresh and thawed TIL products (p=0.9809). A similar trend was observed in fresh re-REP TILs compared to thawed re-REP TILs (p=0.0912). B) Fresh re-REP showed a slight decrease in PD-1 expression in the CD4+ TIL population. All other conditions maintained comparable PD-1 expression patterns. A decrease or change in PD-1 expression was observed in fresh re-REP products compared to all other conditions. Increased PD-1 expression was observed in the thawed re-REP products M1062T, M1063T (CD4+), and EP11001T (CD8+). All other thawed re-REP products showed results comparable to the thawed re-REP products. [Figure 54]

[0155] CD8+PD-1+ cells. A) The CD8+ population from fresh TIL products exhibited a more exhausted phenotype associated with increased PD-1 expression. An exception was observed for EP11001T, where CD8+ thawed TIL products exhibited slightly increased PD-1 expression compared to fresh TIL products. There was a slight, but not significant, difference in PD-1 expression in fresh TILs compared to thawed TILs (p=0.3144). B) Fresh TIL products exhibited a slight, but not significant, increase in PD-1 expression compared to thawed TILs (6.74%, or 1.2-fold greater than thawed TILs), suggesting that thawed TIL products were comparable based on phenotypic pattern. [Figure 55]

[0156] CD4+LAG3+ cells. Exhausted T cells express high levels of the inhibitory receptor LAG3 along with PD-1. A) CD4+ thawed TILs showed slightly higher, but not significantly higher, levels of LAG3 expression compared to fresh TILs (p=0.52). An exception was observed for M1063T. In experiments where LAG3 expression was measured in CD4+ fresh re-REP TILs and fresh re-REP TILs, decreased LAG3+ expression was observed in fresh re-REP samples compared to fresh TILs. B) Overall, LAG3 expression is slightly decreased in fresh re-REP TIL products. Note that for consistency in Figure B, M1061T, M1062T, and M1064T have been excluded because LAG3 expression was not measured in the fresh products. [Figure 56]

[0157] CD8+LAG3+ cells. A) CD8+ LAG3-expressing TILs showed a slight decrease in this experiment, except for M1063T, where a significant decrease in LAG3 expression was observed in fresh re-REP TILs. Overall, thawed re-REP TILs showed a significant 1.5-fold increase in LAG3 expression compared to fresh re-REP TILs (p=0.0154). However, no significant difference was observed between fresh and thawed TIL products (p=0.0884). B) A roughly 30% decrease in LAG3 expression was observed in CD8+ TILs from fresh re-REP compared to thawed TIL products. Fresh and thawed TILs showed a slight increase comparable to thawed TILs. (This figure excludes M1061T, M1062T, and M1064T, as LAG3 expression was not measured in either fresh or fresh re-REP TIL samples.) [Figure 57]

[0158] CD4+TIM-3+ cells. A) As previously observed for PD-1 and LAG3, decreased TIM-3 expression was observed in fresh re-REP TILs compared to thawed re-REP TILs. Nevertheless, there was no significant difference between fresh and thawed re-REP TILs (p=0.2007). B) No significant changes in TIM-3 expression were observed in thawed TILs and thawed re-REP TIL products. A slight decrease of 9.2% in TIM-3 expression was observed in fresh re-REP TILs compared to thawed re-REP products. [Figure 58]

[0159] CD8+TIM-3+ cells. A) The same trend in TIM-3 expression seen in the CD4+ population was also observed in CD8+ TILs. Fresh re-REP TILs had the least TIM-3 expression, a significant difference compared to thawed re-REP TILs (p=0.0147). Comparison of PD-1, LAG3, and TIM-3 suggests that the fresh re-REP TIL phenotype is not exhaustive, with an increased CM phenotype. B) Compared to thawed re-REP TILs, fresh re-REP TILs showed a significant 22% decrease in TIM-3 expression. Both fresh and thawed TILs show similar TIM-3 expression patterns. [Figure 59]

[0160] Cytotoxic potential of TILs against P815 target cell lines. [Figure 60]

[0161] Metabolic respiratory profiles of fresh TILs, fresh re-REP TILs, and thawed re-REP TILs: basal OCR (A), manifest SRC (B), SRC2DG (C), latent SRC (D), basal ECAR (E), and glycolytic reserve (F). [Figure 61]

[0162] Flow FISH technology was used to measure the average telomere repeat length of nine TIL products thawed during Post-REP Process 2A. A) Data represent telomere length measured by qPCR comparing TILs to 1301 cells. B) Data represent telomere length measured by flow FISH assay of TILs compared to 1301 cells. Data used for the graphs are provided in tabular form (Table 25) in Appendix Section 10. Overall, there was a broad similarity in the pattern of results from the two telomere length assays; however, experiments continue to determine methods to more accurately reflect the actual telomere length of TILs. This technology can be applied to future clinical samples to determine the relationship between telomere length and patient response to TIL therapy. [Figure 62]

[0163] Serum-free medium provider's choice (serum substitute). Each fragment was cultured in quadruplicate in a single well of a G-Rex 24-well plate. On day 11, REP was initiated using 45 TILs and 10 feeders, mimicking the 2A process. A) Bar graph showing the average viable cell count recorded on day 11 (pre-REP) for each condition. B) Bar graph showing the average viable cell count recorded on day 22 (post-REP). P values ​​were calculated using Student's t test. *P<0.05, **P<0.01, ***P<0.001, respectively. [Figure 63]

[0164] Serum-free medium donor's choice (platelet lysate serum). Each fragment was cultured in triplicate in a single well of a G-Rex 24-well plate. On day 11, REP was initiated using 4e5 TILs and 10e6 feeders, mimicking process 2A. A) Bar graph showing the average viable cell count recorded on day 11 (pre-REP) for each condition. B) Bar graph showing the average viable cell count recorded on day 22 (post-REP). P values ​​were calculated using Student's t test. *P<0.05, **P<0.01, ***P<0.001, respectively. "#" tumor fragment missing. [Figure 64A]

[0165] A small-scale 2A process (G-Rex 5M) was used to compare the effectiveness of the CTS Optimizer with standard conditions. Two fragments / G-Rex 5M were cultured in triplicate and REP was initiated using 26 TILs on 506 feeders to mimic the 2A process. The numbers above are the average viable cell counts obtained on day 11 (A) or day 22 (B). [Figure 64B] A small-scale 2A process (G-Rex 5M) was used to compare the effectiveness of the CTS Optimizer with standard conditions. Two fragments / G-Rex 5M were cultured in triplicate, and REP was initiated using 26 TILs on 506 feeders to mimic the 2A process. The numbers above are the average viable cell counts obtained on day 11 (A) or day 22 (B). [Figure 65A]

[0166] Pre- and post-extrapolated TIL expansion overview comparing standard conditions and CTS Optimizer. A) Pre-REP. B) Post-REP. C) Extrapolated TIL expansion overview for real-world implementation (standard conditions vs. CTS Optimizer + SR). [Figure 65B]

[0166] Pre- and post-estimated summary of TIL expansion comparing standard conditions and CTS Optimizer. A) Pre-REP. B) Post-REP. C) Summary of TIL expansion extrapolated to actual performance (standard conditions vs. CTS Optimizer + SR). [Figure 65C]

[0166] Pre- and post-estimated summary of TIL expansion comparing standard conditions and CTS Optimizer. A) Pre-REP. B) Post-REP. C) Summary of TIL expansion extrapolated to actual performance (standard conditions vs. CTS Optimizer + SR). [Figure 66]

[0167] CD8+ was gated on live cells. Seven of nine tumors show an increase in the absolute CD8+ population in the CTS+SR condition. [Figure 67]

[0168] Comparability of interferon gamma. Interferon gamma ELISA (Quantikine). IFN-γ production was measured using the Quantikine ELISA kit from R&D Systems. CTS+SR produced similar amounts of IFN-γ when compared to standard conditions. [Figure 68]

[0169] Scheme of an exemplary embodiment of the Rapid Expansion Protocol (REP). Upon arrival, tumors are fragmented and placed in G-Rex flasks containing IL-2 for TIL expansion (pre-REP expansion) for 11 days. For triple cocktail studies, IL-2 / IL-15 / IL-21 are added at the beginning of pre-REP. For the Rapid Expansion Protocol (REP), TILs are cultured on feeders and OKT3 for an additional 11 days of REP expansion. [Figure 69]

[0170] Melanoma-derived TILs (n=4) and lung (n=7) were assessed for CD4+ and CD8+ cell phenotype using flow cytometry post-pre-REP. *P values ​​represent differences in IL-2 and IL-12 / IL-15 / IL-21 expression in CD8+ cells using Student's unpaired t-test. [Figure 70]

[0171] Melanoma-derived TILs (n=4) and lung (n=7) were assessed for CD27+ and CD28+ phenotype of CD4+ and CD8+ cells using flow cytometry post-pre-REP. [Figure 71]

[0172] TILs were assessed for phenotype of effector / memory subsets of CD8+ cells (CD45RA and CCR7) and CD4+ (data not shown) in melanoma (n = 4) (A) and lung (n = 8) (B). CXCR3 expression was assessed in melanoma and lung. All phenotypic expression was assessed using flow cytometry post-pre-REP. TCM = central memory, TSCM = stem cell-like memory, TEMRA (effector T cells), TEM = effector memory. [Figure 72]

[0173] (A) Melanoma (n = 4) and (B) lung (n = 5) derived TILs were assessed by flow cytometry for CD107a expression in CD4+ and CD8+ cells in response to 4 h of PMA stimulation. (C) Pre-REP TILs (n = 5) were stimulated with soluble OKT3 (30 ng / ml) for 24 h, and supernatants were assessed for IFnγ by ELISA. [Figure 73A]

[0174] The TCRvβ repertoire (24 specificities) was assessed in TILs derived from melanoma (A) and lung (B) using a Beckman Coulter kit for flow cytometry. [Figure 73B]

[0174] The TCRvβ repertoire (24 specificities) was assessed in TILs derived from melanoma (A) and lung (B) using a Beckman Coulter kit for flow cytometry. [Figure 74]

[0175] Typical process for producing cryopreserved TILs (approximately 22 days). [Figure 75]

[0176] On day 22, the reduced volume cell product was pooled and sampled to determine culture performance before washing and formulation. Samples were analyzed using an NC-200 automated cell counter as described previously. Total viable cell density was determined by averaging duplicate counts from four independent samples. The Generation 2 (Gen2:Gen2) process produced a similar dose of TIL product to Generation 1 (Gen1:Gen1; Gen1 mean = 4.10 x 1010 ± 2.92 x 1010, Gen2 mean = 3.12 x 1010 ± 2.19 x 1010). B) Fold expansion was calculated for the REP stage as the dividend of the final viable cell density relative to the initial viable TIL seeding density. Gen2 TIL product exhibited a lower fold expansion compared to Gen1 (Gen1 mean = 1.40 x 103 ± 9.86 x 102, Gen2 mean = 5.11 x 102 ± 2.95 x 102). [Figure 76]

[0177] Freshly formulated product was assayed for identity by flow cytometry for release. The Gen1 and Gen2 processes produce highly pure T cell cultures defined by a CD45, CD3 double-positive phenotype (Gen1#±SD, Gen2#±SD). P values ​​were calculated using the Mann-Whitney "t" test. [Figure 77]

[0178] Cryopreserved satellite vials of the formulated product were thawed and assayed for expansion phenotype by flow cytometry as previously described. Gen1 and Gen2 products represent similar proportions of CD8 and CD4 T cell subtypes. P values ​​were calculated using the Mann-Whitney "t" test. [Figure 78]

[0179] Cryopreserved satellite vials of the formulated product were thawed and assayed for expansion phenotype by flow cytometry as previously described. Gen1 and Gen2 products express similar levels of the costimulatory molecules CD27 and CD28 on T cell subsets. P values ​​were calculated using a Mann-Whitney "t" test. Costimulatory molecules such as CD27 and CD28 are required to provide the secondary and tertiary signals necessary for effector cell growth upon T cell receptor engagement. [Figure 79]

[0180] Flow FISH technology was used to measure the average length of telomere repeats as described above. The RTL values ​​above indicate that the control cell line, Gen1 (embodiment of the 1C process), had an average telomere fluorescence per chromosome / genome of #%±SD%, and Gen2 (1301 leukemia cell line) had an average telomere fluorescence per chromosome / genome of #%±SD%. The data indicate that the Gen2 product has, on average, telomere length at least equivalent to that of the Gen1 product. Telomere length is a surrogate measurement of length in ex vivo cell cultures. [Figure 80]

[0181] The Gen2 (Process Embodiment 2A) formulation product exhibits increased capacity to produce IFN-γ compared to the Gen1 formulation product. The ability of the formulation product to be reactivated and secrete cytokines is a surrogate for in vivo function upon TCR binding to cognate antigen in the context of HLA. [Figure 81]

[0182] T Cell Receptor Diversity: RNA from 10 x 10 TILs from Gen1 (embodiment of Process 1C) and Gen2 (embodiment of Process 2A) formulation products was assayed to determine the total number and frequency of unique CDR3 sequences present in each product. A) Total number of unique CDR3 sequences present in each product (Gen1 n=#, mean ± SD; Gen2 n=#, mean ± SD). B) Unique CDR3 sequences were indexed against their frequency in each product to obtain a score representing the relative diversity of T cell receptors within the product. TIL products from both processes are composed of polyclonal populations of T cells with different antigen specificities and avidities. The breadth of the total T cell repertoire can be an indicator of the number of actionable epitopes on tumor cells. [Figure 82]

[0183] 2A shows a schematic diagram of an embodiment of Process 2A, a 22-day process for TIL fabrication. [Figure 83]

[0184] 1 is a comparison table of steps A-F from exemplary embodiments of Process 1C and Process 2A. [Figure 84]

[0185] Detailed comparison of the Process 1C embodiment with the Process 2A embodiment. [Figure 85]

[0186] Detailed scheme of an embodiment of the TIL therapy process. [Figure 86A]

[0187] Phenotypic characterization of TIL products using a 10-color flow cytometry assay. (A) The proportions of T cell and non-T cell subsets were defined by CD45+CD3+ and CD45- (non-lymphocytes) / CD45+CD3- (non-T cell lymphocytes), respectively. Overall, >99% of the TIL products assayed consisted of T cells (CD45+CD3+). Shown is the mean of TIL products (n=10). [Figure 86B]Phenotypic characterization of TIL products using a 10-color flow cytometry assay. (B) Percentages of two T-cell subsets, including CD45+CD3+CD8+ (blue open circles) and CD45+CD3+CD4+ (pink open circles). Using Student's unpaired T-test, no statistical difference was observed between the percentages of both subsets (P=0.68). [Figure 86C]

[0187] Phenotypic characterization of TIL products using a 10-color flow cytometry assay. (C) The non-T cell population was characterized into four distinct subsets, including: 1) non-lymphocytes (CD45-), 2) NK cells (CD45+CD3-CD16+ / 56+), 3) B cells (CD45+CD19+), and 4) non-NK / B cells (CD45+CD3-CD16-CD56-CD19-). [Figure 87A]

[0188] Characterization of T cell subsets in CD45+CD3+CD4+ and CD45+CD3+CD8+ cell populations. Naive, central memory (TCM), effector memory (TEF), and effector memory RA+ (EMRA) T cell subsets were defined using CD45RA and CCR7. Figures show representative T cell subsets from 10 final TIL products in both CD4+ (A) and CD8+ (B) cell populations. The effector memory T cell subset (blue open circles) is the predominant population (>93%) in both the CD4+ and CD8+ subsets in the final TIL product. Fewer than 7% of cells in the TIL product are central memory subsets (pink open circles). EMRA (gray open circles) and naive (black open circles) subsets are barely detectable in the TIL product (<0.02%). P values ​​represent the difference between EM and CM using Student's unpaired t-test. [Figure 87B]Characterization of T cell subsets in CD45+CD3+CD4+ and CD45+CD3+CD8+ cell populations. Naive, central memory (TCM), effector memory (TEF), and effector memory RA+ (EMRA) T cell subsets were defined using CD45RA and CCR7. The figures show representative T cell subsets from 10 final TIL products in both the CD4+ (A) and CD8+ (B) cell populations. The effector memory T cell subset (blue open circles) is the predominant population (>93%) in both the CD4+ and CD8+ subsets in the final TIL product. Fewer than 7% of cells in the TIL product are central memory subsets (pink open circles). The EMRA (gray open circles) and naive (black open circles) subsets are barely detectable (<0.02%) in the TIL product. P values ​​represent the difference between EM and CM using Student's unpaired t-test. [Figure 88A]

[0189] Detection of MCSP and EpCAM expression in melanoma tumor cells. Melanoma tumor cell lines (WM35, 526, and 888), patient-derived melanoma cell lines (1028, 1032, and 1041), and a colorectal adenoma carcinoma cell line (HT29 as a negative control) were characterized by MCSP (melanoma (chondroitin sulfate proteoglycan)) and EpCAM (epithelial cell adhesion molecule) markers. (A) An average of 90% of melanoma tumor cells express MCSP. [Figure 88B] Detection of MCSP and EpCAM expression in melanoma tumor cells. Melanoma tumor cell lines (WM35, 526, and 888), patient-derived melanoma cell lines (1028, 1032, and 1041), and a colorectal adenoma carcinoma cell line (HT29 as a negative control) were characterized with MCSP (melanoma chondroitin sulfate proteoglycan) and EpCAM (epithelial cell adhesion molecule) markers. (B) No EpCAM expression was detected in melanoma tumor cell lines compared with the positive control HT29, an EpCAM+ tumor cell line. [Figure 89A]

[0190] Detection of spiked controls to determine tumor detection accuracy. The assay was performed by spiking a known amount of tumor cells into a PBMC suspension (n = 10). MCSP+526 melanoma tumor cells were diluted at 1:10, 1:100, and 1:1,000 ratios and then mixed with PBMCs, stained with anti-MCSP and anti-CD45 antibodies and live / dead dye, and analyzed by flow cytometry. (A) Approximately 3,000, 300, and 30 cells were detected at dilutions of 1:10, 1:100, and 1:1,000, respectively. [Figure 89B] Detection of spiked controls to determine tumor detection accuracy. The assay was performed by spiking a known amount of tumor cells into a PBMC suspension (n = 10). MCSP+526 melanoma tumor cells were diluted at 1:10, 1:100, and 1:1,000 ratios and then mixed with PBMCs, stained with anti-MCSP and anti-CD45 antibodies and live / dead dye, and analyzed by flow cytometry. (B) The mean (AV) and standard deviation (SD) of cells obtained in each condition were used to define the upper and lower limits of the criteria. [Figure 90A]

[0191] Reproducibility study of upper and lower limits of the contamination control. Three independent experiments were performed in triplicate to determine the reproducibility of the contamination assay. (A) The number of tumor cells detected with MCSP+ was consistently within the upper and lower limits of the criteria. [Figure 90B] (B) Reproducibility study of upper and lower limits of the spiked control. Three independent experiments were performed in triplicate to determine the reproducibility of the spiked assay. (C) Linear regression plot shows the correlation between MCSP + cells and spiked dilution (R = 0.99), with the solid black line demonstrating the optimum. The green and gray dashed lines represent the 95% prediction limits of the standard curve and samples (Exp#1-3), respectively. [Figure 91A]

[0192] Detection of residual melanoma tumor in TIL products. TIL products were evaluated for residual tumor contamination using the developed assay (n=15). (A and B) The median and percentage of detectable MCSP+ events were 2 and 0.0002%, respectively. [Figure 91B]

[0192] Detection of residual melanoma tumor in TIL products. TIL products were evaluated for residual tumor contamination using the developed assay (n=15). (A and B) The median and rate of detectable MCSP+ events were 2 and 0.0002%, respectively. [Figure 92]

[0193] Potency assessment of TIL products after T cell activation. IFNγ secretion after restimulation with anti-CD3 / CD28 / CD137 in TIL products assessed in duplicate by ELISA (n=5). IFNγ secretion by TIL products was significantly higher than unstimulated controls using the Wilcoxon signed-rank test (P=0.02) and was consistently >1000 pg / ml. IFNγ secretion of >200 pg / ml is considered potent. A p-value <0.05 is considered statistically significant. [Figure 93]

[0194] Description of an embodiment of the cryopreserved TIL manufacturing process (22 days). [Figure 94]

[0195] Table of process improvements from Gen1 to Gen2. [Figure 95A]

[0196] Total viable cells, growth rate, and viability. On day 22, the reduced volume cell product is pooled and sampled to determine culture performance before washing and formulation. (A) Samples are analyzed on an NC-200 automated cell counter as described above. Total viable cell density is determined by the grand average of duplicate counts from four independent samples. The Gen2 process produces a TIL product with a dose equivalent to Gen1 (Gen1 mean = 4.10 x 10 ± 2.8 x 10 ± 2.5 x 10 ± 2.1 ... [Figure 95B] Total viable cells, growth rate, and viability. On day 22, the reduced volume cell product is pooled and sampled to determine culture performance before washing and formulation. (B) Growth rate during the REP stage is calculated as gr = ln(N(t) / N(0) / t). [Figure 95C]Total viable cells, growth rate, and viability. On day 22, the reduced volume cell product is pooled and sampled to determine culture performance prior to washing and formulation. (C) Cellometer K2 was used to assess cell viability from nine process development lots, as previously described. No significant decrease in cell viability was observed after a single freeze-thaw cycle of the formulated product. The average loss in viability upon thawing and sampling is 2.19%. [Figure 96A]

[0197] The Gen2 product is a highly pure T cell culture that expresses costimulatory molecules at levels comparable to Gen1. (A) Freshly formulated product was assayed for identity by flow cytometry for release. The Gen1 and Gen2 processes produce highly pure T cell cultures defined by a CD45+, CD3+ (double positive) phenotype. [Figure 96B] The Gen2 product is a highly purified T cell culture that expresses costimulatory molecules at levels comparable to those of Gen1. (B and C) Cryopreserved satellite vials of the formulated product were thawed and assayed for expansion phenotype by flow cytometry as described above. The Gen1 and Gen2 products express comparable levels of the costimulatory molecules CD27 and CD28 on T cell subsets. Costimulatory molecules such as CD27 and CD28 are required to provide the secondary and tertiary signals necessary for effector cell growth upon T cell receptor engagement. P values ​​were calculated using the Mann-Whitney "t" test. [Figure 96C] The Gen2 product is a highly purified T cell culture that expresses costimulatory molecules at levels comparable to those of Gen1. (B and C) Cryopreserved satellite vials of the formulated product were thawed and assayed for expansion phenotype by flow cytometry as described above. The Gen1 and Gen2 products express comparable levels of the costimulatory molecules CD27 and CD28 on T cell subsets. Costimulatory molecules such as CD27 and CD28 are required to provide the secondary and tertiary signals necessary for effector cell growth upon T cell receptor engagement. P values ​​were calculated using the Mann-Whitney "t" test. [Figure 97]

[0198] Gen2 products show similar telomere lengths, although some TIL populations may be prone to longer relative telomeres. [Figure 98]

[0199] The Gen2 formulation product secretes IFNγ in response to engagement of CD3, CD28, and CD137. [Figure 99A]

[0200] T cell receptor diversity. (A) Unique CDR3 sequences were indexed against their frequency in each product to obtain a score representing the overall diversity of T cell receptors within the product. [Figure 99B] T cell receptor diversity. (B) Mean total number of unique CDR3 sequences present in each infusion product. [Figure 100]

[0201] 1 illustrates an embodiment of the TIL manufacturing process of the present invention. [Figure 101]

[0202] Enhancement of expansion cultures during pre-REP by IL-2 / IL-15 / IL-21 in multiple tumor tissues. [Figure 102A]

[0203] IL-2 / IL-15 / IL-21 increased the percentage of CD8+ cells in lung cancer but not melanoma. (A) Melanoma (n=4) and (B) lung (n=7) derived TILs were assessed for CD4+ and CD8+ cell phenotype using flow cytometry post-REP. [Figure 102B] IL-2 / IL-15 / IL-21 increased the percentage of CD8+ cells in lung cancer but not melanoma. (A) Melanoma-derived TILs (n=4) and (B) lung (n=7) were assessed for CD4+ and CD8+ cell phenotype using flow cytometry post-pre-REP. [Figure 103A]

[0204] CD27 expression was slightly upregulated on CD8+ cells in IL-2 / IL-15 / IL-21-treated cultures. (A) Melanoma-derived TILs (n=4) and (B) lung (n=7) were assessed for CD27+ and CD28+ phenotype of CD4+ and CD8+ cells using flow cytometry post-REP. [Figure 103B] CD27 expression was slightly elevated in CD8+ cells from IL-2 / IL-15 / IL-21-treated cultures. (A) Melanoma-derived TILs (n=4) and (B) lungs (n=7) were assessed for CD27+ and CD28+ phenotypes of CD4+ and CD8+ cells using flow cytometry post-REP. [Figure 104A]

[0205] T cell subsets were unchanged by the addition of IL-15 / IL-21. TILs were assessed for effector / memory subsets (CD45RA and CCR7) of CD8+ and CD4+ (data not shown) cells from melanoma (n = 4) (A) and lung (n = 8) phenotypes via flow cytometry post-REP (B). [Figure 104B] T cell subsets were unchanged by the addition of IL-15 / IL-21. TILs were assessed for effector / memory subsets (CD45RA and CCR7) of CD8+ and CD4+ (data not shown) cells from melanoma (n=4) (A) and lung (n=8) phenotypes via flow cytometry post-REP (B). [Figure 105A]

[0206] The functional capacity of TILs was differentially enhanced with IL-2 / IL-15 / IL-21. (A) Melanoma (n=4) and (B) lung (n=5) derived TILs were assessed by flow cytometry for CD107a+ expression in CD4+ and CD8+ cells in response to 4-hour PMA stimulation. [Figure 105B]The functional capacity of TILs was differentially enhanced with IL-2 / IL-15 / IL-21. (A) Melanoma (n=4) and (B) lung (n=5) derived TILs were assessed by flow cytometry for CD107a expression in CD4+ and CD8+ cells in response to 4-hour PMA stimulation. [Figure 105C]

[0206] The functional capacity of TILs was differentially enhanced by IL-2 / IL-15 / IL-21. (C) Melanoma- and lung-derived pre-REP TILs were stimulated with soluble anti-CD3 antibody for 24 h, and supernatants were assessed for IFNγ by ELISA. [Figure 106A]

[0207] The TCRvβ repertoire (24 specificities) was assessed in TILs derived from (A) melanoma and (B) lung tumors using a Beckman Coulter kit for flow cytometry. [Figure 106B]

[0207] The TCRvβ repertoire (24 specificities) was assessed in TILs derived from (A) melanoma and (B) lung tumors using a Beckman Coulter kit for flow cytometry. [Figure 107]

[0208] Scheme of the Gen2 cryopreserved LN-144 manufacturing process. [Figure 108]

[0209] Scheme of study design for a multicenter phase 2 clinical trial of novel cryopreserved TILs administered to patients with metastatic melanoma. [Figure 109]

[0210] Table showing a comparison of patient characteristics between Cohort 1 (ASCO2017) and Cohort 2. [Figure 110]

[0211] Table showing treatment-emergent adverse events (≥30%). [Figure 111]

[0212] Efficacy of infusion products and TIL therapy. [Figure 112]

[0213] Clinical status of response in evaluable patients showing SD or better response. [Figure 113]

[0214] The percentage change in total diameter. [Figure 114]

[0215] Increased HMGB1 levels were observed upon TIL treatment. [Figure 115]

[0216] An increase in the biomarker IL-10 was observed after LN-144 infusion. [Figure 116]

[0217] Updated patient characteristics for Cohort 2 of the metastatic melanoma phase 2 clinical trial from the second data cut (N=17 patients). [Figure 117]

[0218] Treatment adverse events in cohort 2 (≥30%) from the second data cut (N=17 patients). [Figure 118]

[0219] Response time for evaluable patients (stable disease or better) in Cohort 2 from the second data cut (N=17 patients). Of the 10 patients in the efficacy set, one patient (patient 10) was unevaluable due to melanoma-related death before the first tumor assessment, not shown in the figure. [Figure 119]

[0220] Updated efficacy data for Cohort 2 from the second data cut (N=17 patients). The mean number of TILs infused was 34 x 109. The median number of previous lines of therapy was 4.5. Patients with BRAF mutations responded similarly to patients with wild-type BRAF (* indicates patients with BRAF mutations). One patient (patient 10) was not evaluated due to melanoma-related death before the first tumor evaluation but was still considered in the efficacy set. Abbreviations: PR, partial response; SD, stable disease; PD, progressive disease. [Figure 120]

[0221] Updated efficacy data for evaluable patients from Cohort 2 from the second data cut (N=17 patients). * indicates non-evaluable patients who did not reach initial assessment. All efficacy-evaluable patients had previously received anti-PD-1 and anti-CTLA-4 checkpoint inhibitor therapy. [Figure 121]

[0222] Representative computed tomography scans of patients with PR from cohort 2 (003-015), second data cut. [Figure 122]

[0223] Correlation between IFN-γ induction by TIL products before infusion and clinical reduction in tumor size 42 days after TIL infusion. [Figure 123]

[0224] IP-10 (CXCL10) levels (pg / mL, log10) before and after infusion of an embodiment of the Gen2 TIL product. IP-10 is a marker of cell adhesion and homing. [Figure 124]

[0225] IP-10 (CXCL10) levels (pg / mL, log10) before and after infusion of an embodiment of the Gen1 TIL product. [Figure 125]

[0226] MCP-1 levels (pg / mL, log10) before and after infusion of an embodiment of the Gen2 TIL product. MCP-1 is a marker of cell adhesion and homing. [Figure 126]

[0227] MCP-1 levels (pg / mL, log10) before and after infusion of an embodiment of the Gen1 TIL product. [Figure 127]

[0228] Data from phase 2 trials in cervical cancer and head and neck squamous cell carcinoma (HNSCC). SD = stable disease. PR = progressive disease. PR = partial response. DETAILED DESCRIPTION OF THE INVENTION

[0103] Brief description of the sequence listing

[0229] SEQ ID NO: 1 is the amino acid sequence of the heavy chain of muromonab.

[0104]

[0230] SEQ ID NO: 2 is the amino acid sequence of the light chain of muromonab.

[0105]

[0231] SEQ ID NO: 3 is the amino acid sequence of recombinant human IL-2 protein.

[0106]

[0232] SEQ ID NO: 4 is the amino acid sequence of aldesleukin.

[0107]

[0233] SEQ ID NO: 5 is the amino acid sequence of recombinant human IL-4 protein.

[0108]

[0234] SEQ ID NO: 6 is the amino acid sequence of recombinant human IL-7 protein.

[0109]

[0235] SEQ ID NO: 7 is the amino acid sequence of recombinant human IL-15 protein.

[0110]

[0236] SEQ ID NO: 8 is the amino acid sequence of recombinant human IL-21 protein.

[0111] Detailed Description of the Invention I. Introduction

[0237] Rapid Expansion Protocol (REP) Adoptive cell therapy utilizing ex vivo cultured TILs has produced successful adoptive cell therapy in melanoma patients following host immunosuppression. Current infusion eligibility parameters rely on readings of TIL composition (e.g., CD28, CD8, or CD4 positivity) and numerical values ​​of fold expansion and survival of the REP product.

[0112]

[0238] The current REP protocol does not guarantee the health of the TILs that will be infused into patients. T cells undergo a significant metabolic shift during their maturation from naive to effector T cells (Chang, et al., Nat. Immunol. 2016, 17, 364 (hereby expressly incorporated in its entirety), and in particular for the discussion and markers of anaerobic and aerobic metabolism. For example, naive T cells rely on mitochondrial respiration for ATP production, while mature, healthy effector T cells, such as TILs, rely on highly It is primarily glycolytic and relies on aerobic glycolysis to provide the bioenergetic substrates it requires for proliferation, migration, activation, and antitumor efficacy.

[0113]

[0239] Previous studies have shown that cells that rely heavily on glycolysis experience nutrient deprivation during adoptive transfer. Because TILs are susceptible to glycolysis and promote mitochondrial metabolism before transplantation, it is desirable to limit glycolysis and promote mitochondrial metabolism before transplantation. Therefore, the art teaches that promoting mitochondrial metabolism may promote in vivo lifespan, and in fact, it has been proposed to use glycolysis inhibitors before inducing an immune response. See Chang et al. (Chang, et al., Nat. Immunol. 2016, 17(364)).

[0114]

[0240] The present invention further provides, in some embodiments, a method for determining and evaluating this increased metabolic health. Accordingly, the present invention provides methods for assaying the relative health of a TIL population using one or more common metabolic determinations, including, but not limited to, the rate and amount of glycolysis, oxidative phosphorylation, spare respiratory capacity (SRC), and glycolytic reserve.

[0115]

[0241] Moreover, the present invention further provides that, in some embodiments, this increased metabolic health can be achieved by Accordingly, the present invention provides methods for assaying the relative health of a TIL population using one or more common metabolic measures, including, but not limited to, the rate and amount of glycolysis, oxidative phosphorylation, spare respiratory capacity (SRC), and glycolytic reserve.

[0116]

[0242] Additionally, optional additional determinations include, but are not limited to, ATP production, These include mitochondrial mass and glucose uptake.

[0117] II. Definition

[0243] Unless otherwise defined, all technical and scientific terms used herein Terms have the same meaning as commonly understood by one of ordinary skill in the art to which this invention belongs. All patents and publications mentioned herein are hereby incorporated by reference in their entirety.

[0118]

[0244] The term "in vivo" refers to an event that takes place inside a subject's body.

[0119]

[0245] The term "in vitro" refers to events that occur outside a subject's body. The assays include cell-based assays that utilize live or dead cells, and may also include cell-free assays that do not utilize intact cells.

[0120]

[0246] The term "ex vivo" refers to cells, tissues and / or organs that have been removed from a subject's body. "Transplantation" refers to an event that involves the administration of a procedure or treatment. Suitably, the cells, tissues, and / or organs are returned to the subject's body by surgical or procedural means.

[0121]

[0247] The term "rapid expansion culture" refers to a culture that expands at least about three-fold (or four-fold, five-fold, or six-fold) over a one-week period. By rapid expansion is meant an increase in the number of antigen-specific TILs by at least about 10-fold (or 20-fold, 30-fold, 40-fold, 50-fold, 60-fold, 70-fold, 80-fold, or 90-fold) over a one week period, or most preferably at least about 100-fold over a one week period. Several rapid expansion protocols are described below.

[0122]

[0248] As used herein, "tumor infiltrating lymphocytes" or "TILs" refer to cells that migrate through the bloodstream of a subject. TILs refer to a population of cells originally acquired as white blood cells that have migrated away from and into a tumor. TILs include, but are not limited to, CD8 + Cytotoxic T cells (lymphocytes), Th1 and Th17 CD4 + These include T cells, natural killer cells, dendritic cells, and M1 macrophages. TILs include both primary and secondary TILs. "Primary TILs" refers to "Secondary TILs" are those obtained from patient tissue samples as outlined herein (sometimes referred to as "freshly harvested"), and "secondary TILs" are any TIL cell populations that have been expanded or propagated as discussed herein, including, but not limited to, bulk TILs, expanded TILs ("REP TILs" or "post-REP TILs"). TIL cell populations may include genetically modified TILs.

[0123]

[0249] As used herein, a "cell population" (including TILs) refers to a group of cells that share a common trait. refers to a large number of cells. Generally, a population is roughly 1 x 10 6 ~1×10 10 The range is 1 x 10, with different TIL populations containing different numbers. For example, the initial growth of primary TILs in the presence of IL-2 is approximately 1 x 10 8 REP expansion cultures typically yield a bulk TIL population of 1.5 x 10 cells. 9 ~1.5×10 10 This is done to provide a population of cells for injection.

[0124]

[0250] As used herein, "cryopreserved TIL" refers to TILs that have been cultured in primary, bulk, or expanded cultures ( Cryopreservation refers to the processing and storage of TILs, either cryopreserved or cryopreserved (e.g., cryopreserved TILs, cryopreserved TILs, or cryopreserved TILs), at temperatures ranging from about -150°C to -60°C. General cryopreservation methods are also described elsewhere herein, including in the Examples. For clarity, "cryopreserved TILs" are distinguishable from frozen tissue samples that may be used as a source of primary TILs.

[0125]

[0251] As used herein, "thawed cryopreserved TILs" refers to TILs that have been previously cryopreserved. By "TILs" is meant a population of TILs that has been cultured at room temperature and then treated to return to room temperature or above, including but not limited to, cell culture temperature or a temperature at which the TILs may be administered to a patient.

[0126]

[0252] TILs can also be generally defined biochemically using cell surface markers. TILs can also be functionally defined by their ability to infiltrate tumors and achieve treatment. TILs can generally be classified by expressing one or more of the following biomarkers: CD4, CD8, TCRαβ, CD27, CD28, CD56, CCR7, CD45Ra, CD95, PD-1, and CD25. Additionally and alternatively, TILs can be functionally defined by their ability to infiltrate solid tumors upon reintroduction into patients.

[0127]

[0253] The term "cryopreservation media" or "cryopreservation medium" refers to any medium that can be used for cryopreserving cells. Such media can include media containing 7%-10% DMSO. Exemplary media include CryoStor CS10, Hyperthermasol, and combinations thereof. The term "CS10" refers to cryopreservation media obtained from Stemcell Technologies or Biolife Solutions. CS10 medium is commercially available. It is sometimes called "CryoStor (registered trademark) CS10." CS10 medium is a serum-free, animal component-free medium containing DMSO.

[0128]

[0254] The term "central memory T cells" refers to cells that are CD45R0+ in humans and CCR7 (CCR7 hi ) and CD62L (CD62 hi Central memory T cells are a subset of T cells that constitutively express CD4 receptors (CD4, CD127, CD127R, and CD15R). The surface phenotype of central memory T cells also includes TCR, CD3, CD127 (IL-7R), and IL-15R. Transcription factors of central memory T cells include BCL-6, BCL-6B, MBD2, and BMI1. After TCR triggering, central memory T cells primarily secrete IL-2 and CD40L as effector molecules. Central memory T cells are predominant in the CD4 compartment of the blood and are proportionally concentrated in lymph nodes and tonsils in humans.

[0129]

[0255] The term "effector memory T cells" is similar to that of central memory T cells. CD45R0+ but lack constitutive expression of CCR7 (CCR7 lo ), and and heterogeneous or low CD62L expression (CD62L lo ), a subset of human or mammalian T cells. The surface phenotype of central memory T cells also includes TCR, CD3, CD127 (IL-7R), and IL-15R. Transcription factors on central memory T cells include BLIMP1. After antigen stimulation, effector memory T cells rapidly secrete high levels of inflammatory cytokines, including interferon-γ, IL-4, and IL-5. Effector memory T cells predominate in the CD8 compartment in the blood and, in humans, are proportionally enriched in the lungs, liver, and intestine. CD8+ effector memory T cells possess high amounts of perforin.

[0130]

[0256] The term "closed system" refers to a system that is closed to the outside environment. The method can use any closed system suitable for cell culture methods, including, but not limited to, a closed G-container. After tumor segments are added to the closed system, the system is not open to the external environment until immediately prior to administration of the TILs to a patient.

[0131]

[0257] The terms "fragment," "fragment," and "fragmented" refer to the destruction process of a tumor. As used herein to describe processes, includes mechanical fragmentation methods such as crushing, slicing, splitting, and mincing tumor tissue, as well as any other method that disrupts the physical structure of tumor tissue.

[0132]

[0258] The terms "peripheral blood mononuclear cells" and "PBMCs" refer to lymphocytes (T cells, B cells, N cells, etc.). PBMC refers to peripheral blood cells with round nuclei, including PBMCs (K cells) and monocytes. Preferably, the peripheral blood mononuclear cells are irradiated allogeneic peripheral blood mononuclear cells. PBMCs are a type of antigen-presenting cell.

[0133]

[0259] The term "anti-CD3 antibody" refers to an antibody or variant thereof, e.g., a monoclonal antibody. and includes human, humanized, chimeric, or murine antibodies directed against the CD3 receptor in the T cell antigen receptor of mature T cells. Anti-CD3 antibodies include OKT-3, also known as muromonab. Anti-CD3 antibodies also include the UHCT1 clone, also known as T3 and CD3ε. Other anti-CD3 antibodies include, for example, otelixizumab, teplizumab, and visilizumab.

[0134]

[0260] The term "OKT-3" (also referred to herein as "OKT3") refers to the mature T "CD3" refers to monoclonal antibodies, including human, humanized, chimeric, or murine antibodies, directed against the CD3 receptor in the T cell antigen receptor of T cells, or biosimilars or variants thereof, including commercially available forms such as OKT-3 (30 ng / mL, MACS GMP CD3 pure, Miltenyi Biotech, Inc., San Diego, CA, USA) and muromonab or its variants, conservative amino acid substitutions, glycoforms, or biosimilars. The amino acid sequences of the heavy and light chains of muromonab are provided in Table 1 (SEQ ID NO: 1 and SEQ ID NO: 2). A hybridoma capable of producing OKT-3 has been deposited with the American Type Culture Collection and has been assigned ATCC accession number CRL 8001. A hybridoma capable of producing OKT-3 has also been deposited with the European Collection of Authenticated Cell Cultures (ECACC) and has been assigned catalog number 86022706.

[0135] [Table 1]

[0136]

[0261] The term "IL-2" (also referred to herein as "IL2") refers to interleukin-2 (IL-2). "IL-2" refers to the T cell growth factor known as IL-2, and includes all forms of IL-2, including its human and mammalian forms, conservative amino acid substitutions, glycoforms, biosimilars, and variants. IL-2 is described, for example, in Nelson, J. Immunol. 2004, 172, 3983-88 and Malek, Annu. Rev. Immunol. 2008, 26, 453-79, the disclosures of which are incorporated herein by reference. The amino acid sequence of recombinant human IL-2 suitable for use in the present invention is provided in Table 2 (SEQ ID NO: 3). For example, the term IL-2 includes human recombinant forms of IL-2, such as aldesleukin (PROLEUKIN, 22 million I / O per single-use vial). (commercially available in the U.S. from multiple suppliers), as well as forms of recombinant IL-2 commercially supplied by CellGenix, Inc., Portsmouth, NH, USA (CELLGRO GMP) or ProSpec-Tany TechnoGene Ltd., East Brunswick, NJ, USA (catalog no. CYT-209-b) and other commercially available Other commercially available equivalents from commercial vendors are encompassed. Aldesleukin (des-alanyl-1, serine-125 human IL-2) is a non-glycosylated human recombinant form of IL-2 with a molecular weight of approximately 15 kDa. The amino acid sequence of an aldesleukin suitable for use in the present invention is provided in Table 2 (SEQ ID NO: 4). The term IL-2 also includes the pegylated IL2 prodrug NKTR-214, available from Nektar Therapeutics, South San Francisco, CA, USA, and is used herein. Also encompassed are pegylated forms of IL-2, as described in U.S. Patent Application Publication Nos. 2014 / 0328791 A1 and WO 2012 / 065086 A1, the disclosures of which are incorporated herein by reference. Other forms of conjugated IL-2 suitable for use in the present invention are described in U.S. Patent Nos. 4,766,106, 5,206,344, 5,089,261, and 4902,502, the disclosures of which are incorporated herein by reference. Formulations of IL-2 suitable for use in the present invention are described in U.S. Patent No. 6,706,289, the disclosure of which is incorporated herein by reference.

[0137] [Table 2]

[0138]

[0262] The term "IL-4" (also referred to herein as "IL4") refers to interleukin-4 (IL-4). IL-4 refers to a cytokine known as IL-4, which is produced by Th2 T cells, as well as by eosinophils, basophils, and mast cells. IL-4 regulates the differentiation of naive helper T cells (Th0 cells) into Th2 T cells. Steinke and Borish, Respir. Res. 2001, 2, 66-70. Activation by IL-4 subsequently leads to the differentiation of Th2 T cells. The cells produce additional IL-4 in a positive feedback loop. IL-4 also stimulates B cell proliferation and class II MHC expression, and induces class switching from B cells to IgE and IgG1 expression. Recombinant human IL-4 suitable for use in the present invention is available from ProSpec-Tany TechnoGene Ltd., East Brunswick, NJ, USA (catalog number CYT-211) and ThermoFisher Scientific, Inc., Waltham, MA, USA (human IL-15 recombinant protein, catalog number CYT-212). It is commercially available from several suppliers, including Gibco (Log No. CTP0043). Use in the present invention The amino acid sequence of a suitable recombinant human IL-4 is provided in Table 2 (SEQ ID NO: 5).

[0139]

[0263] The term "IL-7" (also referred to herein as "IL7") refers to an interleukin-7 (IL7) It refers to a glycosylated tissue-derived cytokine known as ikine 7, which is available from stromal and epithelial cells, as well as dendritic cells. Fry and Mackall, Blood 2002, 99, 3892-904. IL-7 can stimulate T cell development. IL-7 binds to the heterodimeric IL-7 receptor, consisting of the IL-7 receptor α and the common γ chain receptor, which provides a series of signals important for T cell development in the thymus and survival in the periphery. Recombinant human IL-7 suitable for use in the present invention is available from ProSpec-Tany TechnoGene Ltd., East Brunswick, NJ, USA (Catalog). It is commercially available from several suppliers, including ThermoFisher Scientific, Inc., Waltham, MA, USA (human IL-15 recombinant protein, catalog number CYT-254) and ThermoFisher Scientific, Inc., Waltham, MA, USA (human IL-15 recombinant protein, catalog number Gibco PHC0071). The amino acid sequence of a recombinant human IL-7 suitable for use in the present invention is provided in Table 2 (SEQ ID NO: 6).

[0140]

[0264] The term "IL-15" (also referred to herein as "IL15") refers to an interferon-like protein (IL-15). The term "IL-2" refers to the T-cell growth factor known as IL-15, and includes all forms of IL-2, including its human and mammalian forms, conservative amino acid substitutions, glycoforms, biosimilars, and variants. IL-15 is described, for example, in Fehniger and Caligiuri, Blood 2001, 97, 14-32, the disclosure of which is incorporated herein by reference. IL-15 shares the β and γ signaling receptor subunits with IL-2. Recombinant human IL-15 is a single, non-glycosylated polypeptide chain containing 114 amino acids (and an N-terminal methionine) with a molecular mass of 12.8 kDa. Recombinant human IL-15 is commercially available from several suppliers, including ProSpec-Tany TechnoGene Ltd., East Brunswick, NJ, USA (catalog number CYT-230-b) and ThermoFisherScientific, Inc., Waltham, MA, USA (human IL-15 recombinant protein, catalog number 34-8159-82). The amino acid sequence of recombinant human IL-15 suitable for use in the present invention is provided in Table 2 (SEQ ID NO: 7).

[0141]

[0265] The term "IL-21" (also referred to herein as "IL21") refers to an interleukin-21 (IL-21) "IL-21" refers to the pleiotropic cytokine protein known as IL-21, and includes all forms of IL-21, including its human and mammalian forms, conservative amino acid substitutions, glycoforms, biosimilars, and variants. IL-21 is described, for example, in Spolski and Leonard, Nat. Rev. Drug. Disc. 2014, 13, 379-95, the disclosure of which is incorporated by reference. IL-21 is primarily a cytotoxic agent that stimulates natural killer T cells and activated human CD4 + It is produced by T cells. Recombinant human IL-21 is a single, non-glycosylated polypeptide chain containing 132 amino acids with a molecular mass of 15.4 kDa. Recombinant human IL-21 is commercially available from several suppliers, including ProSpec-Tany TechnoGene Ltd., East Brunswick, NJ, USA (catalog number CYT-408-b) and ThermoFisherScientific, Inc., Waltham, MA, USA (human IL-21 recombinant protein, catalog number 14-8219-80). The amino acid sequence of recombinant human IL-21 suitable for use in the present invention is provided in Table 2 (SEQ ID NO: 8).

[0142]

[0266] When an "antitumor effective amount," "tumor inhibiting effective amount," or "therapeutic amount" is indicated The exact dosage of the compositions of the present invention can be determined by a physician, taking into account individual differences in age, weight, tumor size, degree of infection or metastasis, and condition of the patient (subject). Generally, pharmaceutical compositions comprising tumor-infiltrating lymphocytes (e.g., secondary TILs or genetically modified cytotoxic lymphocytes) described herein are administered in a dose of 100 mg / kg or more. 4 ~10 11 cells / kg body weight (e.g., 10 5 ~10 6 , 10 5 ~10 10 , 10 5 ~10 11 , 10 6 ~10 10 , 10 6 ~10 11 , 10 7 ~10 11 , 10 7 ~10 10 , 10 8 ~10 11 , 10 8 ~10 10 , 10 9 ~10 11 , or 10 9 ~10 10 It can be said that the tumor-infiltrating lymphocyte (optionally including genetically modified cytotoxic lymphocytes) compositions may be administered at a dosage of 1000 mg / kg body weight (cells / kg body weight) (including all integer values ​​within these ranges). The tumor-infiltrating lymphocyte (optionally including genetically modified cytotoxic lymphocytes) compositions may also be administered multiple times at these dosages. The tumor-infiltrating lymphocytes (including genetically modified ones in some cases) may be administered by using injection techniques commonly known in immunotherapy (see, e.g., Rosenberg et al., New Eng. J. of Med. 319: 1676, 1988). Optimal Dosage for a Particular Patient and treatment regimes can be readily determined by one skilled in the medical arts by monitoring the patient for signs of disease and adjusting treatment accordingly.

[0143]

[0267] The term "hematological malignancies" includes, but is not limited to, tumors of the blood, bone marrow, lymph nodes, The term "B-cell hematological malignancies" refers to cancers and tumors of hematopoietic and lymphoid tissues in mammals, including hematopoietic and lymphoid tissues. Hematological malignancies are also referred to as "liquid tumors." Hematological malignancies include, but are not limited to, acute lymphoblastic leukemia (ALL), chronic lymphocytic lymphoma (CLL), small lymphocytic lymphoma (SLL), acute myeloid leukemia (AML), chronic myelocytic leukemia (CML), acute monocytic leukemia (AMoL), Hodgkin's lymphoma, and non-Hodgkin's lymphoma. The term "B-cell hematological malignancies" refers to hematological malignancies affecting B cells.

[0144]

[0268] The term "solid tumor" refers to an abnormal mass of tissue that usually does not contain cysts or fluid areas. Solid tumors can be benign or malignant. The term "solid tumor cancer" refers to a malignant, neoplastic, or cancerous solid tumor. Solid tumor cancers include, but are not limited to, lung cancer, breast cancer, prostate cancer, These include sarcomas, carcinomas, and lymphomas, such as colon, rectum, and bladder cancer. The histology of solid tumors comprises interdependent tissue compartments, including parenchyma (cancer cells) and supporting stromal cells, which may provide a supportive microenvironment within which the cancer cells are dispersed.

[0145]

[0269] The term "liquid tumor" refers to an abnormal mass of cells that is fluid in nature. Examples include, but are not limited to, leukemia, myeloma, and lymphoma, as well as other hematological malignancies. TILs obtained from liquid tumors may also be referred to herein as bone marrow infiltrating lymphocytes (MILs).

[0146]

[0270] The term "microenvironment" as used herein refers to the microenvironment of a solid tumor or a hematological tumor. The term "tumor microenvironment" as used herein may refer to the entire tumor microenvironment or individual cell subsets within the microenvironment. Tumor microenvironment, as used herein, is defined as described in Swartz, et al., Cancer Res., 2012, 72, 2473. As described in [the text], tumor clearance refers to a complex mixture of "cells, soluble factors, signaling molecules, extracellular matrix, and mechanical cues that promote neoplastic transformation, support tumor growth and invasion, protect tumors from host immunity, foster therapeutic resistance, and provide a niche for the development of overt metastases." Tumors express antigens that must be recognized by T cells, but tumor clearance by the immune system is rare due to the immunosuppressive microenvironment.

[0147]

[0271] In one embodiment, the invention includes a method of treating cancer with a TIL population, comprising: wherein the patient is conditioned with non-myeloablative chemotherapy prior to the infusion of TILs of the present invention. In some embodiments, a TIL population may be provided, wherein the patient is conditioned with non-myeloablative chemotherapy prior to the infusion of TILs of the present invention. In one embodiment, the non-myeloablative chemotherapy is cyclophosphamide 60 mg / kg / day for two days (days 27 and 26 prior to TIL infusion) and fludarabine 25 mg / m2 / day for five days (days 27-23 prior to TIL infusion). In one embodiment, after non-myeloablative chemotherapy and TIL infusion of the present invention (day 0), the patient receives an intravenous infusion of 720,000 IU / kg of IL-2 intravenously every 8 hours to a physiologically tolerated dose.

[0148]

[0272] Experimental findings suggest that lymphodepletion prior to adoptive transfer of tumor-specific T lymphocytes may be beneficial for the preparation of tumor-specific T lymphocytes. It has been suggested that removal of nodal T cells and competing elements of the immune system ("cytokine sinks") plays an important role in enhancing therapeutic efficacy. Accordingly, some embodiments of the present invention utilize a lymphodepletion step (also referred to as "immunosuppressive conditioning") on patients prior to introducing the rTILs of the present invention.

[0149]

[0273] The terms "co-administration," "co-administer," "administered in conjunction with," and "concurrently with" "Administered in combination with," "simultaneously," and "concurrently," as used herein, encompass administration of two or more active pharmaceutical ingredients (e.g., at least one potassium channel agonist in combination with multiple TILs) to a subject such that both active pharmaceutical ingredients and / or their metabolites are present in the subject at the same time. Co-administration includes simultaneous administration in separate compositions, administration at different times in separate compositions, or administration in a composition in which two or more active pharmaceutical ingredients are present. Simultaneous administration in separate compositions and administration in a composition in which both agents are present are preferred.

[0150]

[0274] The term "effective amount" or "therapeutically effective amount" refers to any amount of a compound used in the treatment of a disease, including but not limited to the treatment of a disease. "Therapeutically effective" refers to an amount of a compound or combination of compounds as described herein sufficient to achieve the intended application. A therapeutically effective amount may vary depending on the intended application (in vitro or in vivo), or the subject and disease condition under treatment (e.g., the subject's weight, age, and sex), the severity of the disease condition, or the method of administration. The term also applies to a dose that will produce a particular response in target cells (e.g., reduced platelet adhesion and / or cell migration). Specific doses will depend on the particular compound selected, the dosing regimen to be followed, and the compound's interaction with other compounds. It will depend on whether they are administered in combination, the timing of administration, the tissue to which they are administered, and the physical delivery system by which the compounds are carried.

[0151]

[0275] The terms "treatment," "treating," "treat," and the like refer to the desired pharmacological and "Treatment" refers to achieving a therapeutic and / or physiological effect. The effect may be prophylactic, meaning that a disease or its symptoms are completely or partially prevented, and / or therapeutic, meaning that a disease and / or adverse effects resulting from the disease are partially or completely cured. "Treatment," as used herein, encompasses any treatment of disease in a mammal, particularly a human, and includes (a) preventing the occurrence of a disease in a subject who may be predisposed to the disease but has not yet been diagnosed as having it; (b) inhibiting the disease, i.e., halting its onset or progression; and (c) palliating the disease, i.e., causing regression of the disease and / or alleviating one or more disease symptoms. "Treatment" is also intended to encompass the delivery of an agent to provide a pharmacological effect even in the absence of a disease or condition. For example, "treatment" encompasses the delivery of a composition capable of eliciting an immune response or conferring immunity in the absence of a disease state, e.g., in the case of a vaccine.

[0152]

[0276] The term "heterologous" when used in reference to a portion of a nucleic acid or protein Indicates that the nucleic acid or protein comprises two or more subsequences that are not found in the same relationship to each other in nature. For example, nucleic acids are typically produced recombinantly, having two or more sequences from unrelated genes arranged to create a novel functional nucleic acid, e.g., a promoter from one source and a coding region from another source, or coding regions from different sources. Similarly, a heterologous protein indicates that the protein comprises two or more subsequences that are not found in the same relationship to each other in nature (e.g., a fusion protein).

[0153]

[0277] The terms "sequence identity," "percent identity," and "sequence identity" in the context of two or more nucleic acids or polypeptides are used interchangeably. "Sequence identity," "sequence identity," and "sequence percent identity" (or their equivalents, e.g., "99% identity") refer to two or more sequences or subsequences that are the same or have a specified percentage of identical nucleotides or amino acid residues when compared and aligned (introducing gaps as necessary) for maximum correspondence, not considering any conservative amino acid substitutions as part of the sequence identity. Percent identity can be measured using sequence comparison software or algorithms or by visual inspection. A variety of algorithms and software are known in the art that can be used to achieve alignment of amino acid or nucleotide sequences. Suitable programs for determining percent sequence identity include, for example, the BLAST program available from the U.S. government's National Center for Biotechnology Information BLAST website. Comparisons between two sequences can be performed using either the BLASTN or BLASTP algorithm. BLASTN is used to compare nucleic acid sequences, while BLASTP is used to compare amino acid sequences. ALIGN, ALIGN-2 (Genentech, South San Francisco, California), or MegAlign available from DNASTAR can be used to align sequences. Further publicly available software programs are available for use with the alignment software. Those skilled in the art can determine appropriate parameters to maximize alignment with a particular alignment software. In certain embodiments, the default parameters of the alignment software are used.

[0154]

[0278] As used herein, the term "variant" includes, but is not limited to, a variant of a reference antigen. Variants include antibodies or fusion proteins containing an amino acid sequence that differs from the amino acid sequence of a reference antibody by one or more substitutions, deletions, and / or additions at specific positions within or adjacent to the amino acid sequence. A variant may contain one or more conservative substitutions in its amino acid sequence compared to the amino acid sequence of the reference antibody. Conservative substitutions may include, for example, substitutions of similarly charged or uncharged amino acids. A variant retains the ability of the reference antibody to specifically bind to the antigen. The term variant also includes pegylated antibodies or proteins.

[0155]

[0279] As used herein, "tumor infiltrating lymphocytes" or "TILs" refer to cells that migrate through the bloodstream of a subject. TILs refer to a population of cells originally acquired as white blood cells that have migrated away from and into a tumor. TILs include, but are not limited to, CD8 + Cytotoxic T cells (lymphocytes), Th1 and Th17 CD4 + These include T cells, natural killer cells, dendritic cells, and M1 macrophages. TILs include both primary and secondary TILs. "Primary TILs" are those obtained from a patient tissue sample as outlined herein (sometimes referred to as "freshly harvested"), and "secondary TILs" are any TIL cell populations expanded or grown as discussed herein, including, but not limited to, bulk TILs, expanded TILs ("REP TILs"), and "reREP TILs" as discussed herein. reREP TILs can include, for example, second expanded TILs or second additional expanded TILs (e.g., such as those described in step D of FIG. 27, which include TILs designated as reREP TILs).

[0156]

[0280] TILs can also be generally defined biochemically using cell surface markers. TILs can also be functionally defined by their ability to infiltrate tumors and achieve treatment. TILs can generally be classified by expressing one or more of the following biomarkers: CD4, CD8, TCRαβ, CD27, CD28, CD56, CCR7, CD45Ra, CD95, PD-1, and CD25. Additionally and alternatively, TILs can be functionally defined by their ability to infiltrate solid tumors upon reintroduction into a patient. TILs may also be characterized by potency—for example, TILs can be considered potent if, for example, interferon (IFN) release is greater than about 50 pg / mL, greater than about 100 pg / mL, greater than about 150 pg / mL, or greater than about 200 pg / mL.

[0157]

[0281] The term "pharmaceutically acceptable carrier" or "pharmaceutically acceptable excipient" means a This includes any solvents, dispersion media, coatings, antibacterial and antifungal agents, isotonic and absorption delaying agents, and inactive ingredients. The use of such pharmaceutically acceptable carriers or excipients for active pharmaceutical ingredients is well known in the art. Except insofar as any conventional pharmaceutically acceptable carrier or excipient is incompatible with the active pharmaceutical ingredient, its use in the therapeutic compositions of the present invention is contemplated. Additional active pharmaceutical ingredients, such as other drugs, may also be incorporated into the compositions and methods described.

[0158]

[0282] The terms "about" and "approximately" refer to within a statistically significant range of values. The ranges described may be within one order of magnitude of a given value or range, preferably within 50%, more preferably within 20%, even more preferably within 10%, and even more preferably within 5%. The allowable variation encompassed by the term "about" or "approximately" depends on the particular system under study and is readily apparent to one of ordinary skill in the art. Furthermore, as used herein, the terms "about" and "approximately" mean that dimensions, sizes, formulations, parameters, shapes, and other quantities and characteristics are not exact, but may be approximate and / or larger or smaller, as appropriate, reflecting tolerances, conversion factors, rounding, measurement error, etc., and other factors known to those of ordinary skill in the art. In general, a dimension, size, formulation, parameter, shape, or other quantity or characteristic is "about" or "approximately" whether or not explicitly stated as such. It should be noted that embodiments of widely differing sizes, shapes, and dimensions may employ the described terminology.

[0159]

[0283] As used in the appended claims, the terms "comprising," "consisting essentially of" and "composing" are intended to be used interchangeably. The transitional terms "comprising" and "consisting of" are used in conjunction with the original and modified forms to exclude additional unrecited claim elements or steps, if any, from the scope of the claim. The claims are defined in a format similar to the one described above. The term "comprising" is intended to be inclusive or open-ended and does not exclude additional, unrecited elements, methods, steps, or materials. The term "consisting of" excludes elements, steps, or materials other than those specified in the claim, and in the latter case, excludes normal impurities associated with the specified materials. The term "consisting essentially of" limits the claim to the specified elements, steps, or materials and to those that do not materially affect the basic and novel characteristics of the claimed invention. All compositions, methods, and kits described herein that embody the present invention may, in alternative embodiments, be more specifically defined by any of the transitional terms "comprising," "consisting essentially of," and "consisting of."

[0160] III. TIL manufacturing method

[0284] The TIL process known as Process 2A includes some of these characteristics: An example of Process 2A is shown in Figure 1, and some of the advantages of this embodiment of the invention over Process 1C are shown in Figure 2, as well as Figure 84. Process 1C is shown for comparison in Figure 3. Two alternative timelines for TIL treatment based on Process 2A are shown in Figure 4 (high cell number) and Figure 5 (low cell number). An embodiment of Process 2A is shown in Figures 6 and 27. Figures 83 and 84 further provide an exemplary 2A process compared to the exemplary 1C process.

[0161]

[0285] As discussed herein, the present invention provides a method for cryopreserving cells prior to transplantation into a patient. This may include steps related to restimulating TILs to increase their metabolic activity, and thus their relative health, and methods for testing said metabolic health. As generally outlined herein, TILs are generally obtained from patient samples and manipulated to expand their numbers prior to transplantation into the patient. In some embodiments, TILs may optionally be genetically engineered as discussed below.

[0162]

[0286] In some embodiments, the TILs may be cryopreserved. After thawing, the TILs may be: Before infusion into the patient, it may be restimulated to enhance its metabolism.

[0163]

[0287] In some embodiments, as discussed in detail below and in the examples and figures, , the first expansion culture (including a process referred to as Pre-REP and shown as step A in FIG. 27) is shortened to 3-14 days, and the second expansion culture (including a process referred to as REP and shown as step B in FIG. 27) is shortened to 7-14 days. In some embodiments, as discussed in the Examples and shown in FIGS. 4, 5, and 27, the first expansion culture (e.g., the expansion culture described as step B in FIG. 27) is shortened to 11 days, and the second expansion culture (e.g., the expansion culture described as step D in FIG. 27) is shortened to 11 days. In some embodiments, as discussed in detail below and in the Examples and Figures, the combined first and second expansion cultures (e.g., the expansion cultures described as steps B and D in FIG. 27) are shortened to 22 days.

[0164]

[0288] The following "step" names A, B, C, etc. refer to FIG. 27 and are described herein. With reference to the embodiments described above, the order of steps below and in Figure 27 is exemplary, and any combination or order of steps, as well as additional steps, repeated steps, and / or omission of steps, is contemplated by the present application and methods disclosed herein.

[0165] A. Step A: Obtain a patient tumor sample

[0289] Generally, TILs are initially obtained from patient tumor samples ("primary TILs") and then The TILs are expanded into larger populations for further manipulation as described herein, optionally cryopreserved, restimulated as outlined herein, and optionally evaluated for phenotypic and metabolic parameters as indicators of TIL health.

[0166]

[0290] Patient tumor samples are generally obtained by surgical extraction using methods known in the art. The sample may be obtained by resection, needle biopsy, or other means for obtaining a sample containing a mixture of tumor and TIL cells. Generally, the tumor sample may be from any solid tumor, including primary, invasive, or metastatic tumors. The tumor sample may also be from a liquid tumor, such as a tumor obtained from a hematological malignancy. The solid tumor may be of any cancer type, including, but not limited to, breast cancer, pancreatic cancer, prostate cancer, colorectal cancer, lung cancer, brain cancer, renal cancer, gastric cancer, and skin cancer (including, but not limited to, squamous cell carcinoma, basal cell carcinoma, and melanoma). In some embodiments, useful TILs are obtained from malignant melanoma tumors, which have been reported to have particularly high levels of TILs.

[0167]

[0291] The term "solid tumor" refers to an abnormal mass of tissue that usually does not contain cysts or fluid areas. Solid tumors can be benign or malignant. The term "solid tumor cancer" refers to a malignant, neoplastic, or cancerous solid tumor. Solid tumor cancers include, but are not limited to, sarcomas, carcinomas, and lymphomas, such as lung cancer, breast cancer, triple-negative breast cancer, prostate cancer, colon cancer, rectal cancer, and bladder cancer. In some embodiments, the cancer is selected from cervical cancer, head and neck cancer (including head and neck squamous cell carcinoma (HNSCC)), glioblastoma, ovarian cancer, sarcoma, pancreatic cancer, bladder cancer, breast cancer, triple-negative breast cancer, and non-small cell lung cancer. The histology of solid tumors comprises interdependent tissue compartments, including parenchyma (cancer cells) and supporting stromal cells, which may provide a supportive microenvironment within which the cancer cells are dispersed.

[0168]

[0292] The term "hematological malignancies" includes, but is not limited to, tumors of the blood, bone marrow, lymph nodes, The term "B-cell hematological malignancies" refers to cancers and tumors of hematopoietic and lymphoid tissues in mammals, including hematopoietic and lymphoid tissues. Hematological malignancies are also referred to as "liquid tumors." Hematological malignancies include, but are not limited to, acute lymphoblastic leukemia (ALL), chronic lymphocytic lymphoma (CLL), small lymphocytic lymphoma (SLL), acute myeloid leukemia (AML), chronic myelocytic leukemia (CML), acute monocytic leukemia (AMoL), Hodgkin's lymphoma, and non-Hodgkin's lymphoma. The term "B-cell hematological malignancies" refers to hematological malignancies affecting B cells.

[0169]

[0293] Once obtained, tumor samples are typically cut into sections of 1 to approximately 8 mm using sharp scraping. 3 fragmented into small pieces Approximately 2 to 3 mm 3 are particularly useful. TILs are cultured from these fragments using enzymatic tumor digests. Such tumor digests are prepared in an enzymatic medium (e.g., Roswell Park Memorial Institute (RPMI) 1640 buffer, 2 mM glutamic acid, 100 mM erythritol, 100 mM urea ... Tumor digests may be produced by incubation in a 100% soluble medium containing 100% TILs (100% TILs, 10 mcg / mL gentamicin, 30 units / mL DNase, and 1.0 mg / mL collagenase) followed by mechanical dissociation (e.g., using a tissue dissociator). Tumor digests may be produced by placing the tumor in enzyme medium, mechanically dissociating the tumor for approximately 1 minute, followed by incubation at 37°C under 5% CO2 for 30 minutes, and then repeating cycles of mechanical dissociation and incubation under the aforementioned conditions until only small tissue fragments are present. If the cell suspension at the end of this process contains numerous red blood cells or dead cells, density gradient separation using FICOLL branched hydrophilic polysaccharides may be performed to remove these cells. Alternative methods known in the art, such as those described in U.S. Patent Application Publication No. 2012 / 0244133 A1 (the disclosure of which is incorporated herein by reference), may also be used. Any of the aforementioned methods may be used in any of the embodiments described herein for the methods of expanding TILs or treating cancer.

[0170]

[0294] Generally, harvested cell suspensions are referred to as "primary cell populations" or "freshly harvested." These are called cell populations.

[0171]

[0295] In some embodiments, fragmentation can be physical, including, for example, peeling and digestion. In some embodiments, the fragmentation is physical fragmentation. In some embodiments, the fragmentation is by exfoliation. In some embodiments, the fragmentation is by digestion. In some embodiments, the TILs can be initially cultured from enzymatic tumor digests and tumor fragments obtained from the patient. In one embodiment, the TILs can be initially cultured from enzymatic tumor digests and tumor fragments obtained from the patient.

[0172]

[0296] In some embodiments, if the tumor is a solid tumor, e.g., step A( After obtaining a tumor sample (as provided in FIG. 27), the tumor undergoes physical fragmentation. In some embodiments, fragmentation occurs before cryopreservation. In some embodiments, fragmentation occurs after cryopreservation. In some embodiments, fragmentation occurs after obtaining the tumor without any cryopreservation. In some embodiments, the tumor is fragmented and 10, 20, 30, 40 or more fragments or pieces are placed in each container for first expansion culture. In some embodiments, the tumor is fragmented and 30 or 40 fragments or pieces are placed in each container for first expansion culture. In some embodiments, the tumor is fragmented and 40 fragments or pieces are placed in each container for first expansion culture. In some embodiments, the plurality of fragments comprises about 4 to about 50 fragments, each fragment being about 27 mm. 3 In some embodiments, the plurality of pieces has a volume of about 1300 mm 3 ~about 1500mm 3 In some embodiments, the plurality of pieces comprises about 30 to about 60 pieces having a total volume of about 1350 mm 3In some embodiments, the plurality of fragments comprises about 50 fragments having a total mass of about 1 g to about 1.5 g. In some embodiments, the plurality of fragments comprises about 4 fragments.

[0173]

[0297] In some embodiments, the TILs are obtained from tumor fragments. In some embodiments, the tumor fragments are obtained by sharp dissection. 3 ~10mm 3 In some embodiments, the tumor fragment is about 1 mm 3 ~8mm 3 In some embodiments, the tumor fragment is about 1 mm 3 In some embodiments, the tumor fragment is about 2 mm 3 In some embodiments, the tumor fragment is about 3 mm 3 In some embodiments, the tumor fragment is about 4 mm 3 In some embodiments, the tumor fragment is about 5 mm 3 In some embodiments, the tumor fragment is about 6 mm 3 In some embodiments, the tumor fragment is about 7 mm 3 In some embodiments, the tumor fragment is about 8 mm 3 In some embodiments, the tumor fragment is about 9 mm 3 In some embodiments, the tumor fragment is about 10 mm 3 is.

[0174]

[0298] In some embodiments, the TILs are obtained from tumor digests. In this embodiment, tumor digests were generated by incubation in enzyme medium, such as, but not limited to, RPMI 1640, 2 mM GlutaMAX, 10 mg / mL gentamicin, 30 U / mL DNase, and 1.0 mg / mL collagenase, followed by mechanical dissociation (GentleMACS, Miltenyi Biotec, Auburn, CA). Tumors were placed in enzyme medium. After the incubation, the tumor may be mechanically dissociated for about 1 minute. The solution may then be incubated at 37°C under 5% CO2 for 30 minutes, and then it may be mechanically disrupted again for about 1 minute. After again incubating at 37°C under 5% CO2 for 30 minutes, the tumor may be mechanically disrupted a third time for about 1 minute. In some embodiments, if large tissue debris was present after the third mechanical disruption, the sample was subjected to one or two additional rounds of mechanical dissociation, with or without an additional 30-minute incubation at 37°C under 5% CO2. In some embodiments, if the cell suspension at the end of the final incubation contained a large number of red blood cells or dead cells, density gradient separation using Ficoll may be performed to remove such cells.

[0175]

[0299] In some embodiments, the harvested cell suspension prior to the first expansion step The fluid is referred to as a "primary cell population" or a "freshly harvested" cell population.

[0176]

[0300] In some embodiments, the cells are soluble in a medium containing soluble cellulose, as described in more detail below, also shown in FIG. Optionally, the cells may be frozen after sampling and stored frozen before proceeding to expansion as described in step B of 7.

[0177] B. Step B: First Expansion Culture 1.Young TIL

[0301] In some embodiments, the method includes administering to a subject / patient a replication cycle This provides for obtaining immature TILs that can expand the number of TILs in a given cell population, and thus may provide additional therapeutic advantages over mature TILs (i.e., TILs that have undergone more rounds of replication before administration to a subject / patient). Characteristics of immature TILs have been described in the literature. See, for example, Donia, at al., Scandinavian Journal of Immunology, 75:157-167 (2012), Dudley et al., Clin Cancer Res, 16:6122-6131 (2010), Huang et al., J Immunother, 28(3):258-267 (2005), Besser et al., Clin Cancer Res, 19(17):OF1-OF9 (2013), Besser et al., J Immunother 32:415-423 (2009), Robbins, et al., J Immunol 2004; 173:7125-7130, Shen et al., J Immunother, 30:123-129 (2007), Zhou, et al., J Immunother, 28:53-62 (2005), and Tran, et al., J Immunother, 31:742-751 (2008), all of which No. 6,239,693, which is incorporated herein by reference in its entirety.

[0178]

[0302] The diverse antigen receptors of T and B lymphocytes are expressed by a limited but numerous gene segments. These TILs are produced by somatic recombination of chromosomes. These gene segments, V (variable), D (diversity), J (joining), and C (constant), determine the binding specificity and downstream applications of immunoglobulins and T cell receptors (TCRs). The present invention provides methods for generating TILs that exhibit and increase T cell repertoire diversity. In some embodiments, TILs obtained by the present methods exhibit increased T cell repertoire diversity. In some embodiments, TILs obtained by the present methods exhibit increased T cell repertoire diversity compared to freshly harvested TILs and / or TILs prepared using methods other than those provided herein, including, for example, methods other than those embodied in FIG. 27. In some embodiments, as illustrated in FIG. 83, TILs obtained by the present methods exhibit increased T cell repertoire diversity compared to freshly harvested TILs and / or TILs prepared using the method designated Process 1C. In some embodiments, TILs obtained in the first expansion culture exhibit increased T cell repertoire diversity. In some embodiments, the increased diversity is an increase in immunoglobulin diversity and / or T cell receptor diversity. In some embodiments, the immunoglobulin diversity is in immunoglobulin heavy chains. In some embodiments, the immunoglobulin diversity is in immunoglobulin light chains. In some embodiments, the diversity is in T cell receptors. In some embodiments, the diversity is in one of the T cell receptors selected from the group consisting of alpha, beta, gamma, and delta receptors. In some embodiments, expression of T cell receptor (TCR) alpha and / or beta is increased. In some embodiments, expression of T cell receptor (TCR) alpha is increased. In some embodiments, expression of T cell receptor (TCR) beta is increased. In some embodiments, expression of TCRab (i.e., TCR α / β) is increased.

[0179]

[0303] After detachment or digestion of the tumor fragments, e.g., as described in step A of Figure 27, The resulting cells are cultured in serum containing IL-2 under conditions that favor the growth of TILs over tumor and other cells. In some embodiments, tumor digests are incubated in 2 mL wells in medium containing inactivated human AB serum with 6000 IU / mL of IL-2. This primary cell population is cultured for several days, generally 3-14 days, thereby generating a bulk TIL population, generally about 1 x 10 8 In some embodiments, this primary cell population is cultured for a period of 7-14 days, resulting in a bulk TIL population, generally about 1 x 10 8 In some embodiments, this primary cell population is cultured for a period of 10-14 days, resulting in a bulk TIL population, generally about 1 x 10 8 Bulk TIL cells are obtained. In this embodiment, the primary cell population is cultured for a period of about 11 days, thereby generating a bulk TIL population, generally about 1 x 10 8 Bulk TIL cells are obtained.

[0180]

[0304] In a preferred embodiment, as described below and herein, an initial ball Expansion of TILs can be performed using a rapid TIL expansion step (e.g., such as that described in step B of FIG. 27, which may include a process referred to as pre-REP), followed by a second expansion (step D, which includes a process referred to as a rapid expansion protocol (REP) step), as described below under step D and herein, followed by optional cryopreservation, and then a second step D (which includes a process referred to as a restimulation REP step). TILs obtained by this process may optionally be characterized for phenotypic characteristics and metabolic parameters as described herein.

[0181]

[0305] In an embodiment, TIL cultures are performed in 24-well plates, e.g., Costar24 Started using well cell culture clusters, flat bottom (Corning Incorporated, Corning, NY) If used, each well contained 1 × 10 cells in 2 mL of complete medium (CM) containing IL-2 (6000 IU / mL; Chiron Corp., Emeryville, CA). 6 Tumor digest cells or 1 tumor fragment can be seeded. In some embodiments, tumor fragments are about 1 mm 3 ~10mm 3 is.

[0182]

[0306] In some embodiments, the first expansion culture medium is referred to as " In some embodiments, the CM in step B is RPMI containing GlutaMAX supplemented with 10% human AB serum, 25 mM Hepes, and 10 mg / mL gentamicin. 1640. The culture consists of a 40 mL volume and a 10 cm 2 In embodiments initiated in gas-permeable flasks with gas-permeable silicone bottoms (e.g., G-Rex10; Wilson Wolf Manufacturing, New Brighton, MN) (Figure 1), each flask contains 10-40 x 10 cells in 10-40 mL of IL-2-containing CM. 6 Either 50 live tumor digest cells or 5–30 tumor fragments were loaded. Both G-Rex10 and 24-well plates were incubated in a humidified incubator with 5% CO2. The cells were incubated at 37°C under reduced pressure. Five days after the start of culture, half of the medium was removed and replenished with fresh CM and IL-2. From the fifth day onwards, half of the medium was replaced every 2 to 3 days.

[0183]

[0307] After preparation of tumor fragments, the resulting cells (i.e., fragments) are more abundant than tumor and other cells. The primary cells are cultured in serum containing IL-2 under conditions favorable for TIL growth. In some embodiments, tumor digests are incubated in 2 mL wells in medium containing inactivated human AB serum with 6000 IU / mL of IL-2 (or in some cases in the presence of an aAPC cell population, as outlined herein). This primary cell population is cultured for several days, generally 10-14 days, thereby generating a bulk TIL population, generally about 1 x 10 8 In some embodiments, the growth medium during the first expansion culture contains IL-2 or a variant thereof. In some embodiments, the IL is recombinant human IL-2 (rhIL-2). In some embodiments, the IL-2 stock solution contains 20-30 x 10 cells per 1 mg vial. 6 IU / mg specific activity. In some embodiments, the IL-2 stock solution contains 20-10 IU per 1 mg vial. 6 IU / mg specific activity. In some embodiments, the IL-2 stock solution contains 25×10 6 IU / mg specific activity. In some embodiments, the IL-2 stock solution contains 30×10 6 IU / mg specific activity. In some embodiments, the IL-2 stock solution contains 4-8 x 10 6 In some embodiments, the IL-2 stock solution has a final concentration of 5-7 x 10 IU / mg of IL-2. 6 In some embodiments, the IL-2 stock solution has a final concentration of 6×10 IU / mg of IL-2. 6 In some embodiments, the IL-2 stock solution is prepared as described in Example 4. In some embodiments, the first expansion culture medium contains about 10,000 IU / mL of IL-2, about 9,000 IU / mL of IL-2, about The first expansion culture medium contains 8,000 IU / mL of IL-2, about 7,000 IU / mL of IL-2, about 6,000 IU / mL of IL-2, or about 5,000 IU / mL of IL-2. In some embodiments, the first expansion culture medium contains about 9,000 IU / mL of IL-2 to about 5,000 IU / mL of IL-2. In some embodiments, the first expansion culture medium contains about 8,000 IU / mL of IL-2 to about 6,000 IU / mL of IL-2. In some embodiments, the first expansion culture medium contains about 7,000 IU / mL of IL-2 to about 6,000 IU / mL of IL-2. In some embodiments, the first expansion culture medium contains about 6,000 IU / mL of IL-2. In certain embodiments, the cell culture medium further contains IL-2. In some embodiments, the cell culture medium contains about 3,000 IU / mL of IL-2. In some embodiments, the cell culture medium further comprises IL-2. In some embodiments, the cell culture medium comprises about 3000 IU / mL of IL-2. In some embodiments, the cell culture medium comprises about 1000 IU / mL, about 1500 IU / mL, about 2000 IU / mL, about 2500 IU / mL, about 3000 IU / mL, about 3500 IU / mL, about 4000 IU / mL, about 4500 IU / mL, about 5000 IU / mL, about 5500 IU / mL, about 6000 IU / mL, about 6500 IU / mL, about 7000 IU / mL, about 7500 IU / mL, or about 8000 IU / mL of IL-2. In certain embodiments, the cell culture medium contains between 1000-2000 IU / mL, 2000-3000 IU / mL, 3000-4000 IU / mL, 4000-5000 IU / mL, 5000-6000 IU / mL, 6000-7000 IU / mL, 7000-8000 IU / mL, or about 8000 IU / mL of IL-2.

[0184]

[0308] In some embodiments, the first expansion culture medium contains about 500 IU / mL of IL-1 In some embodiments, the first expansion culture medium contains about 500 IU / mL to about 100 IU / mL of IL-15. In some embodiments, the first expansion culture medium contains about 400 IU / mL to about 100 IU / mL of IL-15. In some embodiments, the first expansion culture medium contains about 300 IU / mL to about 100 IU / mL of IL-15. In some embodiments, the first expansion culture medium comprises about 200 IU / mL of IL-15. In some embodiments, the cell culture medium comprises about 180 IU / mL of IL-15. In one embodiment, the cell culture medium further comprises IL-15. In one preferred embodiment, the cell culture medium comprises about 180 IU / mL of IL-15.

[0185]

[0309] In some embodiments, the first expansion culture medium contains about 20 IU / mL of IL- In some embodiments, the first expansion culture medium contains about 20 IU / mL to about 0.5 IU / mL of IL-21. In some embodiments, the first expansion culture medium contains about 15 IU / mL to about 0.5 IU / mL of IL-21. In some embodiments, the first expansion culture medium contains about 12 IU / mL to about 0.5 IU / mL of IL-21. In some embodiments, the first expansion culture medium contains about 10 IU / mL to about 0.5 IU / mL of IL-21. In some embodiments, the first expansion culture medium contains about 5 IU / mL to about 1 IU / mL of IL-21. In some embodiments, the first expansion culture medium contains about 2 IU / mL of IL-21. In some embodiments, the cell culture medium contains about 1 IU / mL of IL-21. In some embodiments, the cell culture medium contains about 0.5 IU / mL of IL-21. In certain embodiments, the cell culture medium further contains IL-21. Preferred In a preferred embodiment, the cell culture medium comprises about 1 IU / mL of IL-21.

[0186]

[0310] In some embodiments, the first expansion culture medium is referred to as "CM," an abbreviation for culture media. In some embodiments, it is referred to as CM1 (culture medium 1). In some embodiments, the CM consists of RPMI 1640 with GlutaMAX supplemented with 10% human AB serum, 25 mM Hepes, and 10 mg / mL gentamicin. The cultures are grown in a 40 mL volume and 10 cm 2 In embodiments initiated in gas-permeable flasks with gas-permeable silicone bottoms (e.g., G-Rex10; Wilson Wolf Manufacturing, New Brighton, MN) (Figure 1), each flask contains 10-40 x 10 cells in 10-40 mL of IL-2-containing CM. 610 live tumor digest cells or 5-30 tumor fragments were loaded. Each 4-well plate was incubated at 37°C under 5% CO in a humidified incubator. Five days after the start of culture, half of the medium was removed and replenished with fresh CM and IL-2. From day 5 onwards, half of the medium was replaced every 2-3 days. In some embodiments, the CM is CM1 as described in the Examples (see Example 5). In some embodiments, the first expansion culture is performed in the initial cell culture medium or the first cell culture medium. In some embodiments, the initial cell culture medium or the first cell culture medium contains IL-2.

[0187]

[0311] In some embodiments, as discussed in the examples and figures, a first expansion The large-scale culture (e.g., including the process described in step B of FIG. 27, which may include what is sometimes referred to as pre-REP) process is shortened to 3-14 days. In some embodiments, the first expansion culture (e.g., including the process described in step B of FIG. 27, which may include what is sometimes referred to as pre-REP), including the expansion culture discussed in the Examples and described in FIGS. 4 and 5 and, e.g., step B of FIG. 27, is shortened to 7-14 days. In some embodiments, as discussed in the Examples and shown in FIGS. 4 and 5, the first expansion culture of step B is shortened to 10-14 days. In some embodiments, the first expansion culture, including the expansion culture discussed in the Examples and described in FIGS. 4 and 5 and, e.g., step B of FIG. 27, is shortened to 11 days.

[0188]

[0312] In some embodiments, the first TIL expansion culture is 1 day, 2 days, 3 days, 4 days In some embodiments, the first TIL expansion culture may be continued for 1 to 14 days. In some embodiments, the first TIL expansion culture may be continued for 2 to 14 days. In some embodiments, the first TIL expansion culture may be continued for 3 to 14 days. In some embodiments, the first TIL expansion culture may be continued for 4 to 14 days. In some embodiments, the first TIL expansion culture may be continued for 5 to 14 days. In some embodiments, the first TIL expansion culture may be continued for 6 to 14 days. In some embodiments, the first TIL expansion culture may be continued for 7 to 14 days. In some embodiments, the first TIL expansion culture may be continued for 8 to 14 days. In some embodiments, the first TIL expansion culture may be continued for 9 to 14 days. In some embodiments, the first TIL expansion culture may be continued for 10 to 14 days. In some embodiments, the first TIL expansion culture may be continued for 11 to 14 days. In some embodiments, the first TIL expansion culture may be continued for 12 to 14 days. In some embodiments, the first TIL expansion culture may be continued for 13 to 14 days. In some embodiments, the first TIL expansion culture may be continued for 14 days. In some embodiments, the first TIL expansion culture may be continued for 1 to 11 days. In some embodiments, the first TIL expansion culture may be continued for 2 to 11 days. In some embodiments, the first TIL expansion culture may be continued for 3 to 11 days. In some embodiments, the first TIL expansion culture may be continued for 4 to 11 days. In some embodiments, the first TIL expansion culture may be continued for 5 to 11 days. In some embodiments, the first TIL expansion culture may be continued for 6 to 11 days. In some embodiments, the first TIL expansion culture may be continued for 7 to 11 days. In some embodiments, the first TIL expansion culture may be continued for 1 to 11 days. The L expansion culture may be continued for 8 to 11 days. In some embodiments, the first TIL expansion culture may be continued for 9 to 11 days. In some embodiments, the first TIL expansion culture may be continued for 10 to 11 days. In some embodiments, the first TIL expansion culture may be continued for 11 days.

[0189]

[0313] In some embodiments, IL-2, IL-7, IL-15, and / or IL-1 In some embodiments, a combination of IL-2, IL-7, IL-15, and / or IL-21, and optional combinations thereof, may be included in the first expansion culture, including during step B of the process, e.g., according to Figure 27 and as described herein. In some embodiments, a combination of IL-2, IL-15, and IL-21 is used as a combination during the first expansion culture. In some embodiments, a combination of IL-2, IL-15, and IL-21, and optional combinations thereof, may be included in the step B of the process, e.g., according to Figure 27 and as described herein.

[0190]

[0314] In some embodiments, as discussed in the examples and figures, a first expansion The large-scale culture (including, for example, the process described in step B of FIG. 27, referred to as pre-REP) process is shortened to 3-14 days. In some embodiments, as discussed in the Examples and shown in FIGS. 4 and 5, the first expansion culture in step B is shortened to 7-14 days. In some embodiments, as discussed in the Examples and shown in FIGS. 4, 5, and 27, the first expansion culture in step B is shortened to 10-14 days. In some embodiments, as discussed in the Examples and shown in FIGS. 4, 5, and 27, the first expansion culture is shortened to 11 days.

[0191]

[0315] In some embodiments, the first expansion culture, e.g., a step according to FIG. B is performed in a closed bioreactor. In some embodiments, a closed system is used for TIL expansion as described herein. In some embodiments, a single bioreactor is used. In some embodiments, the single bioreactor used is, for example, G-REX-10 or G-REX-100. In some embodiments, a closed bioreactor is used. A chain bioreactor is a single bioreactor.

[0192] C. Step C: Transition from the first expansion culture to the second expansion culture

[0316] In some cases, for example, as shown in Figure 27, The bulk TIL population obtained from the first expansion, including the TIL population obtained from the first expansion, may be immediately cryopreserved using the protocols discussed herein below. Alternatively, the TIL population obtained from the first expansion, referred to as the second TIL population, may be subjected to a second expansion (which may include an expansion sometimes referred to as REP) and then cryopreserved as discussed below. Similarly, when genetically modified TILs are used therapeutically, the first TIL population (which may be referred to as the bulk TIL population) or the second TIL population (which may, in some embodiments, include a population referred to as a REP TIL population) may be subjected to genetic modification for an appropriate treatment before expansion, or after the first expansion but before the second expansion.

[0193]

[0317] In some embodiments, from the first expansion culture (e.g., as shown in FIG. 27), The TILs obtained from the first expansion culture (e.g., from step B as shown in FIG. 27) are stored until phenotyping for selection. In some embodiments, the TILs obtained from the first expansion culture (e.g., from step B as shown in FIG. 27) are not stored and proceed directly to the second expansion culture. In some embodiments, the TILs obtained from the first expansion culture are not cryopreserved after the first expansion culture and before the second expansion culture. In some embodiments, the transition from the first expansion culture to the second expansion culture occurs at about 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, or 14 days from the time fragmentation was performed. In some embodiments, the transition from the first expansion culture to the second expansion culture occurs at about 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, or 14 days from the time fragmentation was performed. The transition from the first expansion culture to the second expansion culture occurs about 3 to 14 days after fragmentation. In some embodiments, the transition from the first expansion culture to the second expansion culture occurs about 4 to 14 days after fragmentation. In some embodiments, the transition from the first expansion culture to the second expansion culture occurs about 4 to 10 days after fragmentation. In some embodiments, the transition from the first expansion culture to the second expansion culture occurs about 7 to 14 days after fragmentation. In some embodiments, the transition from the first expansion culture to the second expansion culture occurs about 14 days after fragmentation.

[0194]

[0318] In some embodiments, the transition from the first expansion culture to the second expansion culture comprises: The transition from the first expansion culture to the second expansion culture is performed 1 day, 2 days, 3 days, 4 days, 5 days, 6 days, 7 days, 8 days, 9 days, 10 days, 11 days, 12 days, 13 days, or 14 days after fragmentation. In some embodiments, the transition from the first expansion culture to the second expansion culture is performed 1 day to 14 days after fragmentation. In some embodiments, the first TIL expansion culture may be continued for 2 days to 14 days. In some embodiments, the transition from the first expansion culture to the second expansion culture is performed 3 days to 14 days after fragmentation. In some embodiments, the transition from the first expansion culture to the second expansion culture is performed 4 days to 14 days after fragmentation. In some embodiments, the transition from the first expansion culture to the second expansion culture is performed 5 days to 14 days after fragmentation. In some embodiments, the transition from the first expansion culture to the second expansion culture is performed 6 days to 14 days after fragmentation. In some embodiments, the transition from the first expansion culture to the second expansion culture is performed 7 days to 14 days after fragmentation. In some embodiments, the transition from the first expansion culture to the second expansion culture is carried out 8 to 14 days after fragmentation. In some embodiments, the transition from the first expansion culture to the second expansion culture is carried out 9 to 14 days after fragmentation. In some embodiments, the transition from the first expansion culture to the second expansion culture is carried out 10 to 14 days after fragmentation. In some embodiments, the transition from the first expansion culture to the second expansion culture is carried out 11 to 14 days after fragmentation. In some embodiments, the transition from the first expansion culture to the second expansion culture is carried out 12 to 14 days after fragmentation. In some embodiments, the transition from the first expansion culture to the second expansion culture is carried out 13 to 14 days after fragmentation. In some embodiments, the transition from the first expansion culture to the second expansion culture is carried out 14 days after fragmentation. In some embodiments, the transition from the first expansion culture to the second expansion culture is carried out 1 to 11 days after fragmentation. In some embodiments, the transition from the first expansion culture to the second expansion culture occurs 2 to 11 days after fragmentation.In some embodiments, the transition from the first expansion culture to the second expansion culture is carried out 3 to 11 days after fragmentation. In some embodiments, the transition from the first expansion culture to the second expansion culture is carried out 4 to 11 days after fragmentation. In some embodiments, the transition from the first expansion culture to the second expansion culture is carried out 5 to 11 days after fragmentation. In some embodiments, the transition from the first expansion culture to the second expansion culture is carried out 6 to 11 days after fragmentation. In some embodiments, the transition from the first expansion culture to the second expansion culture is carried out 7 to 11 days after fragmentation. In some embodiments, the transition from the first expansion culture to the second expansion culture is carried out 8 to 11 days after fragmentation. In some embodiments, the transition from the first expansion culture to the second expansion culture is carried out 9 to 11 days after fragmentation. In some embodiments, the transition from the first expansion culture to the second expansion culture is carried out 10 to 11 days after fragmentation. In some embodiments, the transition from the first expansion culture to the second expansion culture occurs 11 days from the time fragmentation occurs.

[0195]

[0319] In some embodiments, the TILs are cultured in a first expansion culture followed by a second expansion culture. Without prior storage, TILs proceed directly to a second expansion culture (e.g., in some embodiments, , as shown in FIG. 27, no storage occurs during the transition from step B to step D). In some embodiments, the transition occurs in a closed system, as described herein. In some embodiments, the TILs from the first expansion culture are TILs from a second TIL population and proceed directly to the second expansion culture without a transition period.

[0196]

[0320] In some embodiments, the transition from the first expansion culture to the second expansion culture comprises: For example, step C according to Figure 27 is performed in a closed bioreactor. In some embodiments, a closed system is used for TIL expansion as described herein. In some embodiments, a single bioreactor is used. In some embodiments, the single bioreactor used is, for example, a G-REX-10 or G-REX-100. In some embodiments, the closed system bioreactor is a single bioreactor.

[0197] D. Step D: Second Expansion Culture

[0321] In some embodiments, the TIL cell population is, for example, as described in FIG. (The numbers increase after harvest and initial bulk processing, following a transition referred to as steps A and B, and step C). This further expansion is referred to herein as the second expansion, which may include an expansion process commonly referred to in the art as the rapid expansion process (REP, and the process shown in step D of Figure 27). The second expansion can generally be accomplished using culture medium in a gas-permeable vessel containing several components, including feeder cells, a source of cytokines, and an anti-CD3 antibody.

[0198]

[0322] In some embodiments, a second expansion culture of TILs or a second TIL expansion culture The culture (which may include expansion culture, sometimes referred to as REP; and the process shown in step D of FIG. 27) can be carried out using any TIL flask or vessel known to one of skill in the art. In some embodiments, the second TIL expansion culture may continue for 7, 8, 9, 10, 11, 12, 13, or 14 days. In some embodiments, the second TIL expansion culture may continue for about 7 days to about 14 days. In some embodiments, the second TIL expansion culture may continue for about 8 days to about 14 days. In some embodiments, the second TIL expansion culture may continue for about 9 days to about 14 days. In some embodiments, the second TIL expansion culture may continue for about 10 days to about 14 days. In some embodiments, the second TIL expansion culture may continue for about 11 days to about 14 days. In some embodiments, the second TIL expansion culture may continue for about 12 days to about 14 days. In some embodiments, the second TIL expansion culture may continue for about 13 days to about 14 days. In some embodiments, the second TIL expansion culture may proceed for about 14 days.

[0199]

[0323] In one embodiment, the second expansion culture is performed using the methods of the present disclosure (e.g., REP Expansion cultures, including those described as "expansion cultures," as well as the process shown in step D of Figure 27, can be performed in gas-permeable containers. For example, TILs can be rapidly expanded using non-specific T cell receptor stimulation in the presence of interleukin-2 (IL-2) or interleukin-15 (IL-15). Non-specific T cell receptor stimulation can include, for example, about 30 ng / ml of an anti-CD3 antibody, such as OKT3, a mouse monoclonal anti-CD3 antibody (commercially available from Ortho-McNeil, Raritan, NJ, or Miltenyi Biotech, Auburn, CA). (commercially available from BioLegend, San Diego, CA, USA) or UHCT-1 (commercially available from BioLegend, San Diego, CA, USA). The TILs can be expanded to induce further stimulation of the TILs in vitro by including during the second expansion one or more antigens of the cancer, including antigenic portions thereof, such as one or more epitopes, optionally expressed from a vector, such as human leukocyte antigen A2 (HLA-A2) binding peptides, e.g., 0.3 μM MART-1:26-35 (27L) or gpl 00:209-217 (210M), optionally in the presence of a T cell growth factor, such as 300 IU / mL IL-2 or IL-15. Other suitable antigens include, for example, NY-ESO-1, TRP-1, TRPS-1, TRPS-2, and TRPS-3. Examples of antigens that can be used to treat cancer include P-2, tyrosinase cancer antigen, MAGE-A3, SSX-2, and VEGFR2, or antigenic portions thereof. TILs can also be rapidly expanded by restimulation with the same cancer antigen or antigens pulsed onto HLA-A2-expressing antigen-presenting cells. Alternatively, TILs can be further restimulated with, for example, irradiated autologous lymphocytes or irradiated HLA-A2+ allogeneic lymphocytes and IL-2. In some embodiments, restimulation occurs as part of a second expansion culture. In some embodiments, the second expansion culture occurs in the presence of irradiated autologous lymphocytes or with irradiated HLA-A2+ allogeneic lymphocytes and IL-2.

[0200]

[0324] In some embodiments, the cell culture medium further comprises IL-2. In some embodiments, the cell culture medium comprises about 3000 IU / mL of IL-2. In certain embodiments, the cell culture medium further comprises IL-2. In some embodiments, the cell culture medium comprises about 3000 IU / mL of IL-2. In certain embodiments, the cell culture medium comprises about 1000 IU / mL, about 1500 IU / mL, about 2000 IU / mL, about 2500 IU / mL, about 3000 IU / mL, about 3500 IU / mL, about 4000 IU / mL, about 4500 IU / mL, about 5000 IU / mL, about 5500 IU / mL, about 6000 IU / mL, about 6500 IU / mL, about 7000 IU / mL, about 7500 IU / mL, or about 8000 IU / mL of IL-2. In certain embodiments, the cell culture medium comprises between 1000-2000 IU / mL, 2000-3000 IU / mL, 3000-4000 IU / mL, 4000-5000 IU / mL, 5000-6000 IU / mL, 6000-7000 IU / mL, 7000-8000 IU / mL, or 8000 IU / mL of IL-2.

[0201]

[0325] In some embodiments, the cell culture medium comprises an OKT3 antibody. In some embodiments, the cell culture medium comprises about 30 ng / mL of OKT3 antibody. In certain embodiments, the cell culture medium comprises about 0.1 ng / mL, about 0.5 ng / mL, about 1 ng / mL, about 2.5 ng / mL, about 5 ng / mL, about 7.5 ng / mL, about 10 ng / mL, about 15 ng / mL, about 20 ng / mL, about 25 ng / mL, about 30 ng / mL, about 35 ng / mL, about 40 ng / mL, about 50 ng / mL, about 60 ng / mL, about 70 ng / mL, about 80 ng / mL, about 90 ng / mL, about 100 ng / mL, about 200 ng / mL, about 500 ng / mL, and about 1 μg / mL of OKT3 antibody. In certain embodiments, the cell culture medium contains 0.1 ng / mL to 1 ng / mL, 1 ng / mL to 5 ng / mL, 5 ng / mL to 10 ng / mL, 10 ng / mL to 20 ng / mL, 20 ng / mL to 30 ng / mL, 30 ng / mL to 40 ng / mL, 40 ng / mL to 50 ng / mL, and 50 ng / mL to 100 ng / mL of OKT3 antibody.

[0202]

[0326] In some embodiments, IL-2, IL-7, IL-15, and / or IL-1 In some embodiments, a combination of IL-2, IL-7, IL-15, and / or IL-21, and any combination thereof, may be included in the second expansion culture, including during step D of the process, for example, according to Figure 27 and as described herein. In some embodiments, a combination of IL-2, IL-15, and IL-21 is used as a combination in the second expansion culture. In some embodiments, a combination of IL-2, IL-15, and IL-21, and any combination thereof, may be included in the second expansion culture, including during step D of the process, for example, according to Figure 27 and as described herein.

[0203]

[0327] In some embodiments, the second expansion culture comprises IL-2, OKT-3, and The second expansion may be performed in a supplemented cell culture medium containing antigen-presenting feeder cells. In some embodiments, the second expansion is performed in a supplemented cell culture medium. In some embodiments, the supplemented cell culture medium contains IL-2, OKT-3, and antigen-presenting feeder cells. In some embodiments, the second cell culture medium contains IL-2, OKT-3, and antigen-presenting feeder cells (APCs). In some embodiments, the second expansion occurs in a cell culture medium comprising IL-2, OKT-3, and antigen-presenting feeder cells (i.e., antigen-presenting cells).

[0204]

[0328] In some embodiments, the second expansion culture medium contains about 500 IU / mL of IL-1 In some embodiments, the second expansion culture medium contains about 500 IU / mL to about 100 IU / mL of IL-15. In some embodiments, the second expansion culture medium contains about 400 IU / mL to about 100 IU / mL of IL-15. In some embodiments, the second expansion culture medium contains about 300 IU / mL to about 100 IU / mL of IL-15. In some embodiments, the second expansion culture medium comprises about 200 IU / mL of IL-15. In some embodiments, the cell culture medium comprises about 180 IU / mL of IL-15. In one embodiment, the cell culture medium further comprises IL-15. In one preferred embodiment, the cell culture medium comprises about 180 IU / mL of IL-15.

[0205]

[0329] In some embodiments, the second expansion culture medium contains about 20 IU / mL of IL- In some embodiments, the second expansion culture medium contains about 20 IU / mL to about 0.5 IU / mL of IL-21. In some embodiments, the second expansion culture medium contains about 15 IU / mL to about 0.5 IU / mL of IL-21. In some embodiments, the second expansion culture medium contains about 12 IU / mL to about 0.5 IU / mL of IL-21. In some embodiments, the second expansion culture medium contains about 10 IU / mL to about 0.5 IU / mL of IL-21. In some embodiments, the second expansion culture medium contains about 5 IU / mL to about 1 IU / mL of IL-21. In some embodiments, the second expansion culture medium contains about 2 IU / mL of IL-21. In some embodiments, the cell culture medium contains about 1 IU / mL of IL-21. In some embodiments, the cell culture medium contains about 0.5 IU / mL of IL-21. In certain embodiments, the cell culture medium further comprises IL-21. In a preferred embodiment, the cell culture medium contains about 1 IU / mL of IL-21.

[0206]

[0330] In some embodiments, the antigen-presenting feeder cells (APCs) are PBMCs. In some embodiments, the ratio of TILs to PBMCs and / or antigen-presenting cells in the rapid expansion culture and / or second expansion culture is about 1:25, about 1:50, about 1:100, about 1:125, about 1:150, about 1:175, about 1:200, about 1:225, about 1:250, about 1:275, about 1:300, about 1:325, about 1:350, about 1:375, about 1:400, or about 1:500. In some embodiments, the ratio of TILs to PBMCs in the rapid expansion culture and / or second expansion culture is 1:50 to 1:300. In some embodiments, the ratio of TILs to PBMCs in the rapid expansion culture and / or second expansion culture is 1:100 to 1:200.

[0207]

[0331] In one embodiment, the REP and / or second expansion culture comprises bulk TILs. This is done in flasks with a 100-fold or 200-fold excess of inactivated feeder cells, 30 mg / mL OKT3 anti-CD3 antibody, and 3000 IU / mL IL-2 in 150 ml of medium. Media replenishment is performed (typically two-thirds media replenishment via respiration with fresh medium) until the cells are transferred to another growth chamber, which is discussed more fully below. Includes G-REX flasks and gas permeable vessels as shown.

[0208]

[0332] In some embodiments, a second expansion culture (a process called the REP process) is performed. The incubation period (which may include the incubation process) is shortened to 7-14 days, as discussed in the Examples and Figures. In some embodiments, the second expansion culture is shortened to 11 days.

[0209]

[0333] In one embodiment, the REP and / or second expansion culture is (Tran, et al., J. Immunother. 2008, 31, 742-51; Dudley, et al., J. Immunother. 2003, 26, 332-42) This may be performed using a T-175 flask and a gas-permeable bag or gas-permeable culture equipment (G-Rex flask). In some embodiments, the second expansion culture ( Expansion cultures (including those referred to as rapid expansion cultures) were performed in T-175 flasks containing approximately 1 × 10 cells suspended in 150 mL of medium. 6 TILs may be added to each T-175 flask. TILs may be cultured in a 1:1 mixture of CM and AIM-V medium supplemented with 3000 IU / mL of IL-2 and 30 ng / mL of anti-CD3. The T-175 flasks may be incubated for 3 days at 5% CO2. The cells may be incubated at 7°C. Half of the medium may be replaced on day 5 using 50 / 50 medium containing 3000 IU / mL of IL-2. In some embodiments, on day 7, cells from two T-175 flasks may be combined into a 3 L bag, and 300 mL of the TIL suspension may be added to 300 mL of AIM V containing 5% human AB serum and 3000 IU / mL of IL-2. The number of cells in each bag may be counted daily or every two days, and fresh medium may be added. Add the medium and count the cells to 0.5-2.0 x 10 6 cells / mL.

[0210]

[0334] In one embodiment, a second expansion culture (which is referred to as the REP expansion culture; 27) can be performed in a 100 cm gas-permeable silicone-bottomed, 500 mL capacity gas-permeable flask (G-Rex 100, commercially available from Wilson Wolf Manufacturing Corporation, New Brighton, MN, USA) containing 5% human 5 × 10 cells in 400 mL of 50 / 50 medium supplemented with AB serum, 3000 IU / mL IL-2, and 30 ng / mL anti-CD3 (OKT3). 6 or 10 x 10 6 TILs can be cultured with PBMCs. G-Rex 100 flasks can be incubated at 37° C. under 5% CO2. On day 5, 250 mL of supernatant can be removed and placed in a centrifuge bottle and centrifuged at 1500 rpm (491 x g) for 10 minutes. The TIL pellet can be resuspended in 150 mL of fresh medium containing 5% human AB serum, 3000 IU / mL IL-2, and added back to the original G-Rex 100 flask. When serially expanding TILs in G-Rex 100 flasks, on day 7, the TILs from each G-Rex 100 flask may be suspended in the 300 mL of medium present in each flask, and the cell suspension may be divided into three 100 mL aliquots, which are used to seed three G-Rex 100 flasks. The G-Rex 100 flasks may be incubated at 37°C under 5% CO2, and after 4 days, 150 mL of AIM-V containing 3000 IU / mL of IL-2 may be added to each flask. Cells may be harvested on day 14 of culture.

[0211]

[0335] In one embodiment, the second expansion culture (including the expansion culture referred to as REP) This is performed in flasks by mixing bulk TILs with a 100-fold or 200-fold excess of inactivated feeder cells, 30 mg / mL OKT3 anti-CD3 antibody, and 3000 IU / mL IL-2 in 150 ml of medium. In some embodiments, medium changes are performed until the cells are transferred to another growth chamber. In some embodiments, two-thirds of the medium is replaced by respiration with fresh medium. In some embodiments, the other growth chamber includes G-REX flasks and gas-permeable vessels, as discussed more fully below.

[0212]

[0336] In one embodiment, the second expansion culture (including the expansion culture referred to as REP) is performed, which further comprises selecting TILs for superior tumor response. Any selection method known in the art can be used. For example, U.S. Pat. The methods described in Publication No. 2016 / 0010058 A1, the disclosure of which is incorporated herein by reference, may be used to select TILs for superior tumor response.

[0213]

[0337] Optionally, a second expansion culture (including an expansion culture referred to as a REP expansion culture) Following this, cell viability assays can be performed using standard assays known in the art. For example, a sample of bulk TILs can be subjected to a trypan blue dye exclusion assay, which selectively labels dead cells and allows assessment of viability. In some embodiments, TIL samples can be counted and viability determined using a Cellometer K2 automated cell counter (Nexcelom Bioscience, Lawrence, MA). In some embodiments, viability is determined according to the Cellometer K2 Image Cytometer automated cell counter protocol described, for example, in Example 15.

[0214]

[0338] In some embodiments, a second expansion culture of TILs (an expansion called REP) is performed. The second expansion culture (including the culture) can be performed using T-175 flasks and gas-permeable bags (Tran KQ, Zhou J, Durflinger KH, et al., 2008, J Immunother., 31:742-751 and Dudley ME, Wunderlich JR, Shelton TE, et al. 2003, J Immunother., 26:332-342) or gas-permeable G-Rex flasks, as previously described. In some embodiments, the second expansion culture is performed using flasks. In some embodiments, the second expansion culture is performed using gas-permeable G-Rex flasks. In some embodiments, the second expansion culture is performed using T-175 flasks. A second expansion culture is performed, and approximately 1 x 10 6 TILs are suspended in approximately 150 mL of medium and added to each T-175 flask. TILs are cultured at a 1:100 ratio with irradiated (50 Gy) allogeneic PBMCs as "feeder" cells, and the cells are cultured in a 1:100 ratio of CM and AIM-V medium supplemented with 3000 IU / mL IL-2 and 30 ng / mL anti-CD3. The T-175 flasks are incubated at 37°C under 5% CO2. In some embodiments, half of the medium is replaced on day 5 using 50 / 50 medium containing 3000 IU / mL of IL-2. In some embodiments, on day 7, cells from two T-175 flasks are combined into a 3 L bag, and 300 mL of TIL suspension is added with 300 mL of AIM-V containing 5% human AB serum and 3000 IU / mL of IL-2. Cell counts can be performed daily or every two days, and fresh medium is added. Add the cells to the container and the number of cells will be approximately 0.5 to 2.0 × 10 6 cells / mL.

[0215]

[0339] In some embodiments, a second expansion culture (including an expansion culture called REP) Mu) is 100cm 2 The cells were cultured in a 500 mL volumetric flask with a gas-permeable silicone bottom (G-Rex 100, Wilson Wolf) (Fig. 1) and approximately 5 × 10 cells were cultured in 400 mL of 50 / 50 medium supplemented with 3000 IU / mL of IL-2 and 30 ng / mL of anti-CD3. 6 or 10 x 10 6 TILs are cultured at a 1:100 ratio with irradiated allogeneic PBMCs. The G-Rex100 flasks are incubated at 37°C under 5% CO2. In some embodiments, on day 5, 250 mL of supernatant is removed, placed in a centrifuge bottle, and centrifuged at 1500 rpm (491 g) for 10 minutes. The TIL pellet can then be resuspended in 150 mL of fresh 50 / 50 medium containing 3000 IU / mL of IL-2 and added back to the original G-Rex100 flask. In embodiments where TILs are serially expanded in G-Rex100 flasks, on day 7, the TILs in each G-Rex100 are suspended in 300 mL of medium present in each flask, and the cell suspension is divided into three 100 mL aliquots, which are used to seed three G-Rex100 flasks. Next, 150 mL of AIM-V containing 5% human AB serum and 3000 IU / mL of IL-2 is added to each flask. The G-Rex100 flasks were incubated at 37°C under 5% CO2, and after 4 days, 150 mL of AIM-V containing 3000 IU / mL of IL-2 was added to each G-Rex100 flask. are harvested on day 14 of culture.

[0216]

[0340] The diverse antigen receptors of T and B lymphocytes are expressed by a limited but numerous gene segments. These gene segments V (variable), D ( Diversity, J (binding), and C (constant) determine the binding specificity and downstream applications of immunoglobulins and T cell receptors (TCRs). The present invention provides methods for generating TILs that exhibit and increase T cell repertoire diversity. In some embodiments, TILs obtained by this method exhibit increased T cell repertoire diversity. In some embodiments, TILs obtained in the second expansion culture exhibit increased T cell repertoire diversity. In some embodiments, the increased diversity is increased immunoglobulin diversity and / or T cell receptor diversity. In some embodiments, the immunoglobulin diversity is in immunoglobulin heavy chains. In some embodiments, the immunoglobulin diversity is in immunoglobulin light chains. In some embodiments, the diversity is in the T cell receptor. In some embodiments, the diversity is in one of the T cell receptors selected from the group consisting of alpha, beta, gamma, and delta receptors. In some embodiments, expression of T cell receptor (TCR) alpha and / or beta is increased. In some embodiments, expression of T cell receptor (TCR) alpha is increased. In some embodiments, expression of T cell receptor (TCR) beta is increased. In some embodiments, expression of TCRab (ie, TCRα / β) is increased.

[0217]

[0341] In some embodiments, the second expansion culture medium (e.g., CM2 or a second The cell culture medium (sometimes referred to as cell culture medium) contains IL-2, OKT-3, and antigen-presenting feeder cells (APCs), as discussed in more detail below.

[0218]

[0342] In some embodiments, a second expansion culture, e.g., a step according to FIG. D is performed in a closed bioreactor. In some embodiments, a closed system is used for TIL expansion as described herein. In some embodiments, a single bioreactor is used. In some embodiments, the single bioreactor used is, for example, a G-REX-10 or G-REX-100. In some embodiments, A closed bioreactor is a single bioreactor.

[0219] 1. Feeder cells and antigen-presenting cells

[0343] In one embodiment, the second expansion procedure described herein (e.g., the The expansion culture described in step D, as well as the expansion culture referred to as REP, is An excess of feeder cells is required during TIL expansion and / or the second expansion. In many embodiments, the feeder cells are peripheral blood mononuclear cells (PBMCs) obtained from a standard whole blood unit from a healthy donor. PBMCs are obtained using standard methods, such as Ficoll-Paque gradient separation.

[0220]

[0344] Generally, allogeneic PBMCs are inactivated by either irradiation or heat treatment. and used in the REP procedure as described in the Examples, particularly Example 14, which provides an exemplary protocol for assessing the replication incompetence of irradiated allogeneic PBMCs.

[0221]

[0345] In some embodiments, the total number of viable cells at day 14 is the same as that at day 0 and / or 14 of the REP. or on day 0 of the second expansion culture, the PBMCs are considered non-replicating and are acceptable for use in the TIL expansion procedures described herein if the number of viable cells entering the culture (i.e., on the initiation day of the second expansion culture) is less than the initial number of viable cells entering the culture (i.e., on the initiation day of the second expansion culture). See, e.g., Example 14.

[0222]

[0346] In some embodiments, cells cultured in the presence of OKT3 and IL-2 If the total number of cells does not increase on days 7 and 14 from the initial viable cell count entered into culture on REP day 0 and / or second expansion day 0 (i.e., the start of the second expansion), the PBMCs are considered non-replicating and are approved for use in the TIL expansion procedures described herein. In some embodiments, PBMCs are cultured in the presence of 30 ng / ml OKT3 antibody and 3000 IU / ml IL-2. See, e.g., Example 13. .

[0223]

[0347] In some embodiments, cells cultured in the presence of OKT3 and IL-2 If the total number of cells does not increase on days 7 and 14 from the initial viable cell count entered into culture on day 0 of REP and / or day 0 of the second expansion culture (i.e., the start day of the second expansion culture), the PBMCs are considered replication-incompetent and are acceptable for use in the TIL expansion procedures described herein. In some embodiments, PBMCs are cultured in the presence of 5-60 ng / ml OKT3 antibody and 1000-6000 IU / ml IL-2. In some embodiments, PBMCs are cultured in the presence of 10-50 ng / ml OKT3 antibody and 2000-5000 IU / ml IL-2. In some embodiments, PBMCs are cultured in the presence of 20-40 ng / ml OKT3 antibody and 2000-4000 IU / ml IL-2. In some embodiments, PBMCs are cultured in the presence of 25-35 ng / ml OKT3 antibody and 2500-3500 IU / ml IL-2.

[0224]

[0348] In some embodiments, the antigen-presenting feeder cells are PBMCs. In some embodiments, the antigen-presenting feeder cells are artificial antigen-presenting feeder cells. In some embodiments, the ratio of TILs to antigen-presenting feeder cells in the second expansion culture is about 1:25, about 1:50, about 1:100, about 1:125, about 1:150, about 1:175, about 1:200, about 1:225, about 1:250, about 1:275, about 1:300, about 1:325, about 1:350, about 1:375, about 1:400, or about 1:500. In some embodiments, the ratio of TILs to antigen-presenting feeder cells in the second expansion culture is 1:50 to 1:300. In some embodiments, the ratio of TILs to antigen-presenting feeder cells in the second expansion culture is 1:100 to 1:200.

[0225]

[0349] In one embodiment, the second expansion procedure described herein is performed at about 2.5 x10 9 feeder cells and approximately 100 x 10 6 In another embodiment, the second expansion procedure described herein requires a ratio of about 2.5 x 10 TILs. 9 feeder cells and approximately 50 x 10 6 In yet another embodiment, the second expansion procedure described herein requires a ratio of about 2.5 x 10 TILs. 9 feeder cells and approximately 25 x 10 6 TILs are required.

[0226]

[0350] In certain embodiments, the second expansion procedure described herein comprises a second expansion step. Excessive amounts of feeder cells are required during large-scale culture. In many embodiments, the feeder cells are peripheral blood mononuclear cells (PBMCs) obtained from a standard whole blood unit from a healthy donor. PBMCs are obtained using standard methods, such as Ficoll-Paque gradient separation. In some embodiments, artificial antigen-presenting (aAPC) cells are used instead of PBMCs.

[0227]

[0351] Generally, allogeneic PBMCs are inactivated by either irradiation or heat treatment. and used in the TIL expansion procedures described herein, including the exemplary procedures described in Figures 4, 5, and 27.

[0228]

[0352] In one embodiment, artificial antigen presenting cells are used as a replacement for PBMCs or In combination with it, it is used in a second expansion culture.

[0229] 2. Cytokines

[0353] The expansion methods described herein are generally similar to those known in the art. and use culture media containing high doses of cytokines, especially IL-2.

[0230]

[0354] Alternatively, cytokine combinations may be used in rapid or secondary expansion of TILs. It is further possible to use a combination of IL-2, IL-15 and IL-21. These combinations are generally as outlined in WO 2015 / 189356 and WO 2015 / 189357, which are hereby expressly incorporated by reference in their entireties. Thus, possible combinations include IL-2 and IL-15, IL-2 and IL-21, IL-15, IL-21 and IL-2, and IL-15 and IL-21, the latter finding particular use in many embodiments. As described therein, the use of combinations of cytokines is particularly advantageous for the generation of lymphocytes, particularly T cells.

[0231] 3. Anti-CD3 antibody

[0355] In some embodiments, the culture medium used in the expansion methods described herein The culture medium (including what is called REP, see e.g., Figure 27) also contains an anti-CD3 antibody. When combined with IL-2, the anti-CD3 antibody induces T cell activation and cell division in TIL populations. This effect can be seen with full-length antibodies as well as Fab and F(ab')2 fragments, the former being generally preferred; see e.g., Tsoukas et al., J. Immunol. 1985, 135, 1719 (hereby incorporated by reference in its entirety).

[0232]

[0356] As one skilled in the art will appreciate, suitable A number of anti-human CD3 antibodies exist, including anti-human CD3 polyclonal and monoclonal antibodies from various mammals, including, but not limited to, murine, human, primate, rat, and canine antibodies. In a particular embodiment, the OKT3 anti-CD3 antibody is used (commercially available from Ortho-McNeil, Raritan, NJ or Miltenyi Biotech, Auburn, CA). (This is the case.)

[0233] E. Step E: Harvesting TILs

[0357] After the second expansion step, the cells can be harvested. In some embodiments, the TILs are harvested after one, two, three, four or more expansion steps, e.g., as provided in Figure 27. In some embodiments, the TILs are harvested after two expansion steps, e.g., as provided in Figure 27.

[0234]

[0358] The TILs may be collected by any suitable and sterile method, including, for example, by centrifugation. Methods for harvesting TILs are well known in the art, and any such known method can be used with the present process. In some embodiments, TILs are harvested using an automated system.

[0235]

[0359] Cell harvesters and / or cell processing systems are commercially available from a variety of sources, including, for example, Fresenius Kabi, Tomtec Life Science, Perkin Elmer, and Inotech Biosystems International, Inc. Any cell-based harvester can be used in the present methods. In some embodiments, the cell harvester and / or cell processing system is a membrane-based cell harvester. In some embodiments, cell collection is performed via a cell processing system, such as the LOVO system (manufactured by Fresenius Kabi). The term "LOVO cell processing system" also refers to an instrument or device manufactured by any vendor that can pump a cell-containing solution through a membrane or filter, such as a spinning membrane or spinning filter, in a sterile and / or closed environment, allowing for continuous flow and cell processing with removal of supernatant or cell culture medium without pelleting. In some embodiments, the cell harvester and / or cell processing system can perform cell separation, washing, fluid exchange, concentration, and / or other cell processing steps in a closed, sterile system.

[0236]

[0360] In some embodiments, the recovery, e.g., step E according to FIG. 27, is performed by closing In some embodiments, a closed system is used for TIL expansion as described herein. In some embodiments, a single bioreactor is used, for example, a G-REX-10 or a G-REX-100. The monoreactor is a single bioreactor.

[0237] F. Step F: Final Formulation and / or Transfer to Infusion Bag

[0361] As provided in an exemplary sequence in FIG. 27 and as described above and in detail herein. After steps A-E are completed as outlined, the cells are transferred to a container for use in administration to a patient. In some embodiments, once a therapeutically sufficient number of TILs are obtained using the expansion methods described above, the TILs are transferred to a container for use in administration to a patient.

[0238]

[0362] In one embodiment, the TILs expanded using the APCs of the present disclosure: They are administered to patients as a pharmaceutical composition. In one embodiment, the pharmaceutical composition is a suspension of TILs in a sterile buffer. TILs expanded using PBMCs of the present disclosure may be administered by any suitable route as known in the art. In some embodiments, T cells are administered as a single intra-arterial or intravenous infusion, preferably lasting about 30-60 minutes. Other suitable routes of administration include intraperitoneal, intrathecal, and intralymphatic.

[0239] 1. Pharmaceutical Compositions, Dosages, and Administration Regimen

[0363] In one embodiment, the TILs expanded using the methods of the present disclosure are useful for the treatment of medicinal diseases. The T cells are administered to a patient as a pharmaceutical composition. In one embodiment, the pharmaceutical composition is a suspension of TILs in a sterile buffer. The TILs expanded using PBMCs of the present disclosure may be administered by any suitable route as known in the art. In some embodiments, the T cells are administered as a single intra-arterial or intravenous infusion, preferably lasting about 30-60 minutes. Other suitable routes of administration include intraperitoneal, intrathecal, and intralymphatic administration.

[0240]

[0364] Any suitable dose of TILs can be administered. , approximately 2.3 x 10 10 ~Approx. 13.7×10 10 TILs were administered, with an average of approximately 7.8 x 10 10 In one embodiment, about 1.2 x 10 TILs. 10 ~Approx. 4.3×10 10In some embodiments, about 3 x 10 TILs are administered. 10 ~Approx. 12×10 10 In some embodiments, about 4 x 10 TILs are administered. 10 ~About 10×10 10 In some embodiments, about 5 x 10 TILs are administered. 10 ~Approx. 8×10 10 In some embodiments, about 6 x 10 TILs are administered. 10 ~Approx. 8×10 10 In some embodiments, about 7 x 10 TILs are administered. 10 ~Approx. 8×10 10 In some embodiments, the therapeutically effective dosage is about 2.3 x 10 TILs. 10 ~Approx. 13.7×10 10 In some embodiments, a therapeutically effective dosage is about 7.8 x 10, particularly when the cancer is melanoma. 10 In some embodiments, the therapeutically effective dosage is about 1.2 x 10 TILs. 10 ~Approx. 4.3×10 10 In some embodiments, the therapeutically effective dosage is about 3 x 10 TILs. 10 ~Approx. 12×10 10 In some embodiments, the therapeutically effective dosage is about 4 x 10 TILs. 10 ~About 10×10 10 In some embodiments, the therapeutically effective dosage is about 5 x 10 TILs. 10 ~Approx. 8×10 10 In some embodiments, the therapeutically effective dosage is about 6 x 10 TILs. 10 ~Approx. 8×10 10 In some embodiments, the therapeutically effective dosage is about 7 x 10 TILs. 10 ~Approx. 8×10 10 This is the individual TIL.

[0241]

[0365] In some embodiments, the number of TILs provided in the pharmaceutical composition of the present invention is , about 1×10 6 , 2 × 10 6、3×10 6 、4×10 6 、5×10 6 、6×10 6 、7× 10 6 、8×10 6 、9×10 6 、1×10 7 、2×10 7 、3×10 7 、4×10 7 、5×10 7 、6×10 7 、7×10 7 、8×10 7 、9×10 7 、1×10 8 、2×10 8 、3×10 8 、4×10 8 、5×10 8 、6×10 8 、7×10 8 、8×10 8 、9×10 8 、1×10 9 、2×10 9 、3×10 9 、4×10 9 、5×10 9 、6×10 9 、7×10 9 、8×10 9 、9×10 9 、1×10 10 、2×10 10 、3×10 10 、4×10 10 、5×10 10 、6×10 10 、7×10 10 、8×10 10 、9×10 10 、1×10 11 、2×10 11 、3×10 11 、4×10 11 、5×10 11 、6×10 11 、7×10 11 、8×10 11 、9×10 11 、1×10 12 、2×10 12 、3×10 12, 4×10 12 , 5×10 12 , 6×10 12 , 7×10 12 , 8×10 12 , 9×10 12 , 1×10 13 , 2 × 10 13 , 3×10 13 , 4×10 13 , 5×10 13 , 6×10 13 , 7×10 13 , 8×10 13 , and 9×10 13 In one embodiment, the number of TILs provided in the pharmaceutical composition of the present invention is 1 x 10 6 ~5×10 6 , 5×10 6 ~1×10 7 , 1×10 7 ~5×10 7 , 5×10 7 ~1×10 8 , 1×10 8 ~5×10 8 , 5×10 8 ~1×10 9 , 1×10 9 ~5×10 9 , 5×10 9 ~1×10 10 , 1×10 10 ~5×10 10 , 5×10 10 ~1×10 11 , 5×10 11 ~1×10 12 , 1×10 12 ~5×10 12 , and 5 × 10 12 ~1×10 13 It is a range of individuals.

[0242]

[0366] In some embodiments, the concentration of TILs provided in the pharmaceutical composition of the present invention is, for example, 100%, 90%, 80%, 70%, 60%, 50%, 40%, 30%, 20%, 19%, 18%, 17%, 16%, 15%, 14%, 13%, 12%, 11%, 10%, 9%, 8%, 7%, 6%, 5%, 4%, 3%, 2%, 1%, 0.5%, 0.4%, 0.3%, 0.2%, 0.1%, 0.09%, 0.08%, 0.07%, 0.06%, 0.05%, 0.0% of the pharmaceutical composition. less than 4%, 0.03%, 0.02%, 0.01%, 0.009%, 0.008%, 0.007%, 0.006%, 0.005%, 0.004%, 0.003%, 0.002%, 0.001%, 0.0009%, 0.0008%, 0.0007%, 0.0006%, 0.0005%, 0.0004%, 0.0003%, 0.0002% or 0.0001% w / w, w / v or v / v.

[0243]

[0367] In some embodiments, the concentration of TILs provided in the pharmaceutical composition of the present invention represents 90%, 80%, 70%, 60%, 50%, 40%, 30%, 20%, 19.75%, 19.50%, 19.25% 19%, 18.75%, 18.50%, 18.25% 18%, 17.75%, 17.50%, 17.25% 17%, 16.75%, 16.50%, 16.25% 16%, 15.75%, 15.50%, 15.25% of the pharmaceutical composition 15%, 14.75%, 14.50%, 14.25% 14%, 13.75%, 13.50%, 13.25% 13%, 12.75%, 12.50%, 12.25% 12%, 11.75%, 11.50%, 11.25% 11%, 10.75%, 10.50%, 10.25% 10%, 9.75%, 9.50%, 9.25% 9%, 8.75%, 8.50%, 8.25% 8%, 7.75%, 7.50%, 7.25% 7%, 6.75%, 6.50%, 6.25% 6%, 5.75%, 5.50%, 5.25% 5%, 4.75%, 4.50%, 4.25%, 4%, 3.75%, 3.50%, 3.25%, 3%, 2.75%, 2.50%, 2.25%, 2%, 1.75%, 1.50%, 125%, 1%, 0.5%, 0.4%, 0.3%, 0.2%, 0.1%, 0.09%, 0.08%, 0.07%, 0.06%, 0.05%, 0.04%, 0.03%, 0.02%, 0.01%, 0.009%, 0.008%, 0.007%, 0.006%, 0.005%, 0.004%, 0.003%, 0.002%, 0.001%, 0.0009%, 0.0008%, 0.0007%, 0.0006%, 0.0005%, 0.0004%, 0.0003%, 0.0002% or greater than 0.0001% w / w, w / v, or v / v.

[0244]

[0368] In some embodiments, the concentration of TILs provided in the pharmaceutical composition of the present invention is about 0.0001% to about 50%, about 0.001% to about 40%, about 0.01% to about 30%, about 0.02% to about 29%, about 0.03% to about 28%, about 0.04% to about 27%, about 0.05% to about 26%, about 0.06% to about 25%, about 0.07% to about 24%, about 0.08% to about 23%, about 0.0 The range is 9% to about 22%, about 0.1% to about 21%, about 0.2% to about 20%, about 0.3% to about 19%, about 0.4% to about 18%, about 0.5% to about 17%, about 0.6% to about 16%, about 0.7% to about 15%, about 0.8% to about 14%, about 0.9% to about 12%, or about 1% to about 10% w / w, w / v, or v / v.

[0245]

[0369] In some embodiments, the concentration of TILs provided in the pharmaceutical composition of the present invention is in the range of about 0.001% to about 10%, about 0.01% to about 5%, about 0.02% to about 4.5%, about 0.03% to about 4%, about 0.04% to about 3.5%, about 0.05% to about 3%, about 0.06% to about 2.5%, about 0.07% to about 2%, about 0.08% to about 1.5%, about 0.09% to about 1%, or about 0.1% to about 0.9% w / w, w / v, or v / v of the pharmaceutical composition.

[0246]

[0370] In some embodiments, the amount of TILs provided in the pharmaceutical composition of the present invention is , 10g, 9.5g, 9.0g, 8.5g, 8.0g, 7.5g, 7.0g, 6.5g, 6.0g, 5.5g, 5.0g, 4.5g, 4.0g, 3.5g, 3.0g, 2.5g, 2.0g, 1.5g, 1.0g, 0. 95g, 0.9g, 0.85g, 0.8g, 0.75g, 0.7g, 0.65g, 0.6g, 0.55g, 0.5g, 0.45g, 0.4g, 0.35g, 0.3g, 0.25g, 0.2g, 0.15g, 0.1g, 0 0.09g, 0.08g, 0.07g, 0.06g, 0.05g, 0.04g, 0.03g, 0.02g, 0.01g, 0.009g, 0.008g, 0.007g, 0.006g, 0.005g, 0.004g, 0.003g, 0.002g, 0.001g, 0.0009g, 0.0008g, 0.0007g, 0.0006g, 0.0005g, 0.0004g, 0.0003g, 0.0002g, or 0.0001g.

[0247]

[0371] In some embodiments, the amount of TILs provided in the pharmaceutical composition of the present invention is , 0.0001g, 0.0002g, 0.0003g, 0.0004g, 0.0005g, 0.0006g, 0.0007g, 0.0008 g, 0.0009g, 0.001g, 0.0015g, 0.002g, 0.0025g, 0.003g, 0.0035g, 0.004g, 0 .0045g, 0.005g, 0.0055g, 0.006g, 0.0065g, 0.007g, 0.0075g, 0.008g, 0.00 85g, 0.009g, 0.0095g, 0.01g, 0.015g, 0.02g, 0.025g, 0.03g, 0.035g, 0.04g , 0.045g, 0.05g, 0.055g, 0.06g, 0.065g, 0.07g, 0.075g, 0.08g, 0.085g, 0.0 9g, 0.095g, 0.1g, 0.15g, 0.2g, 0.25g, 0.3g, 0.35g, 0.4g, 0.45g, 0.5g, 0.55 g, 0.6 g, 0.65 g, 0.7 g, 0.75 g, 0.8 g, 0.85 g, 0.9 g, 0.95 g, 1 g, 1.5 g, 2 g, 2.5, 3 g, 3.5, 4 g, 4.5 g, 5 g, 5.5 g, 6 g, 6.5 g, 7 g, 7.5 g, 8 g, 8.5 g, 9 g, 9.5 g, or more than 10 g.

[0248]

[0372] The TILs provided in the pharmaceutical compositions of the present invention are effective over a wide dosage range. The exact dosage will depend on the route of administration, the dosage form of the compound, the sex and age of the subject, the body weight of the subject, and the preferred choice and experience of the attending physician. Clinically established dosages of TILs may also be used where appropriate. The amount of the pharmaceutical composition administered using the methods herein, including the dosage of TILs, will depend on the human or mammal being treated, the severity of the disorder or condition, the rate of administration, the nature of the active pharmaceutical ingredient, and the discretion of the prescribing physician.

[0249]

[0373] In some embodiments, the TILs may be administered in a single dose. Administration may be by injection, for example, intravenous injection. In some embodiments, the TILs are administered intravenously. The TILs may be administered in a single dose. Administration may be once, twice, three times, four times, five times, six times, or more than six times per year. Administration may be monthly, once every two weeks, once a week, or once every other day. Administration of the TILs may be continued as long as necessary.

[0250]

[0374] In some embodiments, the effective dosage of TILs is about 1×10 6 , 2 × 10 6 , 3×10 6 , 4×10 6 , 5×10 6 , 6×10 6 , 7×10 6 , 8×10 6 , 9×10 6 , 1×10 7 , 2 × 10 7 , 3×10 7 , 4×10 7 , 5×10 7 , 6×10 7 , 7×10 7 , 8×10 7 , 9×10 7 , 1×10 8 , 2 × 10 8 , 3×10 8 , 4×10 8 , 5×10 8 , 6×10 8 , 7×10 8 , 8×10 8 , 9×10 8 , 1×10 9 , 2 × 10 9 , 3×10 9 , 4×10 9 , 5×10 9 , 6×10 9 , 7×10 9 , 8×10 9 , 9×10 9 , 1×10 10 , 2 × 10 10 , 3×10 10 , 4×10 10 , 5×10 10 , 6×10 10 , 7×10 10 , 8×10 10 , 9×10 10 , 1×1011 , 2 × 10 11 , 3×10 11 , 4×10 11 , 5×10 11 , 6×10 11 , 7×10 11 , 8×10 11 , 9×10 11 , 1×10 12 , 2 × 10 12 , 3×10 12 , 4×10 12 , 5×10 12 , 6×10 12 , 7×10 12 , 8×10 12 , 9×10 12 , 1×10 13 , 2 × 10 13 , 3×10 13 , 4×10 13 , 5×10 13 , 6×10 13 , 7×10 13 , 8×10 13 , and 9×10 13 In some embodiments, the effective dosage of TILs is 1 x 10 6 ~5×10 6 , 5×10 6 ~1×10 7 , 1×10 7 ~5×10 7 , 5×10 7 ~1×10 8 , 1×10 8 ~5×10 8 , 5×10 8 ~1×10 9 , 1×10 9 ~5×10 9 , 5×10 9 ~1×10 10 , 1×10 10 ~5×10 10 , 5×10 10 ~1×10 11 , 5×10 11 ~1×10 12 , 1×10 12 ~5×10 12 , and 5 × 10 12 ~1×10 13 It is a range of individuals.

[0251]

[0375] In some embodiments, the effective dosage of TILs is about 0.01 mg / kg to Approximately 4.3 mg / kg, approximately 0.15 mg / kg to approximately 3.6 mg / kg, approximately 0.3 mg / kg to approximately 3.2 mg / kg, approximately 0.35 mg / kg to approximately 2.85 mg / kg, approximately 0.15 mg / kg to approximately 2.85 mg / kg, approximately 0.3 mg to approximately 2.15 mg / kg, approximately 0.45 mg / kg to approximately 1.7 mg / k g, about 0.15 mg / kg to about 1.3 mg / kg, about 0.3 mg / kg to about 1.15 mg / kg, about 0.45 mg / kg to about 1 mg / kg, about 0.55 mg / kg to about 0.85 mg / kg, about 0.65 mg / kg to about 0.8 mg / kg, about 0.7 mg / kg to about 0.75 mg / kg, about 0.7 mg / kg to about 2.15 mg / kg, about 0.85 mg / kg to about 2 mg / kg, about 1 mg / kg to about 1.85 mg / kg, about 1.15 mg / kg to about 1.7 mg / kg, about 1.3 mg / kg to about 1.6 mg / kg, about 1.35 mg / kg to about 1.5 mg / kg, about 2.15 mg / kg to about 3.6 mg / kg, about 2.3 mg / kg to about 3.4 mg / kg, about 2.4 mg / kg to about 3.3 mg / kg, about 2.6 mg / kg to about 3.15 mg / kg, about 2.7 mg / kg to about 3 mg / kg, about 2.8 mg / kg to about 3 mg / kg, or about 2.85 mg / kg to about 2.95 mg / kg.

[0252]

[0376] In some embodiments, an effective dosage of TILs is from about 1 mg to about 500 mg. , about 10 mg to about 300 mg, about 20 mg to about 250 mg, about 25 mg to about 200 mg, about 1 mg to about 50 mg, about 5 mg to about 45 mg, about 10 mg to about 40 mg, about 1 5mg to about 35mg, about 20mg to about 30mg, about 23mg to about 28mg, about 50mg to about 150mg, about 60mg to about 140mg, about 70mg to about 130mg, about 80m The range is from about 100 mg to about 120 mg, from about 90 mg to about 110 mg, or from about 95 mg to about 105 mg, from about 98 mg to about 102 mg, from about 150 mg to about 250 mg, from about 160 mg to about 240 mg, from about 170 mg to about 230 mg, from about 180 mg to about 220 mg, from about 190 mg to about 210 mg, from about 195 mg to about 205 mg, or from about 198 mg to about 207 mg.

[0253]

[0377] Effective doses of TILs can be administered by intranasal and transdermal routes, intra-arterial injection, intravenous, They may be administered in single or multiple doses by any of the commonly accepted modes of administration for agents having similar utilities, including intraperitoneally, parenterally, intramuscularly, subcutaneously, topically, by implant, or by inhalation.

[0254] G. Optional Cell Viability Analysis

[0378] Optionally, after the first expansion culture of step B, a method known in the art for Cell viability assays can be performed using known standard assays. For example, a sample of bulk TILs can be subjected to a trypan blue dye exclusion assay, which selectively labels dead cells and allows assessment of viability. Other assays used to test viability include, but are not limited to, the Alamar Blue assay; and the MTT assay.

[0255] 1. Cell Count, Viability, and Flow Cytometry

[0379] In some embodiments, cell number and / or viability are measured. Expression of markers such as, but not limited to, CD3, CD4, CD8, and CD56, and any others disclosed or described herein, can be measured by antibody-based flow cytometry, such as, but not limited to, those commercially available from BD Biosciences (BD Biosciences, San Jose, CA) using a FACSCanto™ flow cytometer (BD Biosciences). Cells can be counted manually using a disposable c-chip hemocytometer (VWR, Batavia, IL), and viability can be assessed using any method known in the art, including, but not limited to, trypan blue staining.

[0256]

[0380] In some cases, bulk TIL populations are directly cultured using the protocols discussed below. The bulk TIL population can be immediately cryopreserved. Alternatively, the bulk TIL population can be subjected to REP, as discussed below, and then cryopreserved. Similarly, if the genetically modified TILs are to be used in therapy, the bulk or REP TIL population can be subjected to the appropriate therapeutic genetic modification.

[0257] 2.Cell culture

[0381] In one embodiment, the method for expanding TILs involves culturing a culture medium containing about 5,000 mL to about 2 This may include using 5,000 mL of cell culture medium, about 5,000 mL to about 10,000 mL of cell culture medium, or about 5,800 mL to about 8,700 mL of cell culture medium. In some embodiments, one or less types of cell culture medium are used to expand the number of TILs. Any suitable cell culture medium may be used, for example, AIM-V cell culture medium (L-glutamine, 50 μM streptomycin sulfate, 50 μM ethanol ... and 10 μM gentamicin sulfate) cell culture medium (Invitrogen, Carlsbad CA). In this regard, the methods of the present invention advantageously reduce the amount of medium and number of media types required to expand the number of TILs. In certain embodiments, expanding the number of TILs can involve adding fresh cell culture medium to the cells (also referred to as feeding the cells) no more frequently than every two or three days. Expanding the number of cells in a gas-permeable container simplifies the procedures required to expand the number of cells by reducing the feeding frequency required for cell expansion.

[0258]

[0382] In some embodiments, the cell culture medium in the first and / or second gas permeable container The cell culture medium in the first and / or second gas-permeable containers is unfiltered. The use of unfiltered cell culture medium can simplify the procedures required to expand the number of cells. In some embodiments, the cell culture medium in the first and / or second gas-permeable containers does not contain β-mercaptoethanol (BME).

[0259]

[0383] In one embodiment, obtaining a tumor tissue sample from the mammal; cell culture medium the tumor tissue sample in a first gas-permeable container containing the aAPC; obtaining TILs from the tumor tissue sample; and expanding the number of TILs in a second gas-permeable container containing cell culture medium using the aAPC for about 14 to about 42 days, e.g., about 28 days. period.

[0260]

[0384] In one embodiment, the TILs are expanded in a gas-permeable container. Permeable containers have been used to expand TILs using PBMCs using methods, compositions, and devices known in the art, including those described in U.S. Patent Application Publication No. 2005 / 0106717 A1, the disclosure of which is incorporated herein by reference. In one embodiment, TILs are expanded in gas-permeable bags. In one embodiment, TILs are expanded in gas-permeable bags, such as the Xuri Cell Expansion System W25 (GE Healthcare). The TILs are expanded using a cell expansion system that expands TILs in gas-permeable bags. In one embodiment, the TILs are expanded using a cell expansion system that expands TILs in gas-permeable bags, such as the WAVE Bioreactor System, also known as the Xuri Cell Expansion System W5 (GE Healthcare). In one embodiment, the cell expansion system comprises a gas-permeable cell bag having a volume selected from the group consisting of about 100 mL, about 200 mL, about 300 mL, about 400 mL, about 500 mL, about 600 mL, about 700 mL, about 800 mL, about 900 mL, about 1 L, about 2 L, about 3 L, about 4 L, about 5 L, about 6 L, about 7 L, about 8 L, about 9 L, and about 10 L. In one embodiment, the TILs can be expanded in G-Rex flasks (commercially available from Wilson Wolf Manufacturing). Such an embodiment expands the cell population to about 5 x 10 5 cells / cm 2 From 10 x 10 6 ~30×10 6 cells / cm 2In some embodiments, this expansion is done without adding fresh cell culture medium to the cells (also referred to as feeding the cells). In some embodiments, this is without feeding, as long as there is about 10 cm of medium in the GRex flask. In some embodiments, this is without feeding, but one or more cytokines are added. In some embodiments, the cytokines can be added as a bolus without any need to mix the cytokine with the medium. Such vessels, devices, and methods are known in the art and have been used for the expansion of TILs, including, for example, U.S. Patent Application Publication No. 2014 / 0377739 A1, WO 2014 / 210036 A1, U.S. Patent Application Publication No. 2013 / 0115617 A1, WO 2013 / 188427 A1, U.S. Patent Application Publication No. 2011 / 0136228 A1, U.S. Patent No. 8,809,050 B2, WO 2011 / 072088 A2, U.S. Patent Application Publication No. 2016 / 0208216 A1, U.S. Patent Application Publication No. 2012 / 0244133 A1, WO 2012 / 129201 A1, U.S. Patent Application Publication No. 2013 / 0102075 A1, U.S. Patent No. 8,956,860 B2, and the like. B2, WO 2013 / 173835 A1, and U.S. Patent Application Publication No. 2015 / 0175966 A1, the disclosures of which are incorporated herein by reference. Such processes are also described in Jin et al., J. Immunotherapy, 2012, 35:283-292. Optional TIL Gene Modification

[0261]

[0385] In some embodiments, the TILs are optionally, but not limited to, high affinity TILs. They are genetically engineered to contain additional functionality, including T cell receptors (TCRs), for example, TCRs that target tumor-associated antigens such as MAGE-1, HER2, or NY-ESO-1, or chimeric antigen receptors (CARs) that bind to tumor-associated cell surface molecules (e.g., mesothelin) or lineage-restricted cell surface molecules (e.g., CD19).

[0262] H. Optional TIL cryopreservation

[0386] As discussed above and provided in FIG. 27, in steps A to E As illustrated in Figure 27, cryopreservation can occur at various points throughout the TIL expansion process. In some embodiments, the expanded TIL population may be cryopreserved after the second expansion (e.g., as per step D of Figure 27). Cryopreservation can generally be achieved by placing the TIL population in a freezing solution, such as 85% complement-inactivated AB serum and 15% dimethyl sulfoxide (DMSO). The cells in this solution are placed in a cryogenic vial. The TILs are cryopreserved in 5% DMSO. In some embodiments, the TILs are cryopreserved in cell culture medium + 5% DMSO. In some embodiments, the TILs are cryopreserved according to the methods provided in Examples 8 and 9.

[0263]

[0387] If appropriate, remove the cells from the freezer and place them in a 37°C water bath for approximately 5 minutes. Thaw until a quarter of the cells are thawed. The cells are generally resuspended in complete medium and optionally washed one or more times. In some embodiments, the thawed TILs can be counted and assessed for viability as known in the art.

[0264] I. Phenotypic characteristics of expanded TILs

[0388] In some embodiments, the TILs are expanded and then cultured to produce the desired TILs. The TILs are analyzed for expression of a number of phenotypic markers, including those listed above. In certain embodiments, expression of one or more phenotypic markers is examined. In some embodiments, the phenotypic characteristics of the TILs are analyzed after the first expansion in step B. In some embodiments, the phenotypic characteristics of the TILs are analyzed during the transfer in step C. In some embodiments, the phenotypic characteristics of the TILs are analyzed during the transfer and after cryopreservation in step C. In some embodiments, the phenotypic characteristics of the TILs are analyzed after the second expansion in step D. In some embodiments, the phenotypic characteristics of the TILs are analyzed after two or more expansions in step D. In some embodiments, the markers are selected from the group consisting of TCRab (i.e., TCRα / β), CD57, CD28, CD4, CD27, CD56, CD8a, CD45RA, CD8a, CCR7, CD4, CD3, CD38, and HLA-DR. In some embodiments, the markers are selected from the group consisting of TCRab (i.e., TCRα / β), CD57, CD28, CD4, CD27, CD56, and CD8a. In certain embodiments, the markers are selected from the group consisting of CD45RA, CD8a, CCR7, CD4, CD3, CD38, and HLA-DR. In some embodiments, expression of 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, or 14 markers is examined. In some embodiments, expression from one or more markers from each group is examined. In some embodiments, one or more of HLA-DR, CD38, and CD69 expression is maintained (i.e., does not exhibit a statistically significant difference) in fresh TILs compared to thawed TILs. In some embodiments, the activation state of TILs is maintained in thawed TILs.

[0265]

[0389] In certain embodiments, the expression of one or more regulatory markers is measured. In some embodiments, the regulatory marker is selected from the group consisting of CD137, CD8a, Lag3, CD4, CD3, PD-1, TIM-3, CD69, CD8a, TIGIT, CD4, CD3, KLRG1, and CD154. In some embodiments, the regulatory marker is selected from the group consisting of CD137, CD8a, Lag3, CD4, CD3, PD-1, and TIM-3. In some embodiments, the regulatory marker is selected from the group consisting of CD69, CD8a, TIGIT, CD4, CD3, KLRG1, and CD154. In some embodiments, regulatory molecule expression is decreased in thawed TILs compared to fresh TILs. In some embodiments, expression of regulatory molecules LAG-3 and TIM-3 is decreased in thawed TILs compared to fresh TILs. In some embodiments, there is no significant difference in CD4, CD8, NK, or TCRαβ expression. In some embodiments, there are no significant differences in CD4, CD8, NK, TCRαβ expression, and / or memory markers in fresh TILs compared to thawed TILs. In some embodiments, for example, as illustrated in FIG. 27, there are no significant differences in CD4, CD8, NK, TCRαβ expression between TILs generated by the methods provided herein and / or TILs prepared using other methods other than those provided herein, including, for example, methods other than those embodied in FIG. 27. None.

[0266]

[0390] In some embodiments, the first TIL population, the second TIL population, the third TIL population, Selection of TIL populations, recovered TIL populations, and / or therapeutic TIL populations based on CD4, CD8, and / or NK, TCRαβ expression is not performed during any of the steps discussed above or including the example in FIG. 27 . In some embodiments, selection of a first TIL population based on CD4, CD8, and / or NK, TCRαβ expression is not performed. In some embodiments, selection of a second TIL population based on CD4, CD8, and / or NK, TCRαβ expression is not performed. In some embodiments, selection of a third TIL population based on CD4, CD8, and / or NK, TCRαβ expression is not performed. In some embodiments, selection of recovered TIL populations based on CD4, CD8, and / or NK, TCRαβ expression is not performed. In some embodiments, selection of a therapeutic TIL population based on CD4, CD8, and / or NK, TCRαβ expression is not performed.

[0267]

[0391] In one embodiment, the first TIL population, the second TIL population, the third TIL population, Selection of the L population or recovered TILs based on CD4, CD8, and / or NK, TCRαβ expression is not performed during any of steps (a)-(f) of the method for expanding tumor-infiltrating lymphocytes (TILs) into a therapeutic TIL population, and this step (a) obtaining a first population of TILs from a tumor excised from a patient by processing a tumor sample obtained from the patient into multiple tumor fragments; (b) adding tumor fragments to a closed system; (c) performing a first expansion culture by culturing the first TIL population in a cell culture medium containing IL-2 to generate a second TIL population, wherein the first expansion culture is performed in a closed vessel providing a first gas permeable surface area, the first expansion culture is performed for about 3 to 14 days to obtain a second TIL population, wherein the second TIL population is at least 50-fold more numerous than the first TIL population, and the transition from step (b) to step (c) occurs without opening the system; (d) performing a second expansion culture by adding additional IL-2, OKT-3, and antigen-presenting cells (APCs) to the cell culture medium of the second TIL population to generate a third TIL population, wherein the second expansion culture is performed for about 7 to 14 days to obtain the third TIL population, the third TIL population being a therapeutic TIL population comprising an expanded subpopulation of effector T cells and / or central memory T cells compared to the second TIL population, wherein the second expansion culture is performed in a closed vessel providing a second gas-permeable surface area, and the transition from step (c) to step (d) occurs without opening the system; (e) recovering the therapeutic TIL population obtained from step (d), wherein the transition from step (d) to step (e) occurs without releasing the system; and (f) transferring the TIL population collected in step (e) into an infusion bag, wherein the transition from step (e) to (f) occurs without opening the system. Includes.

[0268]

[0392] In one embodiment, the first TIL population, the second TIL population, the third TIL population, Selection of the recovered TILs based on the L population, or CD4, CD8, and / or NK, TCRαβ expression, is not performed during any of steps (a) to (h) of the method for treating a subject with cancer, and the method includes administering the expanded tumor-infiltrating lymphocytes (TILs): (a) obtaining a first population of TILs from a tumor resected from a patient by processing a tumor sample obtained from the subject into multiple tumor fragments; (b) adding tumor fragments to a closed system; (c) performing a first expansion by culturing the first TIL population in a cell culture medium containing IL-2 to generate a second TIL population, wherein the first expansion is performed in a closed vessel providing a first gas permeable surface area, and wherein the first expansion produces a second TIL population. the second TIL population is at least 50-fold more numerous than the first TIL population, and the transition from step (b) to step (c) occurs without releasing the system; (d) performing a second expansion culture by adding additional IL-2, OKT-3, and antigen-presenting cells (APCs) to the cell culture medium of the second TIL population to generate a third TIL population, wherein the second expansion culture is performed for about 7 to 14 days to obtain the third TIL population, the third TIL population being a therapeutic TIL population comprising an expanded subpopulation of effector T cells and / or central memory T cells compared to the second TIL population, wherein the second expansion culture is performed in a closed vessel providing a second gas-permeable surface area, and the transition from step (c) to step (d) occurs without opening the system; (e) recovering the therapeutic TIL population obtained from step (d), wherein the transition from step (d) to step (e) occurs without releasing the system; and (f) transferring the TIL population collected in step (e) to an infusion bag, wherein the transition from step (e) to (f) occurs without opening the system; (g) optionally cryopreserving the infusion bag containing the TIL population collected in step (f) using a cryopreservation process; and (h) administering a therapeutically effective dose of the third population of TILs from the infusion bag in step (g) to the patient. Includes.

[0269]

[0393] In some embodiments, the memory markers are selected from CCR7 and CD62L. is selected from the group consisting of:

[0270]

[0394] In some embodiments, the viability of fresh TILs compared to thawed TILs is at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, or at least 98%. In some embodiments, the viability of both fresh and thawed TILs is greater than 70%, greater than 75%, greater than 80%, greater than 85%, greater than 90%, greater than 95%, or greater than 98%. In some embodiments, the viability of both fresh and thawed products is greater than 80%, greater than 81%, greater than 82%, greater than 83%, greater than 84%, greater than 85%, greater than 86%, greater than 87%, greater than 88%, greater than 89%, or greater than 90%. In some embodiments, the viability of both fresh and thawed products is greater than 86%.

[0271]

[0395] In one embodiment, restimulated TILs are also assayed using cytokine release assays. In some embodiments, TILs can be assessed for interferon-7 (IFN-7) secretion in response to stimulation with either OKT3 or co-culture with autologous tumor digest. For example, in embodiments using OKT3 stimulation, TILs are washed extensively and plated at 1 x 10 in 0.2 mL CM onto 96-well flat-bottom plates pre-coated with 0.1 or 1.0 μg / mL OKT3 diluted in phosphate-buffered saline. 5 Duplicate wells are prepared with cells. After overnight incubation, the supernatant is collected and IFN-7 in the supernatant is measured by ELISA (Pierce / Endogen, Woburn, MA). For co-culture assays, 1 x 10 cells are used. 5 TIL cells are plated in 96-well plates together with autologous tumor cells (1:1 ratio). After 24 hours of incubation, supernatants can be collected and IFN-7 release can be quantified, for example, by ELISA.

[0272]

[0396] Flow cytometry analysis of cell surface biomarkers: TIL samples were aliquoted for flow cytometry analysis (see, e.g., Examples 7, 8, and 9).

[0273]

[0397] In some embodiments, the TILs are assessed for various regulatory markers. In some embodiments, the regulatory markers are TCRα / β, CD56, CD In some embodiments, the regulatory marker is selected from the group consisting of CD27, CD28, CD57, CD45RA, CD45RO, CD25, CD127, CD95, IL-2R, CCR7, CD62L, KLRG1, and CD122. In some embodiments, the regulatory marker is TCRα / β. In some embodiments, the regulatory marker is CD56. In some embodiments, the regulatory marker is CD27. In some embodiments, the regulatory marker is CD28. In some embodiments, the regulatory marker is CD57. In some embodiments, the regulatory marker is CD45RA. In some embodiments, the regulatory marker is CD45RO. In some embodiments, the regulatory marker is CD25. In some embodiments, the regulatory marker is CD127. In some embodiments, the regulatory marker is CD95. In some embodiments, the regulatory marker is IL-2R. In some embodiments, the regulatory marker is CCR7. In some embodiments, the regulatory marker is CD62L. In some embodiments, the regulatory marker is KLRG1. In some embodiments, the regulatory marker is CD122.

[0274]

[0398] In one embodiment, the expanded TILs are those described herein and in the Examples. In some embodiments, expression of one or more phenotypic markers is examined. In some embodiments, the markers are selected from the group consisting of TCRab (i.e., TCRα / β), CD57, CD28, CD4, CD27, CD56, CD8a, CD45RA, CD8a, CCR7, CD4, CD3, CD38, and HLA-DR. In some embodiments, the markers are selected from the group consisting of TCRab (i.e., TCRα / β), CD57, CD28, CD4, CD27, CD56, and CD8a. In some embodiments, the markers are selected from the group consisting of CD45RA, CD8a, CCR7, CD4, CD3, CD38, and HLA-DR. In some embodiments, expression of 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, or 14 markers is examined. In some embodiments, expression of one or more markers from each group is examined. In some embodiments, one or more of HLA-DR, CD38, and CD69 expression is maintained (i.e., does not exhibit a statistically significant difference) in fresh TILs compared to thawed TILs, hi some embodiments, the activation state of TILs is maintained in thawed TILs.

[0275]

[0399] In certain embodiments, the expression of one or more regulatory markers is measured. In some embodiments, the regulatory marker is selected from the group consisting of CD137, CD8a, Lag3, CD4, CD3, PD1, TIM-3, CD69, CD8a, TIGIT, CD4, CD3, KLRG1, and CD154. In some embodiments, the regulatory marker is selected from the group consisting of CD137, CD8a, Lag3, CD4, CD3, PD1, and TIM-3. In some embodiments, the regulatory marker is selected from the group consisting of CD69, CD8a, TIGIT, CD4, CD3, KLRG1, and CD154. In some embodiments, regulatory molecule expression is decreased in thawed TILs compared to fresh TILs. In some embodiments, expression of regulatory molecules LAG-3 and TIM-3 is decreased in thawed TILs compared to fresh TILs. In some embodiments, there is no significant difference in CD4, CD8, NK, or TCRαβ expression. In some embodiments, there are no significant differences in CD4, CD8, NK, TCRαβ expression, and / or memory markers in fresh TILs compared to thawed TILs.

[0276]

[0400] In some embodiments, the memory markers are selected from CCR7 and CD62L. is selected from the group consisting of:

[0277]

[0401] In some embodiments, the viability of fresh TILs compared to thawed TILs is at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, or at least 98%. In some embodiments, the viability of both the fresh and thawed TILs is greater than 70%, 75%, 80%, 85%, 90%, 95%, or 98%. In some embodiments, the viability of both the fresh and thawed products is greater than 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, or 90%. In some embodiments, the viability of both the fresh and thawed products is greater than 86%.

[0278]

[0402] In one embodiment, restimulated TILs are also assayed using cytokine release assays. In some embodiments, TILs can be assessed for interferon-7 (IFN-7) secretion in response to stimulation with either OKT3 or co-culture with autologous tumor digest. For example, in embodiments using OKT3 stimulation, TILs are washed extensively and plated at 1 x 10 in 0.2 mL CM onto 96-well flat-bottom plates pre-coated with 0.1 or 1.0 μg / mL OKT3 diluted in phosphate-buffered saline. 5 Duplicate wells are prepared with cells. After overnight incubation, the supernatant is collected and IFN-7 in the supernatant is measured by ELISA (Pierce / Endogen, Woburn, MA). For co-culture assays, 1 x 10 cells are used. 5 TIL cells are plated in 96-well plates together with autologous tumor cells (1:1 ratio). After 24 hours of incubation, supernatants can be collected and IFN-7 release can be quantified, for example, by ELISA.

[0279]

[0403] In some embodiments, phenotypic characterization is performed after cryopreservation.

[0280] J. Metabolic Health of Expanded TILs

[0404] Restimulated TILs were either freshly harvested and / or thawed TILs. The resulting TILs are characterized by a significant increase in basal glycolysis compared to the initial TIL population. In certain embodiments, selection of the first TIL population, the second TIL population, the third TIL population, the harvested TIL population, and / or the therapeutic TIL population based on CD8 expression is not performed during any of the steps discussed above or including the example in FIG. 27 . In some embodiments, selection of the first TIL population based on CD8 expression is not performed. In some embodiments, selection of the second TIL population based on CD8 expression is not performed. In some embodiments, selection of the third TIL population based on CD8 expression is not performed. In some embodiments, selection of the harvested TIL population based on CD8 expression is not performed. In some embodiments, selection of the therapeutic TIL population based on CD8 expression is not performed.

[0281]

[0405] In one embodiment, the first TIL population, the second TIL population, the third TIL population, Selection of the L population or recovered TILs based on CD8 expression is not performed during any of steps (a)-(f) of the method for expanding tumor-infiltrating lymphocytes (TILs) into a therapeutic TIL population, and this step (a) obtaining a first population of TILs from a tumor excised from a patient by processing a tumor sample obtained from the patient into multiple tumor fragments; (b) adding tumor fragments to a closed system; (c) performing a first expansion culture by culturing the first TIL population in a cell culture medium containing IL-2 to generate a second TIL population, wherein the first expansion culture is performed in a closed vessel providing a first gas permeable surface area, the first expansion culture is performed for about 3 to 14 days to obtain a second TIL population, wherein the second TIL population is at least 50-fold more numerous than the first TIL population, and wherein the transition from step (b) to step (c) occurs without opening the system; (d) performing a second expansion culture by adding additional IL-2, OKT-3, and antigen-presenting cells (APCs) to the cell culture medium of the second TIL population to generate a third TIL population, wherein the second expansion culture is performed for about 7 to 14 days to obtain the third TIL population, and the third TIL population has a higher number of effector T cells and / or T cells than the second TIL population. or a therapeutic TIL population comprising an expanded subpopulation of central memory T cells, wherein the second expansion is performed in a closed vessel providing a second gas permeable surface area, and the transition from step (c) to step (d) occurs without opening the system; (e) recovering the therapeutic TIL population obtained from step (d), wherein the transition from step (d) to step (e) occurs without releasing the system; and (f) transferring the TIL population collected in step (e) into an infusion bag, wherein the transition from step (e) to (f) occurs without opening the system. Includes.

[0282]

[0406] In one embodiment, the first TIL population, the second TIL population, the third TIL population, Selection of the L population or recovered TILs based on CD8 expression is not performed during any of steps (a) to (h) of the method of treating a subject with cancer, and the method comprises administering expanded tumor-infiltrating lymphocytes (TILs): (a) obtaining a first population of TILs from a tumor resected from a patient by processing a tumor sample obtained from the subject into multiple tumor fragments; (b) adding tumor fragments to a closed system; (c) performing a first expansion culture by culturing the first TIL population in a cell culture medium containing IL-2 to generate a second TIL population, wherein the first expansion culture is performed in a closed vessel providing a first gas permeable surface area, the first expansion culture is performed for about 3 to 14 days to obtain a second TIL population, wherein the second TIL population is at least 50-fold more numerous than the first TIL population, and the transition from step (b) to step (c) occurs without opening the system; (d) performing a second expansion culture by adding additional IL-2, OKT-3, and antigen-presenting cells (APCs) to the cell culture medium of the second TIL population to generate a third TIL population, wherein the second expansion culture is performed for about 7 to 14 days to obtain the third TIL population, the third TIL population being a T cell therapy TIL population comprising an expanded subpopulation of effector T cells and / or central memory T cells compared to the second TIL population, wherein the second expansion culture is performed in a closed vessel providing a second gas-permeable surface area, and the transition from step (c) to step (d) occurs without opening the system; (e) recovering the therapeutic TIL population obtained from step (d), wherein the transition from step (d) to step (e) occurs without releasing the system; and (f) transferring the TIL population collected in step (e) to an infusion bag, wherein the transition from step (e) to (f) occurs without opening the system; (g) optionally cryopreserving the infusion bag containing the TIL population collected in step (f) using a cryopreservation process; and (h) administering a therapeutically effective dose of the third population of TILs from the infusion bag in step (g) to the patient. Includes.

[0283]

[0407] The TILs prepared by the methods described herein may be, for example, freshly harvested TILs. The TILs are characterized by a significant difference in basal glycolysis compared to TILs harvested from the primary TILs and / or TILs prepared using methods other than those provided herein, including, for example, methods other than those embodied in FIG. 27 . In certain embodiments, selection of the first TIL population, the second TIL population, the third TIL population, the harvested TIL population, and / or a therapeutic TIL population based on CD8 expression is not performed during any of the steps discussed above or including the examples in FIG. 27 . In some embodiments, selection of the first TIL population based on CD8 expression is not performed. In some embodiments, selection of the second TIL population based on CD8 expression is not performed. In some embodiments, selection of the third TIL population based on CD8 expression is not performed. In some embodiments, selection of the harvested TIL population based on CD8 expression is not performed. In some embodiments, selection of the therapeutic TIL population based on CD8 expression is not performed. In some embodiments, selection of the first TIL population, the second TIL population, the third TIL population, or the recovered TILs based on CD8 expression is not performed during any of steps (a)-(h).

[0284]

[0408] The spare respiratory capacity (SRC) of TILs expanded using the different methods described herein was The Seahorse XF cell mitochondria stress test can be used to assess mitochondria, mitochondrial function, and glycolytic reserve. Mitochondrial function is measured by directly measuring cellular oxygen consumption rate (OCR) using respiratory regulators that target components of the electron transport chain in mitochondria. Test compounds (oligomycin, FCCP, and a mixture of rotenone and antimycin A, described below) are continuously injected to measure ATP production, maximal respiration, and non-mitochondrial respiration, respectively. These parameters and basal respiration are then used to calculate proton leak and spare respiratory capacity. Each regulator targets a specific component of the electron transport chain. Oligomycin inhibits ATP synthase (complex V), and the decrease in OCR after oligomycin injection correlates with mitochondrial respiration, which is associated with cellular ATP production. Carbonyl cyanide-4(trifluoromethoxy)phenylhydrazone (FCCP) is an uncoupler that disrupts the proton gradient and disrupts the mitochondrial membrane potential. As a result, electron flow through the electron transport chain is uninhibited, allowing maximal oxygen consumption by complex IV. FCCP-stimulated OCR can then be used to calculate spare respiratory capacity, defined as the difference between maximal and basal respiration. Spare respiratory capacity (SRC) is a measure of the ability of cells to respond to increased energy demands. The third injection is a mixture of rotenone, a complex I inhibitor, and antimycin A, a complex III inhibitor. This combination blocks mitochondrial respiration, allowing for the calculation of non-mitochondrial respiration, which is driven by processes outside of the mitochondria. In some embodiments, the comparison is to, for example, freshly harvested TILs and / or TILs prepared using methods other than those provided herein, including, for example, methods other than those embodied in FIG. 27.

[0285]

[0409] In some embodiments, the metabolic assay is basal respiration. The expanded TILs have a basal respiration rate that is at least 50%, at least 55%, at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 97%, at least 98%, or at least 99% of the basal respiration rate of freshly harvested TILs and / or TILs prepared using methods other than those provided herein, including, for example, methods other than those embodied in FIG. 27. In some embodiments, the basal respiration rate is about 50% to about 99% of the basal respiration rate of freshly harvested TILs and / or TILs prepared using methods other than those provided herein, including, for example, methods other than those embodied in FIG. 27. In some embodiments, the basal respiration rate is about 60% to about 99% of the basal respiration rate of freshly harvested TILs and / or TILs prepared using methods other than those provided herein, including, for example, methods other than those embodied in FIG. 27. In some embodiments, the basal respiration rate is about 70% to about 99% of the basal respiration rate of freshly harvested TILs and / or TILs prepared using methods other than those provided herein, including, for example, methods other than those embodied in FIG. 27. In some embodiments, the basal respiration rate is about 80% to about 99% of the basal respiration rate of freshly harvested TILs and / or TILs prepared using methods other than those provided herein, including, for example, methods other than those embodied in FIG. 27. In some embodiments, the basal respiration rate is about 90% to about 99% of the basal respiration rate of freshly harvested TILs and / or TILs prepared using methods other than those provided herein, including, for example, methods other than those embodied in FIG. 27. In some embodiments, the basal respiration rate is about 95% to about 99% of the basal respiration rate of freshly harvested TILs and / or TILs prepared using methods other than those provided herein, including, for example, methods other than those embodied in FIG. 27. In some embodiments, the second expanded TILs or second additional expanded TILs (e.g., those described in step D of Figure 27, including TILs referred to as reREP TILs) are freshly harvested TILs and / or or have a basal respiration rate that is not statistically significantly different from the basal respiration rate of TILs prepared using methods other than those provided herein, including, for example, methods other than those embodied in Figure 27. In some embodiments, the comparison is to, for example, freshly harvested TILs and / or TILs prepared using methods other than those provided herein, including, for example, methods other than those embodied in Figure 27.

[0286]

[0410] In some embodiments, the metabolic assay is spare respiratory capacity. The second expanded TILs or the second additional expanded TILs (e.g., including TILs referred to as reREP TILs, such as those described in step D of FIG. 27 ) have a spare respiratory capacity that is at least 50%, at least 55%, at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 97%, at least 98%, or at least 99% of the basal respiration rate of freshly harvested TILs and / or TILs prepared using methods other than those provided herein, including, for example, methods other than those embodied in FIG. 27 . In some embodiments, the spare respiratory capacity is about 50% to about 99% of the basal respiration rate of freshly harvested TILs. In some embodiments, the spare respiratory capacity is about 50% to about 99% of the basal respiration rate of freshly harvested TILs and / or TILs prepared using methods other than those provided herein, including, for example, methods other than those embodied in FIG. 27 . In some embodiments, the reserve respiratory capacity is about 60% to about 99% of the basal respiratory rate of freshly harvested TILs and / or TILs prepared using methods other than those provided herein, including, for example, methods other than those embodied in FIG. 27. In some embodiments, the reserve respiratory capacity is about 70% to about 99% of the basal respiratory rate of freshly harvested TILs and / or TILs prepared using methods other than those provided herein, including, for example, methods other than those embodied in FIG. 27. In some embodiments, the reserve respiratory capacity is about 80% to about 99% of the basal respiratory rate of freshly harvested TILs. In some embodiments, the reserve respiratory capacity is about 90% to about 99% of the basal respiratory rate of freshly harvested TILs and / or TILs prepared using methods other than those provided herein, including, for example, methods other than those embodied in FIG. 27. In some embodiments, the spare respiratory capacity is about 95% to about 99% of the basal respiratory rate of freshly harvested TILs and / or TILs prepared using methods other than those provided herein, including, for example, methods other than those embodied in FIG. 27.In some embodiments, the second expanded TILs or the second additional expanded TILs (e.g., including TILs referred to as reREP TILs, such as those described in step D of FIG. 27) have a spare respiratory capacity that is not statistically significantly different from the basal respiration rate of freshly harvested TILs and / or TILs adjusted using other methods other than those provided herein, including, for example, methods other than those embodied in FIG. 27.

[0287]

[0411] Generally, a second expanded culture of TILs or a second additional expanded culture of TILs (e.g., The TILs (such as those described in step D of FIG. 27 , including those referred to as reREP TILs) have a spare respiratory capacity that is at least 50%, at least 55%, at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 97%, at least 98%, or at least 99% of the basal respiration rate of freshly harvested TILs and / or TILs prepared using methods other than those provided herein, including, for example, methods other than those embodied in FIG. 27 . In some embodiments, the metabolic assay measured is glycolytic reserve. In some embodiments, the metabolic assay is spare respiratory capacity. To measure cellular (respiratory) metabolism, cells were treated with inhibitors of mitochondrial respiration and glycolysis to determine a metabolic profile of the TILs consisting of the following measures: baseline oxidative phosphorylation (as measured by OCR), spare respiratory capacity, baseline glycolytic activity (as measured by ECAR), and glycolytic reserve. Metabolic profiling can determine the ability of cells to perform glycolysis upon blockage of mitochondrial ATP production using the Seahorse combination mitochondrial / glycolytic stress test. Assays (including kits commercially available from Agilent®) were used. In some embodiments, the cells are glucose starved and then infused with glucose, followed by infusion of a stress agent. In some embodiments, the stress agent is selected from the group consisting of oligomycin, FCCP, rotenone, antimycin A, and / or 2-deoxyglucose (2-DG), and combinations thereof. In some embodiments, oligomycin is added at 10 mM. In some embodiments, FCCP is added at 10 mM. In some embodiments, rotenone is added at 2.5 mM. In some embodiments, antimycin A is added at 2.5 mM. In some embodiments, 2-deoxyglucose (2-DG) is added at 500 mM. In some embodiments, glycolytic capacity, glycolytic reserve, and / or non-glycolytic acidification are measured. Generally, the TILs have a glycolytic reserve that is at least 50%, at least 55%, at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 97%, at least 98%, or at least 99% of the basal respiration rate of freshly harvested TILs and / or TILs prepared using methods other than those provided herein, including, for example, methods other than those embodied in FIG. 27. In some embodiments, the glycolytic reserve is about 50% to about 99% of the basal respiration rate of freshly harvested TILs and / or TILs prepared using methods other than those provided herein, including, for example, methods other than those embodied in FIG. 27. In some embodiments, the glycolytic reserve is about 60% to about 99% of the basal respiration rate of freshly harvested TILs and / or TILs prepared using methods other than those provided herein, including, for example, methods other than those embodied in FIG. 27. In some embodiments, the glycolytic reserve is about 70% to about 99% of the basal respiration rate for freshly harvested TILs and / or TILs prepared using methods other than those provided herein, including, for example, methods other than those embodied in Figure 27. In some embodiments, the glycolytic reserve is about 80% to about 99% of the basal respiration rate for freshly harvested TILs and / or TILs prepared using methods other than those provided herein, including, for example, methods other than those embodied in Figure 27.In some embodiments, the glycolytic reserve is about 90% to about 99% of the basal respiration rate of freshly harvested TILs and / or TILs prepared using methods other than those provided herein, including, for example, methods other than those embodied in Figure 27. In some embodiments, the glycolytic reserve is about 95% to about 99% of the basal respiration rate of freshly harvested TILs and / or TILs prepared using methods other than those provided herein, including, for example, methods other than those embodied in Figure 27.

[0288]

[0412] In some embodiments, the metabolic assay is basal glycolysis. In the second expanded culture of TILs or a second additional expanded culture of TILs (e.g., reREP The second expanded TILs (e.g., including TILs referred to as reREP TILs, such as those described in step D of FIG. 27) have at least a two-fold, at least a three-fold, at least a four-fold, at least a five-fold, at least a six-fold, at least a seven-fold, at least a eight-fold, at least a nine-fold, or at least a ten-fold increase in basal glycolysis compared to the basal respiration rate of freshly harvested TILs and / or TILs prepared using other methods other than those provided herein, including, for example, methods other than those embodied in FIG. 27. In some embodiments, the second expanded TILs or second additional expanded TILs (e.g., including TILs referred to as reREP TILs, such as those described in step D of FIG. 27) have about a two-fold to about a ten-fold increase in basal glycolysis compared to the freshly harvested TILs and / or TILs prepared using other methods other than those provided herein, including, for example, methods other than those embodied in FIG. 27. In some embodiments, the second expanded TILs or the second additional expanded TILs (e.g., including TILs referred to as reREP TILs, such as those described in step D of FIG. 27) have about a 2-fold to about 8-fold increase in basal glycolysis compared to freshly harvested TILs and / or TILs prepared using other methods other than those provided herein, including, for example, methods other than those embodied in FIG. 27. In some embodiments, the second expanded TILs or the second additional expanded TILs (e.g., including TILs referred to as reREP TILs) have about a 2-fold to about an 8-fold increase in basal glycolysis compared to freshly harvested TILs and / or TILs prepared using other methods other than those provided herein, including, for example, methods other than those embodied in FIG. 27. The cultured TILs (e.g., those described in step D of FIG. 27, including TILs designated reREP TILs) have about a 3-fold to about a 7-fold increase in basal glycolysis compared to freshly harvested TILs and / or TILs prepared using other methods other than those provided herein, including, for example, methods other than those embodied in FIG. 27. In some embodiments, the second expanded cultured TILs or second additional expanded cultured TILs (e.g., those described in step D of FIG. 27, including TILs designated reREP TILs) have about a 2-fold to about a 4-fold increase in basal glycolysis compared to freshly harvested TILs and / or TILs prepared using other methods other than those provided herein, including, for example, methods other than those embodied in FIG. 27. In some embodiments, the second expanded TILs or second additional expanded TILs (e.g., those described in step D of FIG. 27, including TILs referred to as reREP TILs) have about a 2-fold to about a 3-fold increase in basal glycolysis compared to freshly harvested TILs and / or TILs prepared using methods other than those provided herein, including, for example, methods other than those embodied in FIG. 27.

[0289]

[0413] Generally, a second expanded culture of TILs or a second additional expanded culture of TILs (e.g., TILs (including those referred to as reREP TILs, such as those described in step D of FIG. 27 ) have a glycolytic reserve that is at least 50%, at least 55%, at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 97%, at least 98%, or at least 99% of the basal respiration rate of freshly harvested TILs and / or TILs prepared using methods other than those provided herein, including, for example, methods other than those embodied in FIG. 27 . In some embodiments, the glycolytic reserve is about 50% to about 99% of the basal respiration rate of freshly harvested TILs. In some embodiments, the glycolytic reserve is about 60% to about 99% of the basal respiration rate of freshly harvested TILs and / or TILs prepared using methods other than those provided herein, including, for example, methods other than those embodied in FIG. 27 . In some embodiments, the glycolytic reserve is about 70% to about 99% of the basal respiration rate of freshly harvested TILs and / or TILs prepared using methods other than those provided herein, including, for example, methods other than those embodied in FIG. 27. In some embodiments, the glycolytic reserve is about 80% to about 99% of the basal respiration rate of freshly harvested TILs and / or TILs prepared using methods other than those provided herein, including, for example, methods other than those embodied in FIG. 27. In some embodiments, the glycolytic reserve is about 90% to about 99% of the basal respiration rate of freshly harvested TILs and / or TILs prepared using methods other than those provided herein, including, for example, methods other than those embodied in FIG. 27. In some embodiments, the glycolytic reserve is about 95% to about 99% of the basal respiration rate of freshly harvested TILs.

[0290]

[0414] Granzyme B production: Granzyme B enhances the ability of TILs to kill target cells. Granzyme B levels were also measured in culture supernatants restimulated as described above with antibodies against CD3, CD28, and CD137 / 4-1BB using the Human Granzyme B DuoSet ELISA Kit (R & D Systems, Minneapolis, MN). The TILs were used and evaluated according to the manufacturer's instructions. In some embodiments, the second expanded TILs or the second additional expanded TILs (e.g., including TILs designated reREP TILs, such as those described in step D of FIG. 27) have increased granzyme B production. In some embodiments, the second expanded TILs or the second additional expanded TILs (e.g., including TILs designated reREP TILs, such as those described in step D of FIG. 27) have increased cytotoxic activity.

[0291]

[0415] In some embodiments, telomere length is a predictor of cell viability and / or cell function. In some embodiments, the telomeres may be prepared using methods other than those provided herein, including methods other than those embodied in FIG. Surprisingly, the telomere length of TILs produced by the present invention was identical to that of TILs produced by the present invention. Measurement of telomere length: Various methods have been used to measure telomere length in genomic DNA and cytological preparations. Telomere restriction fragment (TRF) analysis is the gold standard for measuring telomere length (de Lange et al., 1990). However, TRF analysis has not been shown to be a reliable method for measuring telomere length. The main limitation of F is the large amount of DNA required (1.5 g). Two widely used techniques for measuring telomere length may be used in the present invention: fluorescent in situ hybridization (FISH; Agilent Technologies, Santa Clara, CA) and quantitative PCR. In some embodiments, there is no change in telomere length between the TILs initially collected in step A and the expanded TILs, e.g., from step D provided in Figure 27.

[0292]

[0416] In some embodiments, the health of TILs is assessed by IFN-gamma (IFN-γ) secretion. IFN-γ secretion is measured by secretion. In some embodiments, IFN-γ secretion is an indicator of active TILs. In some embodiments, a potency assay of IFN-γ production is used. IFN-γ production is another measure of cytotoxic activity. IFN-γ production can be measured by determining the level of the cytokine IFN-γ in the culture medium of TILs stimulated with antibodies to CD3, CD28, and CD137 / 4-1BB. IFN-γ levels in the culture medium from these stimulated TILs can be determined by measuring IFN-γ release. In some embodiments, an increase in IFN-γ production at step D, e.g., TILs shown in FIG. 27, compared to initially harvested TILs at step A, e.g., is an indicator of increased cytotoxic activity of the TILs at step D. In some embodiments, IFN-γ secretion increases by 1-fold, 2-fold, 3-fold, 4-fold, or 5-fold or more. In some embodiments, IFN-γ secretion increases by 1-fold. In some embodiments, IFN-γ secretion increases by 2-fold. In some embodiments, IFN-γ secretion increases by 3-fold. In some embodiments, IFN-γ secretion is increased 4-fold. In some embodiments, IFN-γ secretion is increased 5-fold. In some embodiments, IFN-γ is measured using a Quantikine ELISA kit. In some embodiments, IFN-γ is measured in TIL ex vivo. In some embodiments, IFN-γ is measured in TIL ex vivo, including TILs generated by the methods of the invention, including, for example, the method of FIG. 27, as well as freshly harvested TILs, such as those examples provided in FIG. 83 (such as TILs from Example Process 1C), or TILs generated by other methods.

[0293]

[0417] In some embodiments, the cytotoxic ability of TILs to lyse target cells is increased by The cytotoxic potential of TILs was assessed using a co-culture assay of TILs with the bioluminescent cell line P815 (clone G6) according to the bioluminescent redirected lysis assay for TILs (potency assay), which measures the cytotoxic potential of TILs in a dose-dependent manner.

[0294]

[0418] In some embodiments, the method comprises: , and assays for assessing TIL viability. In some embodiments, TILs are expanded as discussed above, including, for example, as provided in FIG. 27. In some embodiments, TILs are cryopreserved prior to assessment of viability. In some embodiments, viability assessment includes thawing the TILs prior to performing the first expansion, the second expansion, and the additional second expansion. In some embodiments, the method provides assays for assessing cell proliferation, cytotoxicity, cell death, and / or other viability-related features of a TIL population. Viability can be measured by any of the TIL metabolic assays described above as well as any method known in the art for assessing cell viability. In some embodiments, the method provides assays for assessing cell proliferation, cytotoxicity, cell death, and / or other viability-related features of TILs expanded using the methods described herein, including as illustrated in FIG. 27.

[0295]

[0419] The present invention also provides an assay method for determining TIL viability. In some embodiments, the TILs have comparable viability compared to freshly harvested TILs and / or TILs prepared using methods other than those provided herein, including, for example, methods other than those embodied in Figure 27. In some embodiments, the TILs have increased viability compared to freshly harvested TILs and / or TILs prepared using methods other than those provided herein, including, for example, methods other than those embodied in Figure 27. The present disclosure also provides methods of assaying tumor infiltrating lymphocyte (TIL) viability by expanding TILs into a larger TIL population, the methods comprising: (i) Obtaining a first population of pre-expanded TILs; (ii) performing a first expansion by culturing the first TIL population in a cell culture medium containing IL-2 to generate a second TIL population; and (iii) performing a second expansion culture by adding additional IL-2, OKT-3, and antigen-presenting cells (APCs) to the cell culture medium of the second TIL population to generate a third TIL population, wherein the third TIL population is at least 100-fold more numerous than the second TIL population, and wherein the second expansion culture is performed for at least 14 days to obtain the third TIL population, wherein the third TIL population comprises increased effector T cell and / or central memory T cell subpopulations compared to the second TIL population, and wherein the third population is further assayed for viability. Includes.

[0296]

[0420] In some embodiments, the method comprises: (iv) further comprising performing an additional second expansion culture by adding additional IL-2, additional OKT-3, and additional APCs to the cell culture medium of the third TIL population, wherein the additional second expansion culture is performed for at least 14 days to obtain a larger TIL population than that obtained in step (iii), wherein the larger TIL population comprises increased effector T cell and / or central memory T cell subpopulations compared to the third TIL population, and wherein the third population is further assayed for viability.

[0297]

[0421] In some embodiments, prior to step (i), the cells are cryopreserved.

[0298]

[0422] In some embodiments, the cells are thawed prior to performing step (i).

[0299]

[0423] In some embodiments, to obtain enough TILs for analysis, steps ( iv) is repeated 1 to 4 times.

[0300]

[0424] In some embodiments, steps (i)-(iii) or (iv) are performed for about 4 It will be carried out within a period of 0 to approximately 50 days.

[0301]

[0425] In some embodiments, steps (i)-(iii) or (iv) are performed for about 4 It will be carried out within a period of 2 to approximately 48 days.

[0302]

[0426] In some embodiments, steps (i)-(iii) or (iv) are performed for about 4 It will be carried out within a period of 2 to approximately 45 days.

[0303]

[0427] In some embodiments, steps (i)-(iii) or (iv) are performed for about 4 This will be carried out within four days.

[0304]

[0428] In some embodiments, the cells from step (iii) or (iv) They express CD4, CD8, and TCRαβ to the same extent as freshly harvested cells.

[0305]

[0429] In some embodiments, the antigen-presenting cells are peripheral blood mononuclear cells (PBMCs). .

[0306]

[0430] In some embodiments, the PBMCs are 9 to 17 It is added to the cell culture on either day 1 or day 2.

[0307]

[0431] In some embodiments, the enzymes in the larger TIL population of step (iv) The effector T cells and / or central memory T cells exhibit one or more characteristics selected from the group consisting of CD27 expression, CD28 expression, longer telomeres, increased CD57 expression, and decreased CD56 expression compared to the effector T cells and / or central memory T cells in the third cell population.

[0308]

[0432] In some embodiments, effector T cells and / or central memory -T cells exhibit increased CD57 expression and decreased CD56 expression.

[0309]

[0433] In some embodiments, the APC is an artificial APC (aAPC).

[0310]

[0434] In some embodiments, the method comprises administering to a subject a nucleic acid encoding a high affinity T cell receptor. The method further comprises transducing the first TIL population with an expression vector comprising the acid.

[0311]

[0435] In some embodiments, the transduction step occurs before step (i). do.

[0312]

[0436] In some embodiments, the method comprises administering to a subject a subject a T cell signaling molecule comprising at least one of: The method further comprises transducing the first TIL population with an expression vector comprising a nucleic acid encoding a chimeric antigen receptor (CAR) comprising a single-chain variable fragment antibody fused to one endodomain.

[0313]

[0437] In some embodiments, the transduction step occurs before step (i). do.

[0314]

[0438] In some embodiments, the TILs are assayed for viability.

[0315]

[0439] In some embodiments, the TILs are assayed for viability after cryopreservation. can be.

[0316]

[0440] In some embodiments, the TILs are cryopreserved and after step (iv). Assayed for viability.

[0317]

[0441] The diverse antigen receptors of T and B lymphocytes are expressed by a limited but numerous gene segments. These TILs are produced by somatic recombination of T cells. These gene segments, V (variable), D (diversity), J (joining), and C (constant), determine the binding specificity and downstream applications of immunoglobulins and T cell receptors (TCRs). The present invention provides methods for generating TILs that exhibit and increase the diversity of their T cell repertoire (sometimes referred to as polyclonality). In some embodiments, the increase in diversity of the T cell repertoire is associated with increased survival compared to freshly harvested TILs and / or TILs prepared using methods other than those provided herein, including, for example, methods other than those embodied in FIG. 27. In some embodiments, TILs obtained by the present methods exhibit increased T cell repertoire diversity. In some embodiments, TILs obtained in the first expansion culture exhibit increased T cell repertoire diversity. In some embodiments, the increase in diversity is associated with increased immunoglobulin diversity. and / or increased T cell receptor diversity. In some embodiments, the immunoglobulin diversity is in immunoglobulin heavy chains. In some embodiments, the immunoglobulin diversity is in immunoglobulin light chains. In some embodiments, the diversity is in T cell receptors. In some embodiments, the diversity is in one of the T cell receptors selected from the group consisting of alpha, beta, gamma, and delta receptors. In some embodiments, expression of T cell receptor (TCR) alpha and / or beta is increased. In some embodiments, expression of T cell receptor (TCR) alpha is increased. In some embodiments, expression of T cell receptor (TCR) beta is increased. In some embodiments, expression of TCRab (i.e., TCR α / β) is increased.

[0318]

[0442] According to the present disclosure, methods for assaying the viability of TILs and / or administering them to a subject In some embodiments, the method for assaying tumor infiltrating lymphocytes (TILs) comprises: (i) Obtaining the first TIL population; (ii) performing a first expansion by culturing the first TIL population in a cell culture medium containing IL-2 to generate a second TIL population; and (iii) performing a second expansion by adding additional IL-2, OKT-3, and antigen-presenting cells (APCs) to the cell culture medium of the second TIL population to generate a third TIL population, wherein the third TIL population is at least 50-fold more numerous than the second TIL population; (iv) harvesting, washing, and cryopreserving the third TIL population; (v) cryopreserved TILs are stored at cryogenic temperatures; (vi) thawing the third TIL population to provide a thawed third TIL population; and (vii) performing an additional second expansion of a portion of the thawed third TIL population for an additional expansion period of at least 3 days (sometimes referred to as a reREP period) by adding IL-2, OKT-3, and APC to the cell culture medium of the third population, wherein the third expansion is performed to obtain a fourth TIL population, and comparing the number of TILs in the fourth TIL population with the number of TILs in the third TIL population to determine a ratio; (viii) determining whether the thawed TIL population is suitable for administration to a patient based on the ratio of step (vii); (ix) administering a therapeutically effective dose of the thawed third TIL population to the patient when the ratio of the number of TILs in the fourth TIL population to the number of TILs in the third TIL population is determined to be greater than 5:1 in step (viii). Includes.

[0319]

[0443] In some embodiments, an additional expansion period (referred to as the reREP period) is performed. The subsequent step of repopulation (sometimes referred to as "repopulation 4") is performed until the ratio of the number of TILs in the fourth TIL population to the number of TILs in the third TIL population is greater than 50:1.

[0320]

[0444] In some embodiments, a number of TILs sufficient to provide a therapeutically effective dosage. is approximately 2.3 x 1010 ~Approx. 13.7×10 10 There are individuals.

[0321]

[0445] In some embodiments, steps (i)-(vii) are carried out over a period of about 40 days to about 50 days. In some embodiments, steps (i)-(vii) are performed within a period of about 42 days to about 48 days. In some embodiments, steps (i)-(vii) are performed within a period of about 42 days to about 45 days. In some embodiments, steps (i)-(vii) are performed within about 44 days.

[0322]

[0446] In some embodiments, the cells from step (iii) or (vii) , CD4, CD8, and TCRαβ to the same extent as freshly harvested cells. In some embodiments, the cells are TILs.

[0323]

[0447] In some embodiments, the antigen-presenting cells are peripheral blood mononuclear cells (PBMCs). In some embodiments, the PBMCs are added to the cell culture in step (iii) on any day between 9 and 17.

[0324]

[0448] In some embodiments, the greater of step (iii) or (vii) The effector T cells and / or central memory T cells in the TIL population exhibit one or more characteristics selected from the group consisting of CD27 expression, CD28 expression, longer telomeres, increased CD57 expression, and decreased CD56 expression compared to the effector T cells and / or central memory T cells in the third cell population.

[0325]

[0449] In some embodiments, effector T cells and / or central memory -T cells exhibit increased CD57 expression and decreased CD56 expression.

[0326]

[0450] In some embodiments, the APC is an artificial APC (aAPC).

[0327]

[0451] In some embodiments, the gene encoding the high affinity T cell receptor is Transducing a first population of TILs with the present vector.

[0328]

[0452] In some embodiments, the transduction step occurs before step (i). do.

[0329]

[0453] In some embodiments, at least one end of a T cell signaling molecule Transducing the first TIL population with an expression vector comprising a nucleic acid encoding a chimeric antigen receptor (CAR) comprising a single-chain variable fragment antibody fused to a CAR domain.

[0330]

[0454] In some embodiments, the transduction step occurs before step (i). do.

[0331]

[0455] In some embodiments, the TILs are evaluated for viability after step (vii). Assayed.

[0332]

[0456] The present disclosure also provides additional methods for assaying TILs. The present disclosure provides a method for assaying TILs, the method comprising: (i) Obtaining a portion of the first cryopreserved TIL population; (ii) thawing a portion of the first cryopreserved TIL population; (iii) performing a first expansion culture to generate a second TIL population by culturing a portion of the first TIL population in cell culture medium comprising IL-2, OKT-3, and antigen-presenting cells (APCs) for an additional expansion period (sometimes referred to as the reREP period) of at least 3 days, wherein the portion from the first TIL population is compared with the second TIL population to determine a ratio of TIL numbers, and the ratio of the number of TILs in the second TIL population to the number of TILs in the portion of the first TIL population is greater than 5:1; (iv) determining whether the first population of TILs is suitable for use in therapeutic administration to a patient based on the ratio of step (iii); (v) determining that the first TIL population is suitable for use in therapeutic administration when the ratio of the number of TILs in the second TIL population to the number of TILs in the first TIL population is determined to be greater than 5:1 in step (iv). Includes.

[0333]

[0457] In some embodiments, the number of TILs in the second TIL population is compared to the number of TILs in the first TIL population. The ratio of TIL numbers in some groups is greater than 50:1.

[0334]

[0458] In some embodiments, the method comprises any of the embodiments provided herein. and performing expansion of the entire first cryopreserved TIL population from step (i) by the method as described in 2.

[0335]

[0459] In some embodiments, the method further comprises the step of: It further includes administering the entire TIL population to the patient.

[0336] Closed system for K.TIL production

[0460] The present invention provides for the use of a closed system during the TIL culture process. This allows for prevention and / or reduction of microbial contamination and allows for the use of fewer flasks, resulting in cost savings. In some embodiments, the closed system uses two vessels.

[0337]

[0461] Such closed systems are known in the art and are described, for example, at http: / / www.fda.gov / cber / guidelines.htm and https: / / www.fda.gov / BiologicsBloodVaccines / GuidanceComplianceRegulatoryInformation / Guidances / Blood / ucm076779.htm.

[0338]

[0462] As provided on the FDA website, closed systems with sterilization methods are It is well known and has been described in detail, see https: / / www.fda.gov / BiologicsBloodVaccines / GuidanceComplianceRegulatoryInformation / Guidances / Blood / ucm076779.htm, referenced above and provided in relevant sections below.

[0339] Introduction

[0463] Sterile connecting devices (STCDs) produce a sterile weld between two compatible pieces of tubing. This procedure allows for the sterile connection of containers and tubing of various diameters. This guidance describes recommended practices and procedures for the use of these devices. This guidance does not address the data or information that manufacturers of sterile connecting devices must submit to FDA to obtain marketing approval or authorization. It is also important to note that using an approved or certified sterile connecting device for a purpose not permitted by its labeling may result in the device being considered adulterated and misbranded under the Federal Food, Drug, and Cosmetic Act.

[0340] 1. FDA Recommendations

[0464] Manufacturers of blood products proposing routine use of FDA-approved STCDs Providers must incorporate information regarding such use into the Standard Operating Procedure (SOP) manual for each blood product. These descriptions must include record-keeping, product tracking, tube weld quality control, software, and disposable lot numbers (including vendor of additional elements). Quality control procedures must include integrity testing of each weld.

[0341] 2. Application of STCD

[0465] By using the device, the user is agreeing to comply with all applicable international treaties and regulations, including the United States and Canada, and to comply with all applicable international treaties and regulations. It should be noted that a "new product" may create a new product or significantly change its composition. For a "new product" that is the subject of a license, an application or supplement to the application must be submitted to the FDA in addition to the submission of SOPs. Generally, pooling or blending, including cell composition, represents a product change that requires the submission and approval of a license application or additional application. Such applications and additional applications must be meaningful throughout the period of the date the "new product" is proposed. It must include data demonstrating that it is safe and effective for its intended use and a description of its manufacturing procedures.

[0342]

[0466] The following commentary concerns the more common uses of FDA-cleared or approved STCDs. This document is provided as guidance to:

[0343] L. Adding a new or smaller needle to the blood collection set

[0467] Add needles using STCD before starting the procedure (whole blood collection, platelets Plasmapheresis (or primary plasma collection) is not considered to functionally open a closed system. If a needle is added during the procedure, only STCDs approved for welding of the liquid-filled tubing should be used. Use of an STCD is not considered to functionally open a closed system if it meets the weld integrity test.

[0344]

[0468] Platelets and open-system prepared pheresis are marked with a 24-hour expiration date. Platelets prepared in a functionally closed system, including pheresis products, must be labeled with a 5-day expiration date (see Revised Guideline for Collection of Platelets, Pheresis, October 7, 1988).

[0345]

[0469] The vendor and specifications of the added tubes and needles are consistent with the blood center's SOPs and records. The use of an STCD to add needles does not represent a significant change in manufacturing for which a licensed facility requires prior approval.

[0346] M. Use of STCD for the preparation of components

[0470] When using STCD to attach a component preparation bag, transfer pack Appropriate records must be maintained identifying the distributor of the blood and identifying the blood unit number and proper ABO / Rh verification. All blood and blood components must be properly labeled (21 CFR 606.121). example: · A fourth bag is added to the whole blood collection triple pack to produce cryoprecipitate antihemophilic factor from fresh frozen plasma. · Connection of additive solution to red blood cell unit. ·Addition of an in-line filter approved by the FDA for use in ingredient manufacturing. Addition of a third storage container to the plateletpheresis harness. While procedures must be established and records must be maintained for the above uses, the licensee does not need to obtain FDA approval to establish such procedures.

[0347] 1. Use of STCD to pool blood products

[0471] Appropriate use of STCD to pool platelets prepared from whole blood collections This eliminates potential contamination from commonly used spike and port entrances. Pooling immediately prior to transfusion is an example of such appropriate use. Pooled platelets must be administered within four hours of pooling (see 21 CFR 606.122(l)(2)).

[0348]

[0472] However, pooling and subsequent storage are managed as random donor units. This may increase risk compared to conventional pooling, and if a contaminated unit is pooled and stored with other units before administration, the total administered bacterial inoculum may increase as a result of replication in the additional units. Therefore, the recommended use of STCDs to pool and store platelets for more than 4 hours should be supported by data that adequately resolve whether such pooling is associated with an increased risk.

[0349]

[0473] Such pooling of platelets constitutes the production of a new product.

[0350]

[0474] Pools or mixtures containing platelets should be freshly prepared if stored for more than 4 hours. It is considered to be the manufacture of a product and requires the submission and approval of a license application or supporting documents.

[0351] 2. Use of STCD to prepare aliquots for pediatric use and split units

[0475] Pediatric units of whole blood, red blood cells, and fresh frozen plasma prepared using STCD A biologics license application (BLA) is not considered a new product requiring a supplemental BLA if it meets the following criteria: The manufacturer must have an approved biologics approval or supplemental license for the original (i.e., undivided) product, including approval for each anticoagulant used. Labeling must be submitted for review and approval before distribution. Labeling must be based on the comments section of FDA Form 2567, Transmittal of Labels and Circulars. Finished product containers approved for storing ingredients during preparation must be used.

[0352]

[0476] Licensed platelets contain at least 5.5 × (10) 10 pieces Platelets must contain at least 3.0 × (10) platelets (21 CFR 640.24(c)). 11 platelets (see Revised Guideline for the Collection of Platelets, Pheresis, October 7, 1988).

[0353]

[0477] Whole blood collection and automated hemapheresis procedures using STCD The procedures to be followed when preparing fractionated products from plasma and platelets prepared using the methods described below should include the following instructions: · A method for retrofitting an apheresis harness or collection container with an FDA-cleared STCD. Minimal volume of split plasma or whole blood product. Split plateletpheresis product volume and platelet concentration. Storage time of the preparation. The preparation must be in an approved container and conform to the storage time indicated on the label of such container. ·The method used to label and track divided products in the blood center's records.

[0354]

[0478] NOTE: The aliquot labeling procedure is designed to ensure that all components are tracked and unsatisfactory, if necessary. Procedures sufficient to authorize product recall must be clearly documented in the book.

[0355] 3. Use of the STCD for connection of additional saline or anticoagulant lines during automated plasma exchange procedures

[0479] Procedures must be developed and records maintained in accordance with the equipment manufacturer's instructions for use. However, licensees are not required to obtain FDA approval to establish procedures.

[0356] 4. Use of STCD for installation of processing fluids

[0480] Using the STCD, remove the container containing the washing solution or the processing solution from the frozen red blood cell product. If a product is attached to a food product, the specified shelf life period for the resulting product is 24 hours, unless a longer period is provided in the application for approval or application supplement to CBER (21 CFR 610.53(c)). Any waiver or modification must be approved in writing by the CBER Director (21 CFR 610.53(d)).

[0357] 5. Use of STCD to add an FDA-cleared leukocyte reduction filter

[0481] Some leukocyte reduction filters are not integrally attached to whole blood collection systems. The procedure for using STCD for pre-storage filtration must follow the filter manufacturer's instructions for use. It should not be.

[0358]

[0482] If the white blood cell count drops before delivery, this is considered a significant manufacturing change. Therefore, for new leukocyte-reducing products prepared using STCDs, manufacturers must submit a Biologics License Application (21 CFR 601.2) or a prior approval application supplement to the FDA (21 CFR 601.12). It is necessary.

[0359]

[0483] Use of STCDs to remove test samples from blood product containers (e.g., blood Use of STCD to remove platelet samples from platelet or platelet containers, pheresis for cross-matching, etc.

[0360]

[0484] Product volume and / or cell count after sample collection is stated on the original label or information circular. If the volume and / or cell count differs from that stated, the product labeling must be revised to reflect the new volume and / or cell count. For example, if the number of platelets in a platelet unit is 5.5 x (10), 10 No sample shall be removed that reduces the concentration below 21 CFR 640.24 (c)

[0361] 6. Additional Information from FDA Guidance

[0485] In addition to general guidance, the STCD guidance also provides This document provides specific information and examples regarding specifications for submitting applications and supporting documents to FDA. If you have further questions regarding the appropriate use of STCDs, please contact the Office of Hematology Research and Review in the Center for Biologics Evaluation and Research at the FDA.

[0362]

[0486] In some embodiments, the closed system allows TILs to be isolated from the tumor fragments from the time they are obtained. One container is used until ready for administration to a patient or cryopreservation. In some embodiments, when two containers are used, the first container is a closed G container and the TIL population is centrifuged and transferred to an infusion bag without opening the first closed G container. In some embodiments, when two containers are used, the infusion bag is a HypoThermosol-containing infusion bag. In a closed system or closed TIL cell culture system, once a tumor sample and / or tumor fragment is added, the system is tightly sealed from the outside, forming a closed environment free of bacterial, fungal, and / or other microbial contamination.

[0363]

[0487] In some embodiments, the reduction in microbial contamination is from about 5% to about 100%. In some embodiments, the reduction in microbial contamination is about 5% to about 95%. In some embodiments, the reduction in microbial contamination is about 5% to about 90%. In some embodiments, the reduction in microbial contamination is about 10% to about 90%. In some embodiments, the reduction in microbial contamination is about 15% to about 85%. In some embodiments, the reduction in microbial contamination is about 5%, about 10%, about 15%, about 20%, about 25%, about 30%, about 35%, about 40%, about 45%, about 50%, about 55%, about 60%, about 65%, about 70%, about 75%, about 80%, about 85%, about 90%, about 95%, about 97%, about 98%, about 99%, or about 100%.

[0364]

[0488] The closed system allows growing TILs in the absence and / or greatly reduced microbial contamination. do.

[0365]

[0489] Furthermore, the pH, carbon dioxide tension, and oxygen tension of the TIL cell culture environment are important factors that influence the cells' The temperature and pressure of the culture medium change as the cells are cultured. Therefore, even if a medium suitable for cell culture is available, it is necessary to constantly maintain the closed environment as an optimal environment for TIL growth. For this reason, it is desirable that the physical factors of pH, carbon dioxide partial pressure, and oxygen partial pressure in the culture medium of the closed environment are monitored by a sensor, and the signal from the sensor is used to control a gas exchanger installed at the inlet of the culture environment, and the gas partial pressure in the closed environment is adjusted in real time according to changes in the culture medium to optimize the cell culture environment. In some embodiments, the present invention provides A closed cell culture system is provided that integrates a gas exchanger equipped with a monitoring device at the entrance to the closed environment, which measures the pH, carbon dioxide partial pressure, and oxygen partial pressure of the closed environment and automatically adjusts the gas concentration based on signals from the monitoring device, thereby optimizing the cell culture environment.

[0366]

[0490] In some embodiments, the pressure within the enclosed environment is controlled continuously or intermittently. That is, the pressure within the closed environment can be varied, for example, by a pressure maintenance device, to ensure that the space is suitable for TIL growth under positive pressure conditions, or to promote fluid exudation and thus cell growth under negative pressure conditions. Furthermore, by applying negative pressure intermittently, temporary contraction of the volume of the closed environment can result in uniform and efficient exchange of circulating fluid within the closed environment.

[0367]

[0491] In some embodiments, the optimal culture components for TIL growth are replaced or supplemented. Factors such as IL-2 and / or OKT3, as well as combinations, may also be added.

[0368] C.Cell culture

[0492] In one embodiment, the method for expanding TILs is described above and shown in FIG. Examples include using about 5,000 mL to about 25,000 mL of cell culture medium, about 5,000 mL to about 10,000 mL of cell culture medium, or about 5,800 mL to about 8,700 mL of cell culture medium. In some embodiments, the medium is serum-free, for example, as described in Example 21. In some embodiments, the medium for the first expansion culture is serum-free. In some embodiments, the medium for the second expansion culture is serum-free. In some embodiments, both the medium for the first and second expansion cultures are serum-free. In certain embodiments, one or less types of cell culture medium are used to expand the number of TILs. Any suitable cell culture medium can be used, for example, AIM-V cell culture medium (containing L-glutamine, 50 μM sulfate, 50 μM ATP ... In this regard, the methods of the present invention advantageously utilize a cell culture medium containing 10 μM leptomycin, 10 μM gentamicin sulfate, and 10 μM gentamicin sulfate (Invitrogen, Carlsbad CA) for the expansion of TIL numbers. The amount of medium and number of media types required is reduced. In certain embodiments, expanding the number of TILs can involve feeding the cells no more frequently than once every three or four days. Expanding the number of cells in a gas-permeable container simplifies the procedures required for cell expansion by reducing the feeding frequency required for cell expansion cultures.

[0369]

[0493] In some embodiments, the cell culture medium in the first and / or second gas permeable container The cell culture medium in the first and / or second gas-permeable containers is unfiltered. The use of unfiltered cell culture medium can simplify the procedures required to expand the number of cells. In some embodiments, the cell culture medium in the first and / or second gas-permeable containers does not contain β-mercaptoethanol (BME).

[0370]

[0494] In one embodiment, obtaining a tumor tissue sample from the mammal; cell culture medium The duration of the method includes culturing a tumor tissue sample in a first gas-permeable container containing the TILs; obtaining TILs from the tumor tissue sample; and expanding the number of TILs in a second gas-permeable container containing cell culture medium for about 7-14 days, e.g., about 11 days. In some embodiments, the pre-REP is about 7-14 days, e.g., about 11 days. In some embodiments, the REP is about 7-14 days, e.g., about 11 days.

[0371]

[0495] In one embodiment, the TILs are expanded in a gas-permeable container. Permeable containers have been used to expand TILs using PBMCs using methods, compositions, and devices known in the art, including those described in U.S. Patent Application Publication No. 2005 / 0106717 A1, the disclosure of which is incorporated herein by reference. In one embodiment, TILs are expanded in gas-permeable bags. In one embodiment, TILs are expanded in gas-permeable bags, such as the Xuri Cell Expansion System W25 (GE Healthcare). The TILs are expanded using a cell expansion system that expands the TILs in a gas-permeable bag. In one embodiment, the TILs are expanded using a cell expansion system that expands the TILs in a gas-permeable bag, such as the WAVE Bioreactor System, also known as the Xuri Cell Expansion System W5 (GE Healthcare). In one embodiment, the cell expansion system comprises a gas-permeable cell bag having a volume selected from the group consisting of about 100 mL, about 200 mL, about 300 mL, about 400 mL, about 500 mL, about 600 mL, about 700 mL, about 800 mL, about 900 mL, about 1 L, about 2 L, about 3 L, about 4 L, about 5 L, about 6 L, about 7 L, about 8 L, about 9 L, and about 10 L.

[0372]

[0496] In one embodiment, TILs can be expanded in G-Rex flasks (commercially available from Wilson Wolf Manufacturing). Approximately 5 x 10 5 cells / cm 2 From 10 x 10 6 ~30×10 6 cells / cm 2 In one embodiment, this is done without feeding. In another embodiment, this is done without feeding as long as there is about 10 cm of medium in the G-Rex flask. In some embodiments, there is no feeding, but one or more cytokines are added. In some embodiments, the cytokines can be added as a bolus without any need to mix the cytokines with the medium. Such vessels, devices, and methods are known in the art and have been used for the expansion of TILs, including, for example, U.S. Patent Application Publication No. 2014 / 0377739 A1, WO 2014 / 210036 A1, U.S. Patent Application Publication No. 2013 / 0115617 A1, WO 2013 / 188427 A1, U.S. Patent Application Publication No. 2011 / 0136228 A1, U.S. Patent No. 8,809,050 B2, WO 2011 / 072088 A2, U.S. Patent Application Publication No. 2016 / 0208216 A1, U.S. Patent Application Publication No. 2012 / 0244133 A1, WO 2012 / 129201 A1, U.S. Patent Application Publication No. 2013 / 0102075 A1, U.S. Patent No. 8,956,860 B2, and the like. B2, WO 2013 / 173835 A1, and U.S. Patent Application Publication No. 2015 / 0175966 A1, the disclosures of which are incorporated herein by reference. Such processes are also described in Jin et al., J. Immunotherapy, 2012, 35:283-292. .

[0373] D. Optional TIL Gene Modification

[0497] In some embodiments, the TILs are optionally, but not limited to, high affinity TILs. They are genetically engineered to contain additional functionality, including T cell receptors (TCRs), for example, TCRs that target tumor-associated antigens such as MAGE-1, HER2, or NY-ESO-1, or chimeric antigen receptors (CARs) that bind to tumor-associated cell surface molecules (e.g., mesothelin) or lineage-restricted cell surface molecules (e.g., CD19).

[0374] E. Optional TIL cryopreservation

[0498] Either the bulk TIL population or the expanded cultured population of TILs can be frozen as needed. In some embodiments, cryopreservation is performed on therapeutic TIL populations. In some embodiments, cryopreservation occurs on TILs collected after the second expansion. In some embodiments, cryopreservation is performed on TILs in exemplary step F of FIG. 27. In some embodiments, TILs are cryopreserved in an infusion bag. In some embodiments, TILs are cryopreserved before placement in an infusion bag. In some embodiments, TILs are cryopreserved but not placed in an infusion bag. In some embodiments, cryopreservation is performed using a cryopreservation medium. In some embodiments, the cryopreservation medium contains dimethyl sulfoxide (DMSO). This is generally achieved by placing the TIL population in a freezing solution, such as 85% complement-inactivated AB serum and 15% dimethyl sulfoxide (DMSO). The cells in this solution are placed in a cryogenic vial, stored at -80°C for 24 hours, and optionally transferred to a gas nitrogen freezer for cryopreservation. Sadeghi, et al., Acta See Oncologica 2013, 52, 978-986.

[0375]

[0499] If appropriate, remove the cells from the freezer and place them in a 37°C water bath for approximately 5 minutes. Thaw until a quarter of the cells are thawed. The cells are generally resuspended in complete medium and optionally washed one or more times. In some embodiments, the thawed TILs can be counted and assessed for viability as known in the art.

[0376]

[0500] In a preferred embodiment, the population of TILs is cryopreserved using CS10 cryopreservation medium (CryoStor 10, BioLife Solutions). The population is cryopreserved using a cryopreservation medium (containing dimethyl sulfoxide (DMSO)). In a preferred embodiment, the population of TILs is cryopreserved using a 1:1 (volume:volume) ratio of CS10 and cell culture medium. In a preferred embodiment, the population of TILs is cryopreserved using an approximately 1:1 (volume:volume) ratio of CS10 and cell culture medium, further containing additional IL-2.

[0377]

[0501] As discussed above in steps A-E, the entire TIL expansion process Cryopreservation can occur at various time points throughout the body. In some embodiments, the bulk TIL population after the first expansion according to step B or the expanded TIL population after one or more second expansion rounds according to step D may be cryopreserved. Cryopreservation can generally be achieved by placing the TIL population in a freezing solution, such as 85% complement-inactivated AB serum and 15% dimethyl sulfoxide (DMSO). Cells in this solution are placed in cryovials and stored at -80°C for 24 hours, and optionally transferred to a gas-nitrogen freezer for cryopreservation. See Sadeghi, et al., Acta Oncologica 2013, 52, 978-986.

[0378]

[0502] If appropriate, remove the cells from the freezer and place them in a 37°C water bath for approximately 5 minutes. Thaw until a quarter of the cells are thawed. The cells are generally resuspended in complete medium and optionally washed one or more times. In some embodiments, the thawed TILs can be counted and assessed for viability as known in the art.

[0379]

[0503] In some cases, the TIL population in step B is cultured using the protocols discussed below. Alternatively, the bulk TIL population can be subjected to steps C and D and cryopreserved after step D. Similarly, if the genetically modified TILs are to be used therapeutically, the TIL population from step B or step D can be subject to genetic modification for appropriate treatment.

[0380] F. Optional Cell Viability Analysis

[0504] Optionally, a first expansion culture (sometimes referred to as an initial bulk expansion culture) ) can be followed by a cell viability assay using standard assays known in the art. For example, a sample of bulk TILs can be subjected to a trypan blue dye exclusion assay, which selectively labels dead cells and allows for assessment of viability. Other assays used to test for viability include, but are not limited to, the Alamar Blue assay; and the MTT assay.

[0381] 1. Cell Count, Viability, and Flow Cytometry

[0505] In some embodiments, cell number and / or viability are measured. Expression of markers such as, but not limited to, CD3, CD4, CD8, and CD56, as well as any others disclosed or described herein, can be measured by antibody-based flow cytometry, such as, but not limited to, those commercially available from BD Biosciences (BD Biosciences, San Jose, CA) using a FACSCanto™ flow cytometer (BD Biosciences). Cells are counted using a disposable c-chip hemocytometer (VWR, Batavia, IL). Cells can be counted manually using a cytochrome P4500 (CYP4500) staining kit, and viability can be assessed using any method known in the art, including but not limited to trypan blue staining.

[0382]

[0506] In some cases, bulk TIL populations are directly cultured using the protocols discussed below. The bulk TIL population can be immediately cryopreserved. Alternatively, the bulk TIL population can be subjected to REP, as discussed below, and then cryopreserved. Similarly, where the genetically modified TILs are to be used in therapy, the bulk or REP TIL population can be subjected to genetic modification for the appropriate treatment.

[0383] 2.Cell culture

[0507] In one embodiment, the method for expanding TILs involves culturing a culture medium containing about 5,000 mL to about 2 This may include using 5,000 mL of cell culture medium, about 5,000 mL to about 10,000 mL of cell culture medium, or about 5,800 mL to about 8,700 mL of cell culture medium. In some embodiments, one or less types of cell culture medium are used to expand the number of TILs. Any suitable cell culture medium may be used, for example, AIM-V cell culture medium (L-glutamine, 50 μM streptomycin sulfate, 50 μM ethanol ... and 10 μM gentamicin sulfate) cell culture medium (Invitrogen, Carlsbad CA). In this regard, the methods of the present invention advantageously reduce the amount of medium and number of media types required to expand the number of TILs. In certain embodiments, expanding the number of TILs can involve feeding the cells no more frequently than once every three or four days. Expanding the number of cells in a gas-permeable container simplifies the procedures required to expand the number of cells by reducing the feeding frequency required for cell expansion.

[0384]

[0508] In some embodiments, the cell culture medium in the first and / or second gas permeable container The cell culture medium in the first and / or second gas-permeable containers is unfiltered. The use of unfiltered cell culture medium can simplify the procedures required to expand the number of cells. In some embodiments, the cell culture medium in the first and / or second gas-permeable containers does not contain β-mercaptoethanol (BME).

[0385]

[0509] In one embodiment, obtaining a tumor tissue sample from the mammal; cell culture medium the duration of the method includes culturing the tumor tissue sample in a first gas-permeable container containing aAPC therein; obtaining TILs from the tumor tissue sample; and expanding the number of TILs in a second gas-permeable container containing cell culture medium using aAPCs for about 14 to about 42 days, e.g., about 28 days.

[0386]

[0510] In one embodiment, the TILs are expanded in a gas-permeable container. Permeable containers have been used to expand TILs using PBMCs using methods, compositions, and devices known in the art, including those described in U.S. Patent Application Publication No. 2005 / 0106717 A1, the disclosure of which is incorporated herein by reference. In one embodiment, TILs are expanded in gas-permeable bags. In one embodiment, TILs are expanded in gas-permeable bags, such as the Xuri Cell Expansion System W25 (GE Healthcare). The TILs are expanded using a cell expansion system that expands the TILs in a gas-permeable bag. In one embodiment, the TILs are expanded using a cell expansion system that expands the TILs in a gas-permeable bag, such as the WAVE Bioreactor System, also known as the Xuri Cell Expansion System W5 (GE Healthcare). In one embodiment, the cell expansion system comprises a gas-permeable cell bag having a volume selected from the group consisting of about 100 mL, about 200 mL, about 300 mL, about 400 mL, about 500 mL, about 600 mL, about 700 mL, about 800 mL, about 900 mL, about 1 L, about 2 L, about 3 L, about 4 L, about 5 L, about 6 L, about 7 L, about 8 L, about 9 L, and about 10 L.

[0387]

[0511] In one embodiment, TILs can be expanded in G-Rex flasks (commercially available from Wilson Wolf Manufacturing). Approximately 5 x 10 5 cells / cm 2 From 10 x 10 6 ~30×10 6 cells / cm 2 In one embodiment, this is done without feeding. In another embodiment, this is done without feeding as long as there is about 10 cm of medium in the G-Rex flask. In some embodiments, there is no feeding, but one or more cytokines are added. In some embodiments, the cytokines can be added as a bolus without any need to mix the cytokines with the medium. Such vessels, devices, and methods are known in the art and have been used for the expansion of TILs, including, for example, U.S. Patent Application Publication No. 2014 / 0377739 A1, WO 2014 / 210036 A1, U.S. Patent Application Publication No. 2013 / 0115617 A1, WO 2013 / 188427 A1, U.S. Patent Application Publication No. 2011 / 0136228 A1, U.S. Patent No. 8,809,050 B2, WO 2011 / 072088 A2, U.S. Patent Application Publication No. 2016 / 0208216 A1, U.S. Patent Application Publication No. 2012 / 0244133 A1, WO 2012 / 129201 A1, U.S. Patent Application Publication No. 2013 / 0102075 A1, U.S. Patent No. 8,956,860 B2, and the like. B2, WO 2013 / 173835 A1, and U.S. Patent Application Publication No. 2015 / 0175966 A1, the disclosures of which are incorporated herein by reference. Such processes are also described in Jin et al., J. Immunotherapy, 2012, 35:283-292. Optional TIL genetic modification

[0388]

[0512] In some embodiments, the TILs are optionally, but not limited to, high affinity TILs. They are genetically engineered to contain additional functionality, including T cell receptors (TCRs), for example, TCRs that target tumor-associated antigens such as MAGE-1, HER2, or NY-ESO-1, or chimeric antigen receptors (CARs) that bind to tumor-associated cell surface molecules (e.g., mesothelin) or lineage-restricted cell surface molecules (e.g., CD19).

[0389] IV. Patient Treatment Methods

[0513] The treatment method begins with the initial collection and culture of TILs. Both of these methods have been described in the art, for example, by Jin et al., J. Immunotherapy, 2012, 35(3):283-292, which is incorporated herein by reference in its entirety. Embodiments of methods of treatment are described throughout the following sections, including the examples.

[0390]

[0514] The methods described herein, including those described in steps A-F above, Alternatively, expanded TILs generated according to the methods described in steps A-F above (such as those shown in FIG. 27) find particular use in treating cancer patients (e.g., Goff, et al., J. Clinical Oncology, 2016, 34(20):2389-239, and supplemental content, which are incorporated by reference herein in their entirety). In some embodiments, TILs are used to treat metastatic tumors, as previously described. Fresh tumors were grown from deposited resections of human melanoma (see Dudley, et al., J Immunother., 2003, 26:332-342; incorporated herein by reference in its entirety). Fresh tumors were grown under sterile conditions. Representative samples can be collected for formal pathological analysis. 3 ~3mm 3 A single fragment of 10 ...

[0391]

[0515] In some embodiments, phenotypic analysis (CD3, CD4, CD8, and CD 56) Expanded TILs were sampled and tested against autologous tumors when available. TILs can be considered responsive if overnight co-culture produces interferon gamma (IFN-γ) levels >200 pg / mL and twice background (Goff, et al., J Immunother., 2010, 33:840-847; see references herein). (The text is hereby incorporated by reference in its entirety.) In some embodiments, cultures with evidence of auto-responsiveness or sufficient growth patterns may be selected for a second expansion culture (e.g., a second expansion culture as provided in step D of FIG. 27), including a second expansion culture, sometimes referred to as a rapid expansion culture (REP). In some embodiments, expanded TILs with high auto-responsiveness (e.g., high proliferation during the second expansion culture) are selected for an additional second expansion culture. In some embodiments, TILs with high auto-responsiveness (e.g., high proliferation during the second expansion culture as provided in step D of FIG. 27) are selected for an additional second expansion culture according to step D of FIG. 27.

[0392]

[0516] In some embodiments, the patient is not directly transferred to ACT (adoptive cell transfer), For example, in some embodiments, the cells are not utilized immediately after tumor harvest and / or the first expansion. In some embodiments, the TILs can be cryopreserved and thawed two days before administration to a patient. In some embodiments, the TILs can be cryopreserved and thawed one day before administration to a patient. In some embodiments, the TILs can be cryopreserved and thawed immediately before administration to a patient.

[0393]

[0517] The cell phenotype of the cryopreserved TIL samples from the transfusion bag was determined by the surface markers CD3, C Serum cytokines were measured using standard enzyme-linked immunosorbent assays. Elevated serum IFN-γ was defined as >100 pg / mL and greater than 4 cm baseline levels.

[0394]

[0518] In some embodiments, the methods provided herein, such as those illustrated in FIG. TILs produced by the methods provided herein, e.g., the methods illustrated in FIG. 27, provide surprising improvements in the clinical efficacy of TILs. In some embodiments, TILs produced by the methods provided herein, e.g., the methods illustrated in FIG. 27, exhibit increased clinical efficacy compared to TILs produced by methods other than those described herein, including, e.g., methods other than those illustrated in FIG. 27. In some embodiments, methods other than those described herein include a method referred to as Process 1C and / or Generation 1 (Gen1). In some embodiments, the increased efficacy is measured by DCR, ORR, and / or other clinical response. In some embodiments, TILs produced by the methods provided herein, e.g., the methods illustrated in FIG. 27, exhibit similar response times and safety profiles as TILs produced by methods other than those described herein, including, e.g., the Gen1 process, other than the methods illustrated in FIG. 27.

[0395]

[0519] In some embodiments, IFN-gamma (IFN-γ) is used to improve treatment efficacy and In some embodiments, IFN-γ in the blood of subjects treated with TILs is an indicator of increased activity and / or clinical efficacy. In some embodiments, IFN-γ in the blood of subjects treated with TILs is an indicator of active TILs. In some embodiments, a potency assay of IFN-γ production is used. IFN-γ production is another measure of cytotoxicity. IFN-γ production can be measured by determining the level of the cytokine IFN-γ in the blood, serum, or TILs ex vivo of subjects treated with TILs prepared by the methods of the invention, including those exemplified in FIG. 27. In some embodiments, an increase in IFN-γ is an indicator of treatment efficacy in patients treated with TILs generated by the methods of the invention. In some embodiments, IFN-γ is increased by 1-fold, 2-fold, or more compared to untreated patients and / or compared to patients treated with TILs prepared using methods other than those provided herein, including, for example, methods other than those embodied in FIG. 27. 27. In some embodiments, IFN-γ secretion is increased by 1-fold, 3-fold, 4-fold, 5-fold, or more. In some embodiments, IFN-γ secretion is increased by 1-fold compared to untreated patients and / or compared to patients treated with TILs prepared using a method other than those provided herein, including, for example, a method other than those embodied in FIG. 27. In some embodiments, IFN-γ secretion is increased by 2-fold compared to untreated patients and / or compared to patients treated with TILs prepared using a method other than those provided herein, including, for example, a method other than those embodied in FIG. 27. In some embodiments, IFN-γ secretion is increased by 3-fold compared to untreated patients and / or compared to patients treated with TILs prepared using a method other than those provided herein, including, for example, a method other than those embodied in FIG. 27. In some embodiments, IFN-γ secretion is increased by 4-fold compared to untreated patients and / or compared to patients treated with TILs prepared using a method other than those provided herein, including, for example, a method other than those embodied in FIG. 27. In some embodiments, IFN-γ secretion is increased 5-fold compared to untreated patients and / or compared to patients treated with TILs prepared using methods other than those provided herein, including, for example, methods other than those embodied in FIG. 27. In some embodiments, IFN-γ is measured using a Quantikine ELISA kit. In some embodiments, IFN-γ is measured in TILs ex vivo from subjects treated with TILs prepared by the methods of the invention, including, for example, those described in FIG. 27. In some embodiments, IFN-γ is measured in the blood of subjects treated with TILs prepared by the methods of the invention, including, for example, those described in FIG. 27. In some embodiments, IFN-γ is measured in TIL serum from subjects treated with TILs prepared by the methods of the invention, including, for example, those described in FIG. 27.

[0396]

[0520] In some embodiments, a higher mean IP-10 may indicate treatment efficacy and / or is an indication of increased clinical efficacy. In some embodiments, a higher mean IP-10 in the blood of subjects treated with TILs is an indication of active TILs. IP-10 production can be measured by determining the level of IP-10 in the blood of subjects treated with TILs prepared by the methods of the invention, including those illustrated in FIG. 27. In some embodiments, a higher mean IP-10 is an indication of treatment efficacy in patients treated with TILs made by the methods of the invention. In some embodiments, a higher mean IP-10 correlates with a 1-fold, 2-fold, 3-fold, 4-fold, or 5-fold or greater increase compared to untreated patients and / or compared to patients treated with TILs prepared using methods other than those provided herein, including, for example, methods other than those embodied in FIG. 27. In some embodiments, a higher mean IP-10 correlates with a 1-fold increase compared to untreated patients and / or compared to patients treated with TILs prepared using methods other than those provided herein, including, for example, methods other than those embodied in FIG. 27. In some embodiments, a higher mean IP-10 correlates with a 2-fold increase compared to untreated patients and / or compared to patients treated with TILs prepared using methods other than those provided herein, including, for example, methods other than those embodied in FIG. 27. In some embodiments, a higher mean IP-10 correlates with a 3-fold increase compared to untreated patients and / or compared to patients treated with TILs prepared using methods other than those provided herein, including, for example, methods other than those embodied in FIG. 27. In some embodiments, a higher mean IP-10 correlates with a 4-fold increase compared to untreated patients and / or compared to patients treated with TILs prepared using methods other than those provided herein, including, for example, methods other than those embodied in FIG. 27. In some embodiments, a higher mean IP-10 correlates with a 5-fold increase compared to untreated patients and / or compared to patients treated with TILs prepared using methods other than those provided herein, including, for example, methods other than those embodied in FIG. 27.In some embodiments, IP-10 is measured in the blood of subjects treated with TILs prepared by the methods of the invention, including, for example, those described in Figure 27. In some embodiments, IP-10 is measured in the blood of subjects treated with TILs prepared by the methods of the invention, including, for example, those described in Figure 27. TIL serum from subjects treated with HIV is measured.

[0397]

[0521] In some embodiments, higher mean MCP-1 may increase treatment efficacy and / or is an indication of increased clinical efficacy. In some embodiments, higher mean MCP-1 in the blood of subjects treated with TILs is an indication of active TILs. MCP-1 production can be measured by determining the level of MCP-1 in the blood of subjects treated with TILs prepared by the methods of the invention, including those illustrated in FIG. 27. In some embodiments, higher mean MCP-1 is an indication of treatment efficacy in patients treated with TILs generated by the methods of the invention. In some embodiments, higher mean MCP-1 correlates with a 1-fold, 2-fold, 3-fold, 4-fold, or 5-fold or greater increas...

Claims

1. 1. A therapeutic population of tumor-infiltrating lymphocytes (TILs) obtained by a method for expanding TILs, said method comprising: (a) obtaining a first population of TILs from a tumor resected from a subject with cancer by processing a tumor sample obtained from the tumor resected from the subject with cancer into a plurality of tumor fragments or digesting the tumor sample into a tumor digest; (b) adding the tumor fragments or tumor digests to a closed system; (c) performing a first expansion culture by culturing the first TIL population in a cell culture medium containing IL-2 to generate a second TIL population, wherein the first expansion culture is performed in the closed system providing a first gas permeable surface area, and the first expansion culture is performed for 3 days, 4 days, 5 days, 6 days, 7 days, 8 days, 9 days, 10 days, 11 days, or 12 days to obtain the second TIL population, and the transition from step (b) to step (c) occurs without opening the system; (d) performing a second expansion by adding additional IL-2, OKT-3, and antigen-presenting cells (APCs) to the cell culture medium of the second TIL population to generate a third TIL population, wherein the second expansion is performed for 7 days, 8 days, 9 days, 10 days, 11 days, or 12 days to obtain the third TIL population, the third TIL population being a therapeutic TIL population, the second expansion being performed in the closed system providing a second gas-permeable surface area, and the transition from step (c) to step (d) occurring without opening the system; (e) recovering the therapeutic TIL population obtained from step (d), wherein the transition from step (d) to step (e) occurs without releasing the system; (f) transferring the TIL population collected in step (e) to an infusion bag, wherein the transition from step (e) to (f) occurs without opening the system; and (g) cryopreserving the infusion bag containing the harvested TIL population from step (f) using a cryopreservation process; A therapeutic TIL population comprising:

2. 1. A therapeutic population of tumor-infiltrating lymphocytes (TILs) obtained by a method for expanding TILs, said method comprising: (a) adding to the closed system a tumor digest or tumor fragment, the tumor digest or tumor fragment comprising a first population of TILs obtained from a tumor resected from a subject with cancer; (b) performing a first expansion culture by culturing the first TIL population in a cell culture medium containing IL-2 to generate a second TIL population, wherein the first expansion culture is performed in the closed system providing a first gas permeable surface area, and the first expansion culture is performed for 3 days, 4 days, 5 days, 6 days, 7 days, 8 days, 9 days, 10 days, 11 days, or 12 days to obtain the second TIL population, and the transition from step (a) to step (b) occurs without opening the system; (c) performing a second expansion culture by adding additional IL-2, OKT-3, and antigen-presenting cells (APCs) to the cell culture medium of the second TIL population to generate a third TIL population, wherein the second expansion culture is performed for 7 days, 8 days, 9 days, 10 days, 11 days, or 12 days to obtain the third TIL population, the second expansion culture is performed in the closed system providing a second gas-permeable surface area, and the transition from step (b) to step (c) occurs without opening the system; (d) recovering the third population of TILs obtained from step (c), wherein the transition from step (c) to step (d) occurs without opening the system; (e) transferring the third population of TILs collected in step (d) to an infusion bag, wherein the transition from step (d) to (e) occurs without opening the system; and (f) cryopreserving the infusion bag containing the harvested TIL population from step (e) using a cryopreservation process; A therapeutic TIL population comprising:

3. The therapeutic TIL population of claim 1 or 2, wherein the culture medium in the first expansion culture and / or the second expansion culture does not contain human serum.

4. The therapeutic TIL population of claim 1 or 2, wherein the harvested TIL population comprises a therapeutically effective dosage of TILs.

5. 5. The therapeutic TIL population of claim 4, wherein the therapeutically effective dosage is 1 x 10 9 to 9 x 10 9 TILs.

6. The therapeutic TIL population of any one of claims 1 to 5, wherein the APCs are peripheral blood mononuclear cells (PBMCs).

7. The recovered TIL population is CD8+ compared to the first and / or second TIL population. ＋ The therapeutic TIL population of any one of claims 1 to 6, which exhibits an expansion of a subpopulation of cells.

8. The therapeutic TIL population of claim 6 or 7, wherein the PBMCs are added at a TIL:PBMC ratio of 1:

25.

9. 9. The therapeutic TIL population of any one of claims 1 to 8, wherein the cancer is selected from the group consisting of melanoma (including metastatic melanoma), ovarian cancer, cervical cancer, non-small cell lung cancer (NSCLC), lung cancer, bladder cancer, breast cancer, cancer caused by human papillomavirus, head and neck cancer (including head and neck squamous cell carcinoma (HNSCC)), renal cancer, and renal cell carcinoma.

10. 10. The therapeutic TIL population of claim 9, wherein the cancer is selected from the group consisting of melanoma, metastatic melanoma, HNSCC, cervical cancer, and NSCLC.

11. The therapeutic TIL population of any one of claims 1 to 10, wherein the first expansion culture and the second expansion culture are each performed separately within a period of up to 12 days.

12. The therapeutic TIL population of any one of claims 1 to 10, wherein the first expansion culture and the second expansion culture are each carried out separately for 12 days.

13. The therapeutic TIL population of any one of claims 1 to 10, wherein the first expansion culture and the second expansion culture are each performed separately within a period of up to 11 days.

14. The therapeutic TIL population of any one of claims 1 to 10, wherein the first expansion culture is carried out for a first period of time and the second expansion culture is carried out for a second period of time, the total of the first period and the second period being up to 12 days.

15. The therapeutic TIL population of any one of claims 1 to 10, wherein the first expansion culture is carried out for a first period of time and the second expansion culture is carried out for a second period of time, the total of the first period and the second period being up to 12 days.

16. The therapeutic TIL population of any one of claims 1 to 15, wherein said first TIL population is obtained from a tumor fragment.

17. 16. The therapeutic TIL population of any one of claims 1 to 15, wherein the tumor digest of step (a) is prepared by incubating a sample of tumor excised from the subject in an enzyme medium.

18. The therapeutic TIL population of claim 17, further comprising mechanically disrupting the tumor sample so that the tumor sample dissociates.

19. 20. The therapeutic TIL population of claim 18, further comprising purifying the dissociated tumor sample using density gradient separation.

20. The therapeutic TIL population of claim 17, wherein the enzyme medium comprises DNase or collagenase.

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