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

Abstract

To provide processes for production of tumor infiltrating lymphocytes and uses of the same in immunotherapy.SOLUTION: The present invention provides improved and / or shortened methods for expanding TILs and producing therapeutic populations of TILs, including novel methods for expanding TIL populations in a closed system that lead to improved efficacy, improved phenotype, and increased metabolic health of the TILs in a shorter time period, while allowing for reduced microbial contamination as well as decreased costs. These TILs are found to be useful in therapeutic treatment regimens.SELECTED DRAWING: None

Description

[Technical Field]

[0001] Cross-reference of related applications

[0001] This application claims priority to U.S. Patent Application Publication No. 15 / 940,901, filed on 29 March 2018, which is incorporated herein by reference in whole. [Background technology]

[0002] Background of the Invention

[0002] 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. Large quantities of TILs are required for successful immunotherapy, and robust and reliable methods are needed for commercialization. This has been hampered by technical, logistical, and regulatory issues associated with cell expansion culture. IL-2-based TIL expansion culture and subsequent "rapid expansion culture" (REP) methods are becoming preferred for TIL expansion culture due to their speed and efficiency. 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 TIL over a 14-day period, but this requires a large excess (e.g., 200x) of irradiated allogeneic peripheral blood mononuclear cells (PBMCs, also known as mononuclear cells (MNCs)) from multiple donors as feeder cells, as well as anti-CD3 antibody (OKT3) and high doses of IL-2. Dudley, et al., J. Immunother. 2003, 26, 332-42. [Overview of the project] [Problems that the invention aims to solve]

[0003]

[0003] Current TIL manufacturing processes are limited by length, cost, sterility concerns and other factors described herein, and the potential for commercializing such processes is severely limited. For these and other reasons, there are currently no commercially available processes. There is an urgent need to provide a TIL manufacturing process and a treatment based on such a process that is suitable for commercial-scale manufacturing and regulatory approval for use in human patients at multiple clinical centers. [Means for solving the problem]

[0004]

[0004] The present invention provides an improved and / or shortened method for expanding TIL culture and producing a therapeutic TIL population.

[0005]

[0005] The present invention also provides a method for expanding the culture of tumor-infiltrating lymphocytes (TILs) into a therapeutic TIL population, and this method is (a) Obtaining a first TIL population from tumors excised from patients by processing tumor samples obtained from patients into multiple tumor fragments; (b) Adding tumor fragments to a closed system; (c) Performing a first expansion culture to produce a second TIL population by culturing a first TIL population in a cell culture medium containing IL-2 and optionally OKT-3, wherein the first expansion culture is performed in a closed container providing a first gas-permeable surface area, and the first expansion culture is performed for approximately 3 to 14 days to obtain a second TIL population, the second TIL population being at least 50 times larger in number than the first TIL population, and the transition from step (b) to step (c) occurs without opening the system; (d) A second expansion culture is performed to produce a third TIL population by adding additional IL-2, optionally OKT-3, and antigen-presenting cells (APCs) to the cell culture medium of the second TIL population, the second expansion culture is performed over approximately 7 to 14 days to obtain the third TIL population, the third TIL population being the therapeutic TIL population, the second expansion culture is performed in a closed container 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 opening the system; and (f) Transferring the TIL population recovered from step (e) to an infusion bag, wherein the transition from step (e) to (f) occurs without opening the system. Includes.

[0006]

[0006] In some embodiments, the method further includes the step of freezing the infusion bag containing the TIL population recovered in step (f) using a cryopreservation process.

[0007]

[0007] In some embodiments, the cryopreservation process is carried out using a 1:1 ratio of the recovered TIL population to the cryopreservation medium.

[0008]

[0008] 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 either day 9 to 14. In some embodiments, the antigen-presenting cells are artificial antigen-presenting cells.

[0009]

[0009] In some embodiments, the recovery in step (e) is carried out using a membrane-based cell processing system.

[0010]

[0010] In some embodiments, the recovery in step (e) is carried out using a LOVO cell processing system.

[0011]

[0011] In some embodiments, the multiple fragments consist of about 4 to about 50 fragments, each fragment being about 27 mm 3 It has the volume of .

[0012]

[0012] In some embodiments, the multiple pieces are approximately 1300 mm 3 ~approx. 1500mm 3 It contains approximately 30 to 60 fragments with a total volume.

[0013]

[0013] In some embodiments, the multiple pieces are approximately 1350 mm 3 It contains approximately 50 fragments with a total volume.

[0014]

[0014] In some embodiments, the plurality of fragments comprises about 50 fragments having a total mass of about 1 g to about 1.5 g.

[0015]

[0015] In some embodiments, the cell culture medium is provided in a container selected from the group consisting of G containers and Xuri cell culture bags.

[0016]

[0016] In some embodiments, the cell culture medium of step (d) further comprises IL-15 and / or IL-21.

[0017]

[0017] In some embodiments, the IL-2 concentration is approximately 10,000 IU / mL to approximately 5,000 IU / mL.

[0018]

[0018] In some embodiments, the IL-15 concentration is about 500 IU / mL to about 100 IU / mL.

[0019]

[0019] In some embodiments, the IL-21 concentration is about 20 IU / mL to about 0.5 IU / mL.

[0020] In some embodiments, the infusion bag in step (f) is a HypoThermosol-containing infusion bag.

[0021]

[0021] In some embodiments, the cryopreservation medium contains dimethyl sulfoxide (DMSO). In some embodiments, the cryopreservation medium contains 7% to 10% dimethyl sulfoxide (DMSO).

[0022]

[0022] In some embodiments, the first period in step (c) and the second period in step (e) are each individually carried out within a period of 10 days, 11 days or 12 days.

[0023]

[0023] In some embodiments, the first period in step (c) and the second period in step (e) are each individually carried out within a period of 11 days.

[0024]

[0024] In some embodiments, steps (a) to (f) are carried out within a period of about 10 days to about 22 days.

[0025]

[0025] In some embodiments, steps (a) to (f) are carried out within a period of about 20 days to about 22 days.

[0026] [[ID=2​​​​​​​​​​​​​​​In some embodiments, steps (a)-(f) are performed within 20 days.

[0031] In some embodiments, steps (a)-(f) are performed within 15 days.

[0032] In some embodiments, steps (a)-(f) are performed within 10 days.

[0033] In some embodiments, steps (a)-(f) and cryopreservation are performed within 22 days.

[0034] In some embodiments, the therapeutic TIL population recovered in step (e) contains sufficient TIL to be a therapeutically effective dosage of TIL.

[0035] In some embodiments, the number of TIL sufficient to be a therapeutically effective dosage is from about 2.3×10^10 to about 13.7×10^10.

[0036] In some embodiments, steps (b)-(e) are performed in a single container, and performing steps (b)-(e) in a single container results in an increased TIL yield per resected tumor compared to performing steps (b)-(e) in two or more containers.

[0037] In some embodiments, antigen-presenting cells are added to the TIL during the second period of step (d) without opening the system.

[0038] In some embodiments, the third TIL population of step (d) provides an increase in efficacy, an increase in interferon gamma production, an increase in polyclonality, an increase in mean IP-10, and / or an increase in mean MCP-1 when administered to a subject.

[0039]

[0039] In some embodiments, the third TIL population of step (d) provides at least a 5-fold increase in interferon-gamma production when administered to a subject.

[0040]

[0040] 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, wherein the effector T cells and / or central memory T cells in the therapeutic TIL population exhibit one or more features 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.

[0041]

[0041] In some embodiments, effector T cells and / or central memory T cells obtained from a third TIL population exhibit increased CD57 expression and decreased CD56 expression compared to effector T cells and / or central memory T cells obtained from a second cell population.

[0042]

[0042] In some embodiments, the risk of microbial contamination is reduced compared to open systems.

[0043]

[0043] In some embodiments, the TIL from step (g) is injected into the patient.

[0044]

[0044] In some embodiments, the multiple fragments include about four fragments.

[0045]

[0045] The present invention also provides a method for treating subjects with cancer, which includes administering expanded cultured tumor-infiltrating lymphocytes (TILs), and this method is (a) Obtaining a first TIL population from tumors resected from patients by processing tumor samples obtained from subjects into multiple tumor fragments; (b) Adding tumor fragments to a closed system; (c) Performing a first expansion culture to produce a second TIL population by culturing a first TIL population in a cell culture medium containing OKT-3 with optional selection of IL-2, wherein the first expansion culture is performed in a closed container providing a first gas-permeable surface area, and the first expansion culture is performed for approximately 3 to 14 days to obtain a second TIL population, the second TIL population being at least 50 times larger in number than the first TIL population, and the transition from step (b) to step (c) occurs without opening the system; (d) A second expansion culture is performed to produce a third TIL population by adding additional IL-2, optionally OKT-3, and antigen-presenting cells (APCs) to the cell culture medium of the second TIL population, the second expansion culture is performed over approximately 7 to 14 days to obtain the third TIL population, the third TIL population being the therapeutic TIL population, the second expansion culture is performed in a closed container 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 opening the system; and (f) Transferring the TIL population recovered from step (e) to an infusion bag, wherein the transition from step (e) to (f) occurs without opening the system; (g) optionally cryopreserve the infusion bag containing the TIL population recovered from step (f) using a cryopreservation process; and (h) Administer a third TIL population of a therapeutically effective dose to the patient from the infusion bag in step (g). Includes.

[0046]

[0046] In some embodiments, the therapeutic TIL population recovered in step (e) contains enough TILs to administer a therapeutically effective dose of TILs in step (h).

[0047]

[0047] 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 cells.

[0048]

[0048] In some embodiments, the antigen presenting cell (APC) is PBMC.

[0049]

[0049] In some embodiments, the PBMC is added to the cell culture on any of the 9th to 14th days in step (d).

[0050]

[0050] In some embodiments, a non-myeloablative lymphodepletion regimen has been administered to the patient before administration of a therapeutically effective dosage of TIL cells in step (h).

[0051]

[0051] In some embodiments, the non-myeloablative lymphodepletion regimen includes an administration step of cyclophosphamide at a dosage of 60 mg / m2 / day for 2 days, followed by an administration step of fludarabine at a dosage of 25 mg / m2 / day for 5 days.

[0052]

[0052] In some embodiments, the method further includes treating the patient with a high-dose IL-2 regimen that begins on the day following administration of TIL cells to the patient in step (h).

[0053]

[0053] In some embodiments, the high-dose IL-2 regimen includes 600,000 or 720,000 IU / kg administered as a 15-minute bolus intravenous infusion every 8 hours up to the tolerated volume.

[0054]

[0054] 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, wherein the effector T cells and / or central memory T cells in the therapeutic TIL population exhibit one or more features 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.

[0055]

[0055] In some embodiments, effector T cells and / or central memory T cells from a therapeutic TIL population exhibit increased CD57 expression and decreased CD56 expression compared to effector T cells and / or central memory T cells obtained from a second cell population.

[0056]

[0056] In some embodiments, 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)), kidney cancer and renal cell carcinoma.

[0057]

[0057] In some embodiments, the cancer is selected from the group consisting of melanoma, HNSCC, cervical cancer and NSCLC.

[0058]

[0058] In some embodiments, the cancer is melanoma.

[0059]

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

[0060]

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

[0061]

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

[0062]

[0062] The present invention also provides a method for expanding the culture of tumor-infiltrating lymphocytes (TILs) into a therapeutic TIL population, and this method is (a) Add processed tumor fragments from tumors resected from patients to a closed system to obtain a first TIL population; (b) Performing a first expansion culture to produce a second TIL population by culturing a first TIL population in a cell culture medium containing IL-2 and optionally OKT-3, wherein the first expansion culture is performed in a closed container providing a first gas-permeable surface area, and the first expansion culture is performed for approximately 3 to 14 days to obtain a second TIL population, the second TIL population being at least 50 times larger in number than the first TIL population, and the transition from step (a) to step (b) occurs without opening the system; (c) Performing a second expansion culture of the second TIL population by adding additional IL-2, optionally OKT-3, and antigen-presenting cells (APCs) to the cell culture medium of the second TIL population to produce a third TIL population, the second expansion culture being performed over approximately 7–14 days to obtain the third TIL population, the third TIL population being the therapeutic TIL population, the second expansion culture being performed in a closed container providing a second gas-permeable surface area, and the transition from step (b) to step (c) occurring 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 opening the system; and (e) Transferring the TIL population recovered from step (d) to an infusion bag, wherein the transition from step (d) to (e) occurs without opening the system. Includes.

[0063]

[0063] In some embodiments, the therapeutic TIL population collected in step (d) contains enough TILs to constitute a therapeutically effective dose of TILs.

[0064]

[0064] In some embodiments, the number of TILs sufficient to constitute a therapeutically effective dosage is approximately 2.3 × 10¹⁰ to approximately 13.7 × 10¹⁰.

[0065]

[0065] In some embodiments, the method further includes the step of freezing and preserving the infusion bag containing the recovered TIL population using a cryopreservation process.

[0066]

[0066] In some embodiments, the cryopreservation process is carried out using a 1:1 ratio of the recovered TIL population to the cryopreservation medium.

[0067]

[0067] In some embodiments, the antigen-presenting cells are peripheral blood mononuclear cells (PBMCs).

[0068]

[0068] In some embodiments, the PBMCs are irradiated and are homogeneous.

[0069]

[0069] The method of claim 68, wherein the PBMC is added to the cell culture on any of days 9 to 14 in step (c).

[0070]

[0070] In some embodiments, the antigen-presenting cells are artificial antigen-presenting cells.

[0071]

[0071] In some embodiments, the recovery in step (d) is carried out using a LOVO cell processing system.

[0072]

[0072] In some embodiments, the multiple fragments consist of about 4 to about 50 fragments, each fragment being about 27 mm 3 It has the volume of .

[0073]

[0073] In some embodiments, the multiple pieces are approximately 1300 mm 3 ~approx. 1500mm 3 It contains approximately 30 to 60 fragments with a total volume.

[0074]

[0074] In some embodiments, the multiple pieces are approximately 1350 mm 3 It contains approximately 50 fragments with a total volume.

[0075]

[0075] In some embodiments, the plurality of fragments comprises about 50 fragments having a total mass of about 1 g to about 1.5 g.

[0076]

[0076] In some embodiments, the multiple fragments include about four fragments.

[0077]

[0077] In some embodiments, the second cell culture medium is provided in a container selected from the group consisting of G containers and Xuri cell culture bags.

[0078]

[0078] In some embodiments, the infusion bag of step (e) is a HypoThermosol-containing infusion bag.

[0079]

[0079] In some embodiments, the first period of step (b) and the second period of step (c) are carried out individually within periods of 10, 11, or 12 days, respectively.

[0080]

[0080] In some embodiments, the first period of step (b) and the second period of step (c) are carried out separately within a period of 11 days.

[0081]

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

[0082]

[0082] In some embodiments, steps (a) to (e) are carried out within a period of approximately 10 to 20 days.

[0083]

[0083] In some embodiments, steps (a) to (e) are carried out within a period of approximately 10 to 15 days.

[0084]

[0084] In some embodiments, steps (a) to (e) are carried out within 22 days.

[0085]

[0085] In some embodiments, steps (a) to (e) and cryopreservation are carried out within 22 days.

[0086]

[0086] 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 excised tumor compared to performing steps (b) to (e) in two or more containers.

[0087]

[0087] In some embodiments, antigen-presenting cells are added to the TIL during the second period of step (c) without opening the system.

[0088]

[0088] 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, wherein the effector T cells and / or central memory T cells obtained in the therapeutic TIL population exhibit one or more features 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.

[0089]

[0089] In some embodiments, effector T cells and / or central memory T cells obtained from a therapeutic TIL population exhibit increased CD57 expression and decreased CD56 expression compared to effector T cells and / or central memory T cells obtained from a second cell population.

[0090]

[0090] In some embodiments, the risk of microbial contamination is reduced compared to open systems.

[0091]

[0091] In some embodiments, the TIL from step (e) is injected into the patient.

[0092]

[0092] In some embodiments, the closed container includes a single bioreactor.

[0093]

[0093] In some embodiments, the closed container includes G-REX-10.

[0094]

[0094] In some embodiments, the closed container includes G-REX-100.

[0095]

[0095] 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.

[0096]

[0096] In some embodiments, the cell culture has a ratio of 2.5×10 9 APCs to 100×10 6 TILs.

[0097]

[0097] 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.

[0098]

[0098] In some embodiments, the cell culture has a ratio of 2.5×10 9 APCs to 100×10 6 TILs.

[0099]

[0099] The present invention also provides a population of expanded culture TILs for use in treating a subject having cancer, the population of expanded culture TILs comprising: (a) obtaining a first TIL population from a tumor excised from a patient by processing a tumor sample obtained from the subject into a plurality of tumor fragments; (b) adding the tumor fragments to a closed system; (c) Performing a first expansion culture to produce a second TIL population by culturing a first TIL population in a cell culture medium containing IL-2, wherein the first expansion culture is performed in a closed container providing a first gas-permeable surface area, and the first expansion culture is performed for approximately 3 to 14 days to obtain a second TIL population, the second TIL population being at least 50 times larger in number than the first TIL population, and the transition from step (b) to step (c) occurs without opening the system; (d) A second expansion culture is performed to produce a third TIL population by adding additional IL-2, optionally OKT-3, and antigen-presenting cells (APCs) to the cell culture medium of the second TIL population, the second expansion culture is performed over approximately 7 to 14 days to obtain the third TIL population, the third TIL population being the therapeutic TIL population, the second expansion culture is performed in a closed container 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 opening the system; and (f) Transferring the TIL mass recovered from step (e) to an infusion bag, wherein the transfer from step (e) to (f) occurs without opening the system; and (g) Optionally, freeze-preserve the infusion bags containing the TIL population recovered from step (f) using a cryopreservation process. This is a third TIL population that can be obtained by a method that includes [a specific method].

[0100]

[0100] In some embodiments, a population of TILs is used to treat a subject having cancer in accordance with the methods described above and herein, the method further comprising one or more of the features listed above and herein. In some embodiments, a population of TILs is for use in the treatment of a subject having cancer in accordance with the methods described above and herein, the method further comprising one or more of the features listed above and herein.

[0101]

[0101] The present invention also provides a method for expanding the culture of tumor-infiltrating lymphocytes (TILs) into a therapeutic TIL population, and this method is (a) Obtaining a first TIL population from tumors excised from patients by processing tumor samples obtained from patients into multiple tumor fragments; (b) Adding tumor fragments to a closed system; (c) A first expansion culture is performed to produce a second TIL population by culturing a first TIL population in a cell culture medium containing IL-2, optionally OKT-3, and optionally a tumor necrosis factor receptor superfamily (TNFRSF) agonist, wherein the first expansion culture is performed in a closed container providing a first gas-permeable surface area, the first expansion culture is performed for approximately 3 to 14 days to obtain a second TIL population, the second TIL population being at least 50 times larger in number than the first TIL population, and the transition from step (b) to step (c) occurs without opening the system; (d) A second expansion culture is performed to produce a third TIL population by adding additional IL-2, optionally OKT-3, optionally tumor necrosis factor superfamily (TNFRSF) agonists, and antigen-presenting cells (APCs) to the cell culture medium of the second TIL population, the second expansion culture is performed for approximately 7 to 14 days to obtain the third TIL population, the third TIL population being the therapeutic TIL population, the second expansion culture is performed in a closed container 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 opening the system; and (f) Transferring the TIL population recovered from step (e) to an infusion bag, wherein the transition from step (e) to (f) occurs without opening the system. Includes.

[0102]

[0102] The present invention also provides a method for treating a subject with cancer, comprising administering expanded cultured tumor-infiltrating lymphocytes (TILs), the method being (a) Obtaining a first TIL population from tumors excised from subjects by processing tumor samples obtained from patients into multiple tumor fragments; (b) Adding tumor fragments to a closed system; (c) A first expansion culture is performed to produce a second TIL population by culturing a first TIL population in a cell culture medium containing IL-2, optionally OKT-3, and optionally a tumor necrosis factor receptor superfamily (TNFRSF) agonist, wherein the first expansion culture is performed in a closed container providing a first gas-permeable surface area, the first expansion culture is performed for approximately 3 to 14 days to obtain a second TIL population, the second TIL population being at least 50 times larger in number than the first TIL population, and the transition from step (b) to step (c) occurs without opening the system; (d) A second expansion culture is performed to produce a third TIL population by adding additional IL-2, optionally OKT-3, optionally tumor necrosis factor superfamily (TNFRSF) agonists, and antigen-presenting cells (APCs) to the cell culture medium of the second TIL population, the second expansion culture is performed for approximately 7 to 14 days to obtain the third TIL population, the third TIL population being the therapeutic TIL population, the second expansion culture is performed in a closed container 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 opening the system; and (f) Transferring the TIL population recovered from step (e) to an infusion bag, wherein the transition from step (e) to (f) occurs without opening the system; (g) optionally, use a cryopreservation process to cryopreserve the infusion bag containing the TIL population recovered from step (f); and (h) Administer a third TIL population of a therapeutically effective dose to the patient from the infusion bag in step (g). Includes.

[0103]

[0103] In some embodiments, the tumor necrosis factor receptor superfamily (TNFRSF) agonist is a 4-1BB antibody. In some embodiments, the TNFRSF agonist is a 4-1BB agonist, which is selected from the group consisting of urelumab, utomirumab, EU-101, fusion proteins and their fragments, derivatives, variants, biosimilars and combinations.

[0104]

[0104] The present invention also provides a cryopreservation composition comprising the TIL group described above and herein, a cryoprotective medium containing dimethyl sulfoxide (DMSO), and an electrolyte solution.

[0105]

[0105] In some embodiments, the cryopreservation composition further comprises one or more stabilizers and one or more lymphocyte growth factors. In some embodiments, the one or more stabilizers comprises human serum albumin (HSA), and the one or more lymphocyte growth factors comprises IL-2. In some embodiments, the composition optionally comprises OKT-3.

[0106]

[0106] In some embodiments, DMSO and electrolyte solution are present in a ratio of about 1.1:1 to about 1:1.1. In some embodiments, DMSO and electrolyte solution are present in a ratio of about 1:1.

[0107]

[0107] In some embodiments, 100 mL of the cryopreservation composition is approximately 1 × 10 6 ~Approx. 9×10 14 Each dose contains a TIL population of one unit, approximately 30 mL to 70 mL of cryoprotective medium containing DMSO, approximately 30 mL to 70 mL of electrolyte solution, approximately 0.1 g to 1.0 g of HSA, and approximately 0.001 mg to 0.005 mg of IL-2.

[0108]

[0108] In some embodiments, 100 mL of the cryopreservation composition is approximately 1 × 10 7 ~Approx. 1×10 11 The solution contains a TIL population of one unit; approximately 30 mL to 70 mL of cryoprotective medium containing approximately 10% DMSO; approximately 30 mL to 70 mL of electrolyte solution; approximately 0.3 g to 0.7 g of HSA; and approximately 0.001 mg to 0.003 mg of IL-2.

[0109]

[0109] In some embodiments, 100 mL of the cryopreservation composition is approximately 1 × 10 7 ~Approx. 1×10 11 A dose consisting of a TIL population of approximately 10%; approximately 30 mL to 70 mL of cryoprotective medium, essentially composed of approximately 10% DMSO; approximately 30 mL to 70 mL of electrolyte solution; approximately 0.3 g to 0.7 g of HSA; and approximately 0.001 mg to 0.003 mg of IL-2.

[0110]

[0110] In some embodiments, the composition is intended for use in treating subjects having cancer.

[0111]

[0111] The present invention also provides a cryopreservation composition comprising a TIL group, a cryoprotective medium containing dimethyl sulfoxide (DMSO), and an electrolyte solution, wherein 100 mL of the composition contains about 1 × 10⁶ 7 ~Approx. 1×10 11The solution contains a TIL population of one unit; approximately 30 mL to 70 mL of cryoprotective medium containing approximately 10% DMSO; approximately 30 mL to 70 mL of electrolyte solution; approximately 0.3 g to 0.7 g of HSA; and approximately 0.001 mg to 0.003 mg of IL-2.

[0112]

[0112] The present invention also provides a cryopreservation composition comprising a TIL population, a cryoprotective medium containing dimethyl sulfoxide (DMSO), and an electrolyte solution, wherein 100 mL of the composition is approximately 1 × 10⁶ 7 ~Approx. 1×10 11 A dose consisting of a TIL population of approximately 10%; approximately 30 mL to 70 mL of cryoprotective medium, essentially composed of approximately 10% DMSO; approximately 30 mL to 70 mL of electrolyte solution; approximately 0.3 g to 0.7 g of HSA; and approximately 0.001 mg to 0.003 mg of IL-2.

[0113]

[0113] The present invention also provides an infusion bag containing a cryopreservation composition as described above and herein.

[0114]

[0114] In some embodiments, the infusion bag is a HypoThermosol-containing infusion bag.

[0115]

[0115] The present invention also provides a storage bag containing a cryopreservation composition as described above and herein.

[0116]

[0116] In some embodiments, the storage bag is the storage bag according to claim 54, which is a CryoStore CS750 freezer bag. [Brief explanation of the drawing]

[0117] Brief explanation of the drawing [Figure 1]

[0117] A schematic diagram of an embodiment of Process 2A, a 22-day process for TIL manufacturing is shown. [Figure 2]

[0118] This shows a comparison of the 1C process and 2A process embodiments for TIL manufacturing. [Figure 3]

[0119] This shows the timeline for the 1C process. [Figure 4]

[0120] For higher cell counts, the process of an embodiment of TIL therapy using process 2A for TIL production, including administration and co-therapy steps, is shown. [Figure 5]

[0121] For lower cell counts, the process of an embodiment of TIL therapy using process 2A for TIL production, including administration and co-therapy steps, is shown. [Figure 6]

[0122] A detailed schematic diagram of an embodiment of the 2A process is shown. [Figure 7]

[0123] 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 TILs and thawed TILs. [Figure 8]

[0124] The properties of TIL prepared using an embodiment of the 2A process are shown by examining the CD3 of fresh TIL and thawed TIL. [Figure 9]

[0125] By examining the recovery between fresh TIL and thawed TIL, the characteristics of TIL prepared using an embodiment of the 2A process are demonstrated. [Figure 10]

[0126] The characteristics of TILs prepared using an embodiment of the 2A process are demonstrated by examining the viability of fresh and thawed TILs. [Figure 11A]

[0127] The main steps of an embodiment of the 2A process, which includes a cryopreservation step, are shown below. [Figure 11B]

[0127] The main steps of an embodiment of process 2A, which includes a cryopreservation step, are shown. [Figure 11C]

[0127] The main steps of an embodiment of process 2A, which includes a cryopreservation step, are shown. [Figure 12]

[0128] The number of cells obtained from the 1C process and the 2A process embodiments is shown. [Figure 13]

[0129] The cell viability obtained from embodiments of process 1C and process 2A is shown. [Figure 14]

[0130] The percentages of CD45 and CD3 cells (i.e., T cells) obtained by flow cytometry of TILs obtained in embodiments of the 1C and 2A processes are shown. [Figure 15]

[0131] Figures 80 and 98 show the IFN-γ release obtained in the 1C and 2A process embodiments, measured by assays different from those used to generate the data. [Figure 16]

[0132] Figures 80 and 98 show the IFN-γ release obtained in the 1C and 2A process embodiments, measured by assays different from those used to generate the data. [Figure 17]

[0133] The TCR / b and NK cell ratios obtained from embodiments of the 1C and 2A processes are shown. [Figure 18]

[0134] The percentages of CD8+ and CD4+ cells, as well as the ratios between each subset, are shown as measured by flow cytometry of TILs obtained by embodiments of the 1C and 2A processes. [Figure 19]

[0135] This shows the percentage of memory subsets measured by flow cytometry of TIL obtained from embodiments of the 1C process and 2A process. [Figure 20]

[0136] The flow cytometry-based expression rates of PD-1, LAG-3, and TIM-3 in TILs obtained from the 1C and 2A process embodiments are shown. [Figure 21]

[0137] The flow cytometry results showing the expression rates of 4-1BB, CD69, and KLRG1 in TILs obtained from embodiments of the 1C and 2A processes are shown. [Figure 22]

[0138] The percentage of TIGIT expression obtained by flow cytometry from TIL obtained from embodiments of process 1C and process 2A is shown. [Figure 23]

[0139] The flow cytometry-based CD27 and CD28 expression rates of TILs obtained from the 1C and 2A process embodiments are shown. [Figure 24]

[0140] The results of flow FISH telomere length analysis are shown. [Figure 25]

[0141] The results of the flow FISH telomere length analysis (after removing outlier data points) are shown. [Figure 26]

[0142] The clinical trial design, including cohorts treated in embodiments of Process 1C and Process 2A, is shown. [Figure 27]

[0143] An example 2A process chart providing an overview of steps A through F. [Figure 28A]

[0144] Process flowchart for Process 2A. [Figure 28B]

[0144] Process flowchart for Process 2A. [Figure 28C]

[0144] Process flowchart for Process 2A. [Figure 29]

[0145] Process flowchart for Process 2A: Data acquisition plan [Figure 30]

[0146] Survival rate of fresh TIL and thawed TIL [Figure 31]

[0147] Expanded culture of fresh and thawed TILs in re-REP culture [Figure 32]

[0148] Routine test values ​​for blood metabolites. [Figure 33A]

[0149] Metabolite analysis of pre-REP TIL in process 2A. [Figure 33B]

[0149] Metabolite analysis of pre-REP TIL in process 2A. [Figure 34]

[0150] Quantification of IL-2 in pre-REP TIL cell cultures of process 2A. [Figure 35]

[0151] Release of cytotoxic cytokine IFN-γ upon stimulation of TIL with anti-CD3, anti-CD28, and anti-4-1BB. [Figure 36]

[0152] Granzyme B release following anti-CD3, anti-CD28, and anti-4-1BB stimulation of TIL. [Figure 37A]

[0153] TCR αβ+ TILs. Most human CD3+ T cells express receptors formed by α and β chains that recognize antigens in a MHC-suppressed manner. With the exception of M1061, more than 80% of fresh and thawed TIL products contained 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). [Figure 37B]

[0153] TCR αβ+ TIL. Most human CD3+ T cells express receptors formed by α and β chains that recognize antigens in a MHC-suppressed manner. Fresh RE-REP and thawed RE-REP TILs expressing TCR αβ showed a 9.2% and 15.7% decrease compared to fresh TILs and thawed TILs, respectively. [Figure 38A]

[0154] TCR αβ-CD56+. Tumor-infiltrating natural killer (NK) and NKT cells also have the ability to lyse cells lacking MHC expression and CD1-presenting lipid antigens, and to provide immunomodulatory cytokines. However, strong NK cell infiltration is associated with progressive disease and may promote cancer development. In all cases except M1063, there was a slight, though not statistically significant, decrease in the NK population of thawed TILs compared to fresh TILs (p=0.27). No significant differences were observed between re-REP TIL populations (p=0.88). [Figure 38B]

[0154] TCR αβ-CD56+. Tumor-infiltrating natural killer (NK) and NKT cells also possess the ability to lyse cells lacking MHC expression as well as CD1-presenting lipid antigens and provide immunomodulatory cytokines. However, strong NK cell infiltration is associated with progressive disease and may promote cancer development. Fresh TILs, fresh re-REP TILs, and thawed re-REP TILs demonstrate similar CD56 expression, as shown. Thawed TIL products had fewer NK-expressing cells than fresh TILs (3.0±2.2) (1.9±1.3), likely as a result of the cryopreservation procedure. [Figure 39A]

[0155] CD4+ cells. No substantial differences were observed in the CD4 population under individual conditions. The average CD4 population is shown for each condition. [Figure 39B]

[0155] CD4+ cells. No substantial differences were observed in the CD4 population under individual conditions. The table shows SD and SEM values. There was a slight decrease in the CD4 population in the fresh re-REP population, which is mainly due to the decrease in CD4 in the fresh re-REP population of EP11001T. [Figure 40A]

[0156] CD8+ cells. In all cases except EP11001T, both fresh and thawed TILs showed comparable CD8+ populations (P=0.10, no significant difference). In most experiments, the number of CD8+ expressing TILs was slightly reduced in fresh re-REP TIL products (with the exception of M1061T and M1065T). The number of CD8+ individuals in 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). [Figure 40B]

[0156] CD8+ cells. The mean values ​​of CD8+ cells expressing TIL under all conditions are shown. Both fresh and thawed TILs showed similar results. However, the thawed re-REP TIL product resulted in a 10.8% reduction in the CD8+ population compared to fresh re-REP TIL. [Figure 41A]

[0157] CD4+CD154+ cells. CD154, also known as CD40L, is a marker for activated T cells. No substantial differences were observed in the CD4+CD154+ population under different conditions, but a 34.1% decrease was observed in fresh re-REP CD4+ TILs of EP11001T. CD154 expression was not measured in M1061T and M1062T. This is because these experiments were performed before the expanded phenotypic panel was established. [Figure 41B]

[0157] CD4+CD154+ cells. CD154, also known as CD40L, is a marker of activated T cells. The slight decrease in thawed TIL status may be due to the absence of CD154 in M1061T and M1062T. All conditions show very similar CD154 expression in the CD4 population, suggesting activated CD4+ T cells. [Figure 42A]

[0158] CD8+CD154+ cells. The activation marker CD154 expressed in CD8+ TILs was also analyzed. A) Overall, CD154 expression was low in the CD8+ population of fresh and thawed TIL products. This is expected, as CD154 is mainly expressed in activated CD4+ T cells. When CD154 expression was measured in both fresh and thawed TIL products, no difference in CD154 expression was observed in thawed TIL products, or an increase was observed. Student's t-test showed no significant difference between the two conditions. Increased CD154 expression in thawed re-REP compared to fresh re-REP was shown in all experiments (p=0.02). [Figure 42B]

[0158] CD8+CD154+ cells. The activation marker CD154 expressed in CD8+ TILs was also analyzed. B) Increased CD154 expression was observed in both thawed TILs 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 43A]

[0159] CD4+CD69+ cells. CD69 is an early activation marker for T cells after stimulation or activation. A) In all TILs except EP11001T, both fresh re-REP and thawed re-REP showed a slight increase in CD69 expression, likely due to 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-REP either (p=0.82). [Figure 43B]

[0159] CD4+CD69+ cells. CD69 is an early activation marker for T cells after stimulation or activation. B) A slight increase in CD69 expression is observed in re-REP TIL products. (Note: CD69 staining was not performed on either M1061T or M1062T thawed TIL products. CD69 expression in fresh M1061T TIL products was 33.9%). [Figure 44A]

[0160] CD8+CD69+ cells. As observed in the CD4+ population, Figure A shows increased CD69 expression in CD8+ re-REP TILs. CD69 expression did not show a significant difference between fresh TILs and thawed TILs (p=0.68) or fresh re-REP TILs and thawed re-REP TILs (p=0.76). [Figure 44B]

[0160] CD8+CD69+ cells. Figure B supports the observation that there is a slight increase in CD69 expression of the re-REP TIL product. [Figure 45A]

[0161] CD4+CD137+ cells. CD137(4-11313) is a T cell costimulatory receptor induced by TCR activation. It is activated in CD4+ T cells 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 TILs and thawed TILs or between fresh re-REP TILs and thawed re-REP TILs (p<0.05 in both cases of Figure B supports this observation). Furthermore, thawed TILs showed a slight decrease in CD137 expression. The increase in CD137 expression in re-REP TILs is thought to be due to the second 7-day re-REP stimulation. [Figure 45B]

[0161] CD4+CD137+ cells. CD137 (4-11313) is a T cell costimulatory receptor induced by TCR activation. It is activated in CD4+ T cells and CD8+ T cells. [Figure 46A]

[0162] CD8+CD137+ cells. A) The CD8+ population showed an overall increase in re-REP products. [Figure 46B]

[0162] CD8+CD137+ cells. B) Fresh re-REP products showed a 33.4% increase in CD8+CD137+ expression compared to fresh TIL products. Thawed re-REP products also showed a 33.15% increase in CD137 expression in the CD8+ population compared to thawed TILs. No significant differences were observed between fresh re-REP TILs and thawed re-REP TILs. Similar observations were made when comparing fresh TILs to thawed TIL products. This increase in CD137 expression may be due to the second activation of re-REP. (Note that only 6 TILs were used for analysis because CD137 expression was not measured in three experiments.) [Figure 47A]

[0163] CD4+ CM cells. The central memory (CM) population is defined by the expression of CD45RA- (negative) and CCR7+ (positive). A) An increase in the CM population was observed under re-REP conditions. M1063T and M1064T showed decreased CM expression in the CD4+ population obtained from thawed TILs compared with fresh TIL products. Neither fresh TIL products nor thawed TIL products (p=0.1658), nor fresh re-REP TILs nor thawed re-REP TILs (p=0.5535) showed a significant difference in the CM population. [Figure 47B]

[0163] CD4+ CM cells. The central memory (CM) population is defined by the expression of CD45RA- (negative) and CCR7+ (positive). B) Increases of 14.4% and 15.4% in the CM population were observed in fresh re-REP TILs and thawed re-REP TILs compared to fresh TILs and thawed TILs, respectively. [Figure 48A]

[0164] CD8+ CM cells. A) In the CD8+ population, a dramatic increase in CM expression was observed with fresh TIL products. This was not seen with TIL products. This increase did not affect significance (p=0.3086), suggesting no difference between fresh TIL and thawed TIL. A similar trend was observed with re-REP TIL products. [Figure 48B]

[0164] CD8+ CM cells. Figure 48B) Compared with thawed TILs, an overall increase in the CM population was observed in fresh TILs. The numerical values ​​indicate that the difference between fresh TILs and fresh re-REP TILs was only about 2%. Fresh TILs showed a very high standard deviation attributable to M1064T. When CM expression of M1064T was excluded, very similar CM expression was observed between fresh TIL products and thawed TIL products (not shown). [Figure 49A]

[0165] CD4+ EM cells. The effector memory (EM) population is defined by the absence of CCR7 and CD45RA expression. A) As expected, the CD4+ populations of fresh and thawed TILs had high levels of the effector memory phenotype. A dramatic decrease in effector memory expression was found in the M1056T re-REP TIL population. In addition, five other experiments showed a decrease in the effector memory phenotype in both fresh and thawed re-REP TILs. [Figure 49B]

[0165] CD4+ EM cells. The effector memory (EM) population is defined by the absence of CCR7 and CD45RA expression. B) Both fresh and thawed TILs showed similar effector memory phenotype expression. Comparing fresh Re-REP TILs to fresh Re-REP TILs, the latter showed a 16% reduction. Compared to thawed TILs, a similar reduction was observed in thawed Re-REP TILs (9%). [Figure 50A]

[0166] CD8+ EM cells. A) A similar pattern of increased effector memory in fresh TILs was also observed in the CD8+ population. M1064T was an exception, with only 20% of its fresh TILs exhibiting an effector memory profile. This is attributed to 73% of these TILs having a CM phenotype, as described in A and B. All samples showing a decrease in the effector memory population of CD4+ TILs from re-REP products showed the same trend as CD8+ TILs. [Figure 50B]

[0166] CD8+ EM cells. B) Unlike the CD4+ TIL population, CD8+ TILs showed a similar effector memory phenotype in fresh TILs, thawed TILs, and re-REP products. (Note the high standard deviations for fresh TILs and thawed TILs. This is due to the small effector memory population of fresh TILs from M1064T and the absence of expression in thawed TIL samples from M1061T.) [Figure 51A]

[0167] CD4+CD28+ cells. CD28 expression correlates with a decrease in age in juvenile TILs. A) Despite an increase in the CM population observed in re-REP TILs, the decrease in CD28 expression was observed as a trend suggesting that CM status alone cannot determine the dynamics of TILs. Except for M1061T CD4+ TILs, a decrease in CD28 expression was observed in re-REP products. [Figure 51B]

[0167] CD4+CD28+ cells. CD28 expression correlates with the decrease in juvenile TILs with age. B) Compared to fresh TIL products and thawed TIL products, fresh TILs showed an 8.89% decrease and thawed TILs showed a 5.71% decrease, respectively. [Figure 52A]

[0168] CD8+CD28+ cells. A) CD28 expression was higher in fresh and thawed TILs than in re-REP products in the CD8+ TIL population. In most cases, thawed re-REP TILs showed a dramatic decrease compared to thawed and fresh re-REP TILs. However, Student's t-test did not show significant differences between fresh and thawed TILs (p=0.3668) or between fresh and thawed re-REP products (p=0.7940). [Figure 52B]

[0168] CD8+CD28+ cells. B) As seen in the CD4+ TIL population, the CD8+CD28+ population was reduced in fresh re-REP (21.5%) and thawed re-REP (18.2%) compared to unstimulated cells. [Figure 53A]

[0169] CD4+PD-1+ cells. PD-1 expression in TILs correlates with antigen reactivity and wasted T cells. Therefore, it is not surprising that a wasting phenotype is observed in TILs that have received 11 days of re-REP. A) This wasting phenotype was maintained or increased in thawed TIL products (specifically, EP11001T and M1056T). No significant difference was observed between fresh and thawed TIL products (p=0.9809). A similar trend was shown in fresh re-REP TILs compared to thawed re-REP TILs (p=0.0912). [Figure 53B]

[0169] CD4+PD-1+ cells. PD-1 expression in TILs correlates with antigen reactivity and wasted T cells. That is, it is not surprising that wasted phenotypes are observed in TILs that have received REP for 11 days. B) Fresh re-REP showed a slight decrease in PD-1 expression in the CD4+ TIL population. All other conditions maintained a comparable PD-1 expression pattern. Decreased or altered PD-1 expression was observed in fresh re-REP products compared to all other conditions. Increased PD-1 expression was seen in thawed re-REP products M1062T, M1063T (CD4+) and EP11001T (CD8+). All other thawed re-REP products showed results comparable to thawed re-REP products. [Figure 54A]

[0170] CD8+PD-1+ cells. A) The CD8+ population from fresh TIL products showed a more depletion phenotype associated with increased PD-1 expression. An exception was observed in EP11001T, where thawed CD8+ TIL products showed a slight increase in PD-1 expression compared to fresh TIL products. There was a slight difference in PD-1 expression between fresh TILs and thawed TILs, but it was not statistically significant (p=0.3144). [Figure 54B]

[0170] CD8+PD-1+ cells. B) Fresh TIL products showed a slight increase in PD-1 expression (6.74% or more than 1.2 times that of thawed TILs), which was not significant compared to thawed TILs, suggesting that thawed TIL products were equivalent based on phenotypic patterns. [Figure 55A]

[0171] CD4+ LAG3+ cells. Wasted T cells express high levels of the inhibitory receptor LAG3 along with PD-1. A) CD4+ thawed TILs showed slightly higher LAG3 expression levels compared to fresh TILs, but not statistically significant (p=0.52). An exception was observed in M1063T. In experiments where LAG3 expression was measured in CD4+ fresh re-REP TILs and fresh re-REP TILs, a decrease in LAG3+ expression was observed in fresh re-REP samples compared to fresh TILs. [Figure 55B]

[0171] CD4+LAG3+ cells. Exhausted T cells express high levels of the inhibitory receptor LAG3 along with PD-1. B) Overall, LAG3 expression is slightly reduced in fresh re-REP TIL products. Note that M1061T, M1062T, and M1064T have been excluded because LAG3 expression was not measured in the fresh product, in order to maintain consistency in Figure B. [Figure 56A]

[0172] CD8+LAG3+ cells. A) CD8+ LAG3 expressed in TILs showed a slight decrease in this experiment, except for M1063T, which showed a significant decrease in LAG3 expression 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 TIL products and thawed TIL products (p=0.0884). [Figure 56B]

[0172] CD8+LAG3+ cells. B) Compared to thawed TIL products, a decrease of approximately 30% in LAG3 expression was observed in CD8+ TILs from fresh re-REP. Fresh TILs and thawed TILs showed a slight increase, comparable to that of thawed TILs. (In this figure, M1061T, M1062T, and M1064T have been excluded because LAG3 expression was not measured in either the fresh TIL or fresh re-REP TIL samples.) [Figure 57A]

[0173] CD4+TIM-3+ cells. A) As previously observed in 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 re-REP TILs and thawed re-REP TILs (p=0.2007). [Figure 57B]

[0173] CD4+TIM-3+ cells. B) No significant changes in TIM-3 expression were observed in thawed TILs or thawed reREP 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 58A]

[0174] CD8+ TIM-3+ cells. A) A similar trend in TIM-3 expression observed in the CD4+ population was also seen in CD8+ TILs. The fresh re-REP TIL phenotype was the least frequent, with low TIM-3 expression, and there was 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 comprehensive and that the CM phenotype is increased. [Figure 58B]

[0174] CD8+ TIM-3+ cells. B) Compared to thawed re-REP TIL products, fresh re-REP TILs showed a significant 22% decrease in TIM-3 expression. Both fresh and thawed TILs exhibited similar TIM-3 expression patterns. [Figure 59]

[0175] Cytotoxic activity of TILs against P815 target cell lines. [Figure 60]

[0176] Metabolic and respiratory profiles of fresh TIL, fresh re-REP TIL, and thawed re-REP TIL. Basal OCR (A), overt SRC (B), SRC2DG (C), latent SRC (D), basal ECAR (E), and glycolytic reserve (F). [Figure 61A]

[0177] Using flowFISH technology, the average telomere repeat length of nine TIL products thawed in post-REP process 2A was measured. A) Data represent telomere lengths measured by qPCR, comparing TILs to 1301 cells. [Figure 61B]

[0177] Using flow FISH technology, the average length of telomere repeats of nine TIL products thawed in post-REP process 2A was measured. B) The data show the telomere lengths measured in comparison to 1301 cells in the flow FISH assay of TILs. The data used in the graphs are provided in tabular form (Table 25) in Appendix Section 10. Overall, there was a broad similarity in the patterns of results from the two telomere length assays, but experiments continue to determine how to more accurately reflect the actual telomere length of TILs. This technique can be applied to future clinical samples to determine the relationship between telomere length and patient response to TIL therapy. [Figure 62A]

[0178] Selection of serum-free medium providers (serum substitutes). Each fragment was cultured four times in a single well of a G-Rex 24-well plate. On day 11, REP was initiated using 45 TIL and 106 feeders, mimicking the 2A process. A) Bar graphs showing the average viable cell count recorded on day 11 (pre-REP) for each condition. [Figure 62B]

[0178] Selection of serum-free medium providers (serum substitutes). Each fragment was cultured four times in a single well of a G-Rex 24-well plate. On day 11, REP was initiated using 45 TIL and 106 feeders to mimic the 2A process. B) Bar graph showing the mean number of viable cells 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 63A]

[0179] Selection of serum-free medium providers (platelet lysate serum). Each fragment was cultured three times in a single well of a G-Rex 24-well plate. On day 11, REP was initiated using 4e5 TIL and 10e6 feeders to mimic the 2A process. A) Bar graphs showing the mean viable cell count recorded on day 11 (pre-REP) for each condition. [Figure 63B]

[0179] Selection of serum-free medium providers (platelet lysate serum). Each fragment was cultured three times in a single well of a G-Rex 24-well plate. On day 11, REP was initiated using 4e5 TIL and 10e6 feeders to mimic the 2A process. B) Bar graph showing the mean number of viable cells 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. "#" indicates tumor fragment deficiency. [Figure 64A-1]

[0180] The effectiveness of CTS Optimizer was compared to standard conditions using a small-scale 2A process (G-Rex 5M). Two fragments / G-Rex 5M were cultured three times, and a REP was initiated using 26 TILs in a 506 feeder to mimic the 2A process. The numbers above represent the average viable cell count obtained on day 11 (A) or day 22 (B). [Figure 64A-2]

[0180] The effectiveness of CTS Optimizer was compared to standard conditions using a small-scale 2A process (G-Rex 5M). Two fragments / G-Rex 5M were cultured three times, and REP was started using 26 TILs in a 506 feeder 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-1]

[0180] The effectiveness of CTS Optimizer was compared to standard conditions using a small-scale 2A process (G-Rex 5M). Two fragments / G-Rex 5M were cultured three times, and REP was started using 26 TILs in a 506 feeder 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-2]

[0180] The effectiveness of CTS Optimizer was compared to standard conditions using a small-scale 2A process (G-Rex 5M). Two fragments / G-Rex 5M were cultured three times, and REP was started using 26 TILs in a 506 feeder 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]

[0181] Summary of estimated pre- and post-tissue lithotripsy (TIL) expansion cultures by comparing standard conditions and CTS Optimizer. A) Pre-REP. [Figure 65B]

[0181] Summary of estimated pre- and post-TIL expansion cultures by comparing standard conditions and CTS Optimizer. B) Post-REP. [Figure 65C]

[0181] Summary of TIL expansion culture before and after, estimated by comparing standard conditions and CTS Optimizer. C) Summary of TIL expansion culture extrapolated to be performed as in reality (standard conditions and CTS Optimizer + SR). [Figure 66]

[0182] CD8+ was gated in living cells. Seven of the nine tumors showed an increase in the absolute CD8+ population in the CTS+SR state. [Figure 67]

[0183] Comparability of interferon-gamma. Interferon-gamma ELISA (Quantikine). IFN-y production was measured using the Quantikine ELISA kit from R&D Systems. CTS+SR produced the same amount of IFN-y as standard conditions. [Figure 68]

[0184] A scheme for an exemplary embodiment of the Rapid Expansion Culture Protocol (REP). Upon arrival, tumors are fragmented and placed in a G-Rex flask containing IL-2 for 11 days of TIL expansion culture (pre-REP expansion culture). In triple cocktail studies, IL-2 / IL-15 / IL-21 is added at the start of pre-REP. For the Rapid Expansion Culture Protocol (REP), TILs are cultured in a feeder and OKT3 for an additional 11 days of REP expansion culture. [Figure 69A]

[0185] TILs (n=4) and lung cells (n=7) derived from melanoma were evaluated for phenotypic characteristics of CD4+ and CD8+ cells using post-pre-REP flow cytometry. *P values ​​represent the difference between IL-2 and IL-12 / IL-15 / IL-21 in CD8+ cells using Student's unpaired t-test. [Figure 69B]

[0185] Phenotyping of CD4+ and CD8+ cells in melanoma-derived TILs (n=4) and lung cells (n=7) was evaluated using post-pre-REP flow cytometry. *P values ​​represent the difference between IL-2 and IL-12 / IL-15 / IL-21 in CD8+ cells using Student's non-corresponding t-test. [Figure 70A]

[0186] TILs (n=4) and lung tissues (n=7) derived from melanoma were evaluated for CD27+ and CD28+ phenotypes of CD4+ and CD8+ cells using post-pre-REP flow cytometry. [Figure 70B]

[0186] TILs (n=4) and lung tissue (n=7) derived from melanoma were evaluated for CD27+ and CD28+ phenotypes of CD4+ and CD8+ cells using post-pre-REP flow cytometry. [Figure 71A]

[0187] TILs were evaluated in effector / memory subsets of CD8+ cells (CD45RA and CCR7) and in the phenotypes of CD4+ cells (not shown in data) from melanoma (n=4) (A) and lung (n=8) (B). CXCR3 expression was evaluated in melanoma and lung. All phenotypic expression was evaluated using post-pre-REP flow cytometry. TCM = Central Memory, TSCM = Stem Cell-like Memory, TEMRA (Effector T Cell), TEM = Effector Memory. [Figure 71B]

[0187] TILs were evaluated in the effector / memory subsets of CD8+ cells (CD45RA and CCR7) and in the phenotypes of CD4+ cells (not shown in data) from melanoma (n=4) (A) and lung (n=8) (B). CXCR3 expression was evaluated in melanoma and lung. All phenotypic expression was evaluated using post-pre-REP flow cytometry. TCM = Central Memory, TSCM = Stem Cell-like Memory, TEMRA (Effector T Cell), TEM = Effector Memory. [Figure 71C]

[0187] TILs were evaluated in the effector / memory subsets of CD8+ cells (CD45RA and CCR7) and in the phenotypes of CD4+ cells (not shown in data) from melanoma (n=4) (A) and lung (n=8) (B). CXCR3 expression was evaluated in melanoma and lung. All phenotypic expression was evaluated using post-pre-REP flow cytometry. TCM = Central Memory, TSCM = Stem Cell-like Memory, TEMRA (Effector T Cell), TEM = Effector Memory. [Figure 72A]

[0188] TILs originating from (A) melanoma (n=4) and (B) lung (n=5) were evaluated by flow cytometry to assess CD107a+ expression in CD4+ and CD8+ cells in response to 4 hours of PMA stimulation. [Figure 72B]

[0188] TILs originating from (A) melanoma (n=4) and (B) lung (n=5) were evaluated by flow cytometry to assess CD107a+ expression in CD4+ and CD8+ cells in response to 4 hours of PMA stimulation. [Figure 72C]

[0188] (C) Pre-REP TILs (n=5) were stimulated with soluble OKT3 (30 ng / ml) for 24 hours, and the supernatant was evaluated for IFnγ by ELISA. [Figure 73A]

[0189] The TCRvβ repertoire (24 specificities) was evaluated using the Beckman-Coulter kit for flow cytometry with TILs originating from melanoma (A) and lung (B). [Figure 73B]

[0189] The TCRvβ repertoire (24 specificities) was evaluated using the Beckman-Coulter kit for flow cytometry with TILs originating from melanoma (A) and lung (B). [Figure 74]

[0190] Typical manufacturing process for cryopreserved TIL (approximately 22 days). [Figure 75A]

[0191] On day 22, the reduced volume of the cell product is pooled and sampled to determine the performance of the cultured cells before washing and formulation. The samples are analyzed using the NC-200 automated cell counter as described above. The total viable cell density is determined by the grand average of the overlapping counts from four independent samples. The Generation 2 (Gen2) process produces a similar dose of TIL product as Generation 1 (Gen1; Gen1 mean = 4.10 × 10¹⁰ ± 2.92 × 10¹⁰, Gen2 mean = 3.12 × 10¹⁰ ± 2.19 × 10¹⁰). [Figure 75B]

[0191] B) The expansion factor is calculated for the REP stage as the dividend of the final viable cell density to the initial viable TIL seeding density. Gen2 TIL products have a lower expansion factor compared to Gen1 (Gen1 mean = 1.40 × 10³ ± 9.86 × 10², Gen2 mean = 5.11 × 10² ± 2.95 × 10²). [Figure 76]

[0192] Freshly prepared formulations were assayed for identity by flow cytometry for release. The Gen1 and Gen2 processes produce high-purity T cell cultures defined by CD45, CD3 double-positive phenotypes (Gen1#±SD, Gen2#±SD). P-values ​​were calculated using the Mann-Whitney "t" test. [Figure 77A]

[0193] The vials of the cryopreserved satellite of the compounded product were thawed and assayed for the expanded culture phenotype by flow cytometry as described above. The Gen1 and Gen2 products represent similar ratios of CD8 T cell subtypes and CD4 T cell subtypes. The p-value was calculated using the Mann-Whitney "t" test. [Figure 77B]

[0193] The vials of the frozen satellite of the compounded product were thawed and assayed for the expanded culture phenotype by flow cytometry as described above. The Gen1 and Gen2 products represent similar ratios of CD8 T cell subtypes and CD4 T cell subtypes. The p-value was calculated using the Mann-Whitney "t" test. [Figure 78A]

[0194] The vials of the cryopreserved satellite of the compounded product were thawed and assayed for the expanded culture phenotype by flow cytometry as described above. Gen1 and Gen2 products express the same levels of the costimulatory molecules CD27 and CD28 on T cell subsets. The P-value was calculated using the Mann-Whitney "t" test. Costimulatory molecules such as CD27 and CD28 are required to supply the secondary and tertiary signals necessary for effector cell growth when T cell receptors are involved. [Figure 78B]

[0194] The vials of the cryopreserved satellite of the compounded product were thawed and assayed for the expanded culture phenotype by flow cytometry as described above. Gen1 and Gen2 products express the same levels of the costimulatory molecules CD27 and CD28 on T cell subsets. The P-value was calculated using the Mann-Whitney "t" test. Costimulatory molecules such as CD27 and CD28 are required to supply the secondary and tertiary signals necessary for effector cell growth when T cell receptors are involved. [Figure 79]

[0195] Using flow FISH technology, the mean length of telomere repeats was measured as described above. The above RTL values ​​indicate that the mean telomere fluorescence per chromosome / genome for Gen1 (1C process embodiment) of the control cell line was #%±SD%, and for Gen2, the mean telomere fluorescence per chromosome / genome was #%±SD% (1301 leukemia cell line). The data indicate that Gen2 products have, on average, at least equivalent telomere lengths to Gen1 products. Telomere length is a surrogate measure of length in exo vivo cell cultures. [Figure 80]

[0196] The Gen2 (Process Embodiment 2A) formulation product exhibits increased IFN-γ production capacity compared to the Gen1 formulation product. The ability of the formulation product to be reactivated and secrete cytokines is an alternative to the in vivo function when a TCR binds to an alloantigen in the context of HLA. [Figure 81A]

[0197] T cell receptor diversity: RNA from 10 × 10⁶ TILs of Gen1 (process 1C embodiment) and Gen2 (process 2A embodiment) formulation products was assayed to determine the total number and frequency of unique CDR3 sequences present in each product. Total number of unique CDR3 sequences present in each product (Gen1 n=#, mean ± SD, Gen2 n=#, mean ± SD). [Figure 81B]

[0197] Diversity of T cell receptors: RNA from 10 × 10⁶ TILs of Gen1 (process 1C embodiment) and Gen2 (process 2A embodiment) formulation products was assayed to determine the total number and frequency of unique CDR3 sequences present in each product. 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 consist of polyclonal populations of T cells with different antigen specificities and binding activities. The breadth of the total T cell repertoire may be an indicator of the number of actionable epitopes on tumor cells. [Figure 82]

[0198] A schematic diagram of an embodiment of Process 2A, a 22-day process for TIL manufacturing, is shown. [Figure 83]

[0199] A comparison table of steps A to F from exemplary embodiments of process 1C and process 2A. [Figure 84]

[0200] A detailed comparison of the embodiment of Process 1C and the embodiment of Process 2A. [Figure 85]

[0201] Detailed scheme of an embodiment of the TIL therapy process. [Figure 86A]

[0202] Phenotypic characterization of TIL products using a 10-color flow cytometry assay. (A) The proportions of T cell and non-T cell subsets are defined by CD45+CD3+ and CD45-(non-lymphocytes) / CD45+CD3-(non-T cell lymphocytes), respectively. Overall, >99% of the tested TIL products consisted of T cells (CD45+CD3+). The figures shown are the mean of TIL products (n=10). [Figure 86B]

[0202] Characterization of the phenotypic characteristics of TIL products using a 10-color flow cytometry assay. (B) Proportions of two T cell subsets, including CD45+CD3+CD8+ (blue open circle) and CD45+CD3+CD4+ (pink open circle). No statistically significant difference was observed between the proportions of the two subsets using Student's non-paired T test (P=0.68). [Figure 86C]

[0202] Characterization of the phenotype of TIL products using a 10-color flow cytometry assay. (C) The non-T cell population was characterized by 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]

[0203] 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 figure shows 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 major population (>93%) in both the CD4+ and CD8+ subsets of the final TIL product. The central memory subset (pink open circles) accounts for less than 7% of the cells in the TIL product. 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 87B]

[0203] 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 figure shows 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 major population (>93%) in both the CD4+ and CD8+ subsets of the final TIL product. Less than 7% of the cells in the TIL product are the central memory subset (pink open circles). The 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 88A]

[0204] 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 colorectal adenoma cancer cell line (HT29 as a negative control) were characterized by MCSP (melanoma (chondroitin sulfate proteoglycan)) and EpCAM (epithelial cell adhesion molecule) markers. (A) On average, 90% of melanoma tumor cells expressed MCSP. [Figure 88B]

[0204] 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 colorectal adenoma cancer cell line (HT29 as a negative control) were characterized by MCSP (melanoma (chondroitin sulfate proteoglycan) and EpCAM (epithelial cell adhesion molecule) markers. (B) Compared with the positive control HT29, which is an EpCAM+ tumor cell line, no EpCAM expression was detected in the melanoma tumor cell lines. [Figure 89A]

[0205] Detection of contamination controls to determine tumor detection accuracy. The assay was performed by contaminating a known amount of tumor cells with a PBMC suspension (n=10). MCSP+526 melanoma tumor cells were diluted at 1:10, 1:100, and 1:1,000 ratios, then mixed with PBMCs, stained with anti-MCSP and anti-CD45 antibodies and live / dead dyes, and analyzed by flow cytometry. (A) Approximately 3000, 300, and 30 cells were detected at dilutions of 1:10, 1:100, and 1:1000, respectively. [Figure 89B]

[0205] Detection of contamination controls to determine tumor detection accuracy. The assay was performed by contaminating a known amount of tumor cells in a PBMC suspension (n=10). MCSP+526 melanoma tumor cells were diluted in ratios of 1:10, 1:100, and 1:1,000, then mixed with PBMCs, stained with anti-MCSP and anti-CD45 antibodies and live / dead dyes, and analyzed by flow cytometry. (B) The mean (AV) and standard deviation (SD) of cells obtained under each condition were used to define the upper and lower limits of the criteria. [Figure 90A]

[0206] Reproducibility study of the upper and lower limits of the contamination control. Three independent experiments were repeated three times 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 reference range. [Figure 90B]

[0206] Reproducibility study of upper and lower limits of contamination control. Three independent experiments were repeated three times to determine the reproducibility of the contamination assay. (B) The linear regression diagram shows the correlation between MCSP+ cells and contamination dilution (R2=0.99), with the solid black line demonstrating the optimal value. The green and gray dashed lines represent the standard curve and the 95% prediction limits for the samples (Exp#1~3), respectively. [Figure 91A]

[0207] 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]

[0207] 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 92]

[0208] Evaluation of the efficacy of TIL products after T cell activation. IFNγ secretion from TIL products after restimulation with anti-CD3 / CD28 / CD137 (n=5) was evaluated repeatedly by ELISA. IFNγ secretion from TIL products was significantly higher than that of the unstimulated control using Wilcoxon's signed-rank test (P=0.02), consistently >1000 pg / ml. IFNγ secretion >200 pg / ml is considered potent. A p-value <0.05 is considered statistically significant. [Figure 93]

[0209] Description of an embodiment of the cryopreservation TIL manufacturing process (22 days). [Figure 94]

[0210] A table showing process improvements from Gen1 to Gen2. [Figure 95A]

[0211] Total viable cells, growth rate, and viability. On day 22, the reduced volume of cell product is pooled and sampled to determine the performance of the cultured cells before washing and formulation. (A) The samples are analyzed with an NC-200 automated cell counter as described above. Total viable cell density is determined by the grand average of overlapping counts from four independent samples. The Gen2 process produces the same dose of TIL product as Gen1 (Gen1 mean = 4.10 × 10¹⁰ ± 2.8 × 10¹⁰, Gen2 mean = 4.12 × 10¹⁰ ± 2.5 × 10¹⁰). [Figure 95B]

[0211] Total viable cells, growth rate, and survival rate. On day 22, the reduced volume of the cell product is pooled and sampled to determine the performance of the cultured cells before washing and formulation. (B) The growth rate at the REP stage is calculated as gr = ln(N(t) / N(0) / t). [Figure 95C]

[0211] Total viable cells, growth rate, and viability. On day 22, the reduced volume cell product was pooled and sampled to determine the performance of the cultured cells before washing and formulation. (C) As previously stated, cell viability was evaluated from nine process development lots using Cellometer K2. No significant decrease in cell viability was observed after a single freeze-thaw cycle of the formulated product. The mean decrease in viability at thawing and sampling was 2.19%. [Figure 96A]

[0212] The Gen2 product is a high-purity T cell culture expressing costimulatory molecules at levels equivalent to Gen1. (A) Fresh, prepared formulations were assayed for identity by flow cytometry for release. The Gen1 and Gen2 processes produce high-purity T cell cultures defined by the CD45+, CD3+ (double-positive) phenotype. [Figure 96B]

[0212] The Gen2 product is a high-purity T cell culture expressing costimulatory molecules at levels equivalent to Gen1. (B and C) Vials of cryopreserved satellites of the prepared formulation products were thawed and assayed for expanded culture phenotype by flow cytometry as described above. The Gen1 and Gen2 products express the same levels of costimulatory molecules CD27 and CD28 on T cell subsets. Costimulatory molecules such as CD27 and CD28 are required to supply secondary and tertiary signals necessary for effector cell growth when T cell receptors are involved. P values ​​were calculated using the Mann-Whitney "t" test. [Figure 96C]

[0212] The Gen2 product is a high-purity T cell culture expressing costimulatory molecules at levels equivalent to Gen1. (B and C) Vials of cryopreserved satellites of the prepared formulation products were thawed and assayed for expanded culture phenotype by flow cytometry as described above. The Gen1 and Gen2 products express the same levels of costimulatory molecules CD27 and CD28 on T cell subsets. Costimulatory molecules such as CD27 and CD28 are required to supply secondary and tertiary signals necessary for effector cell growth when T cell receptors are involved. P values ​​were calculated using the Mann-Whitney "t" test. [Figure 97]

[0213] Gen2 products exhibit similar telomere lengths. However, some TIL populations may tend to have longer relative telomeres. [Figure 98]

[0214] Gen2 formulations secrete IFNγ in response to the involvement of CD3, CD28, and CD137. [Figure 99A]

[0215] Diversity of T cell receptors. (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]

[0215] Diversity of T cell receptors. (B) Average total number of unique CDR3 sequences present in each infusion product. [Figure 100]

[0216] An embodiment of the TIL manufacturing process of the present invention. [Figure 101]

[0217] Enhanced expansion culture during pre-REP in multiple tumor tissues induced by IL-2 / IL-15 / IL-21. [Figure 102A]

[0218] IL-2 / IL-15 / IL-21 increased the percentage of CD8+ cells in lung cancer but not in melanoma. (A) TILs originating from melanoma (n=4) and (B) lung (n=7) were evaluated for CD4+ and CD8+ cell phenotypes using post-pre-REP flow cytometry. [Figure 102B]

[0218] IL-2 / IL-15 / IL-21 increased the proportion of CD8+ cells in lung cancer but not in melanoma. (A) TILs derived from melanoma (n=4), and (B) lungs (n=7) were evaluated for CD4+ and CD8+ cell phenotypes using post-pre-REP flow cytometry. [Figure 103A]

[0219] CD27 expression was slightly upregulated in CD8+ cells from cultures treated with IL-2 / IL-15 / IL-21. (A) TILs derived from melanoma (n=4), and (B) lung tissue (n=7) were evaluated for CD27+ and CD28+ phenotypes in CD4+ and CD8+ cells using post-preREP flow cytometry. [Figure 103B]

[0219] CD27 expression was slightly upregulated in CD8+ cells of cultures treated with IL-2 / IL-15 / IL-21. (A) TILs derived from melanoma (n=4), and (B) lung cells (n=7) were evaluated for CD27+ and CD28+ phenotypes in CD4+ and CD8+ cells using post-preREP flow cytometry. [Figure 104A]

[0220] The T cell subset did not change with the addition of IL-15 / IL-21. TILs were assessed by (A) effector / memory subsets (CD45RA and CCR7) of CD8+ and CD4+ (not shown in data) cells from melanoma (n=4), and by (B) lung (n=8) phenotype via flow cytometry post-preREP. [Figure 104B]

[0220] The T cell subset was not altered by the addition of IL-15 / IL-21. TILs were assessed by (A) effector / memory subsets (CD45RA and CCR7) of CD8+ and CD4+ (not shown in data) cells from melanoma (n=4), and by (B) lung (n=8) phenotype via flow cytometry post-preREP. [Figure 105A]

[0221] The functional capacity of TILs was differentially enhanced by IL-2 / IL-15 / IL-21. TILs derived from (A) melanoma (n=4) and (B) lung (n=5) were evaluated by flow cytometry for CD107a+ expression in CD4+ and CD8+ cells in response to 4 hours of PMA stimulation. [Figure 105B]

[0221] The functional capacity of TILs was differentially enhanced by IL-2 / IL-15 / IL-21. TILs derived from (A) melanoma (n=4) and (B) lung (n=5) were evaluated by flow cytometry for CD107a+ expression in CD4+ and CD8+ cells in response to 4 hours of PMA stimulation. [Figure 105C]

[0221] The functional capacity of TILs was differentially enhanced with IL-2 / IL-15 / IL-21. (C) Pre-REP TILs derived from melanoma and lung were stimulated with soluble anti-CD3 antibody for 24 hours, and the supernatant was evaluated for IFNγ by ELISA. [Figure 106A]

[0222] The TCRvβ repertoire (24 specificities) was evaluated using the Beckman-Coulter kit for flow cytometry with TILs originating from (A) melanoma and (B) lung tumors. [Figure 106B]

[0222] The TCRvβ repertoire (24 specificities) was evaluated using the Beckman-Coulter kit for flow cytometry with TILs originating from (A) melanoma and (B) lung tumors. [Figure 107]

[0223] Gen2 cryopreservation LN-144 manufacturing process scheme. [Figure 108]

[0224] A scheme for a multicenter phase 2 clinical trial of a novel cryopreserved intracellular lump (TIL) administered to patients with metastatic melanoma. [Figure 109]

[0225] A table showing a comparison of patient characteristics between Cohort 1 (ASCO2017) and Cohort 2. [Figure 110]

[0226] A table showing emergency adverse events of the procedure (≥30%). [Figure 111]

[0227] The effectiveness of injectable products and TIL therapy. [Figure 112]

[0228] Clinical status of evaluable patient responses showing SD or better response. [Figure 113]

[0229] The rate of change of the total diameter. [Figure 114]

[0230] An increase in HMGB1 levels was observed during TIL treatment. [Figure 115]

[0231] An increase in the biomarker IL-10 was observed after LN-144 injection. [Figure 116]

[0232] Updated patient characteristics (N=17 patients) for cohort 2 of a phase 2 clinical trial of metastatic melanoma from the second data cut. [Figure 117]

[0233] Treatment-related adverse events in Cohort 2 (≥30%) from the second data cut (N=17 patients). [Figure 118]

[0234] Response times (N=17 patients) for evaluable patients (stable or better condition) in Cohort 2 from the second data cut. Of the 10 patients in the efficacy set, one patient (patient 10) could not be evaluated because he died from melanoma-related causes before the initial tumor assessment (not shown in the figure). [Figure 119]

[0235] Updated efficacy data for Cohort 2 from the second data cut (N=17 patients). The mean number of TILs injected was 34 × 10⁹. The median of previous treatment 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 because they died from melanoma-related causes before the initial tumor evaluation, but were still considered in the efficacy set. Abbreviations: PR, partial response; SD, stable disease; PD, progressive disease. [Figure 120]

[0236] Updated efficacy data (N=17 patients) from evaluable patients in Cohort 2 from the second data cut. * indicates unevaluable patients who did not reach the initial evaluation. All efficacy-evaluable patients had previously received anti-PD-1 and anti-CTLA-4 checkpoint inhibitor therapy. [Figure 121]

[0237] Representative computed tomography images from patients (003-015) with PR from Cohort 2, second data cut. [Figure 122]

[0238] Correlation between IFN-γ induction by TIL products prior to injection and clinical reduction of tumor size 42 days after TIL injection. [Figure 123]

[0239] IP-10 (CXCL10) levels (pg / mL, log10) before and after injection of the Gen2 TIL product embodiment. IP-10 is a marker for cell adhesion and homing. [Figure 124]

[0240] IP-10 (CXCL10) levels (pg / mL, log10) before and after injection of the Gen1 TIL product embodiment. [Figure 125]

[0241] MCP-1 levels (pg / mL, log10) before and after injection of the Gen2 TIL product embodiment. MCP-1 is a marker for cell adhesion and homing. [Figure 126]

[0242] MCP-1 levels (pg / mL, log10) before and after injection of the Gen1 TIL product embodiment. [Figure 127]

[0243] Data from Phase 2 trials for cervical cancer and head and neck squamous cell carcinoma (HNSCC). SD = stable disease. PR = progressive disease. PR = partial response. [Figure 128]

[0244] A schematic diagram of an embodiment of Process 2A, a 22-day process for TIL manufacturing, is shown. [Figure 129]

[0245] A schematic diagram shows the sterile welding of the TIL suspension transfer pack to the lower (single line) of the gravity blood filter (see process note 5.11 of Example 30). [Figure 130]

[0245] A schematic diagram of the sterile welding of the red culture medium extraction line from the GRex100MCS to the "supernatant" transfer pack is shown (see process note 5.11 of Example 30). [Figure 131]

[0245] A schematic diagram shows the welding of 4S-4M60 to a CC2 cell connect (see process note 5.11 of Example 30), replacing the single spike (B) of the cell connect device with (G) at the four-spike end of the 4S-4M60 manifold. [Figure 132]

[0246] A schematic diagram shows the welding of a repeater fluid transfer set to one of the male luer ends of a 4S-4M60 (see process note 5.11 of Example 30). [Figure 133]

[0247] A schematic diagram of the sterile welding of the long end of the gravity blood filter to the LOVO raw material bag (see process note 5.11 of Example 30) is shown. [Figure 134]

[0248] A schematic diagram shows the sterile welding of one of the filter's two supply lines to the "pooled TIL suspension" collection bag (see process note 5.11 for Example 30). [Figure 135]

[0249] A schematic diagram shows sterile welding of the 4S-4M60 to the CC2 cell connect (see process note 5.11 of Example 30), replacing the single spike (B) of the cell connect device with the four spike ends of the 4S-4M60 manifold at (G). [Figure 136]

[0250] A schematic diagram shows the sterile welding of the CS750 cryobags to the harness prepared in step 8.14.8 (see process note 5.11 of Example 30), in which one of the four male luer ends (E) is replaced on each bag. [Figure 137]

[0251] A schematic diagram of welding a CS-10 bag to a 4S-4M60 spike (see process note 5.11 of Example 30) is shown. [Figure 138]

[0252] A schematic diagram shows the welding of the “formulated TIL” bag to the remaining spike (A) in the apparatus prepared in step 8.14.10 (see process note 5.11 of Example 30). [Figure 139]

[0253] The diagram shows the removal of the empty retaining fluid bag and CS-10 bag, and the heat sealing at F (see process note 5.12 of Example 30). [Figure 140]

[0254] Structures IA and IB are shown, with the cylinders pointing to the individual polypeptide-binding domains. Structures IA and IB contain three linearly linked TNFRSF-binding domains, for example, derived from an antibody that binds to 4-1BBL or 4-1BB, which fold to form a trivalent protein, which is then linked to a second trivalent protein via IgG1-Fc (containing CH3 and CH2 domains), which is then used to link the two trivalent proteins via disulfide bonds (small elongated ovals), stabilizing the structure and providing an agonist that can combine the intracellular signaling domains and signaling proteins of the six receptors to form a signaling complex. [Figure 141]

[0255] A chart is provided that outlines the three-stage experiment discussed in Example 21. [Modes for carrying out the invention]

[0118] A brief explanation of sequence listings

[0256] Sequence ID 1 is the amino acid sequence of the heavy chain of muromonab.

[0119]

[0257] Sequence ID 2 is the amino acid sequence of the light chain of muromonab.

[0120]

[0258] Sequence ID 3 is the amino acid sequence of recombinant human IL-2 protein.

[0121]

[0259] Sequence ID 4 is the amino acid sequence of aldethleukin.

[0122]

[0260] Sequence ID 5 is the amino acid sequence of recombinant human IL-4 protein.

[0123]

[0261] Sequence ID 6 is the amino acid sequence of recombinant human IL-7 protein.

[0124]

[0262] Sequence ID 7 is the amino acid sequence of recombinant human IL-15 protein.

[0125]

[0263] Sequence ID 8 is the amino acid sequence of recombinant human IL-21 protein.

[0126]

[0264] Sequence ID 9 is the amino acid sequence of human 4-1BB.

[0127]

[0265] Sequence ID 10 is the amino acid sequence of mouse 4-1BB.

[0128]

[0266] Sequence ID 11 is the heavy chain of the 4-1BB agonist monoclonal antibody utomirumab (PF-05082566).

[0129]

[0267] Sequence ID 12 is the light chain of the 4-1BB agonist monoclonal antibody utomirumab (PF-05082566).

[0130]

[0268] Sequence ID 13 is the heavy chain variable region (V) of the 4-1BB agonist monoclonal antibody utomirumab (PF-05082566). H )

[0131]

[0269] Sequence ID 14 is the light chain variable region (V) of the 4-1BB agonist monoclonal antibody utomirumab (PF-05082566). L )

[0132]

[0270] Sequence ID 15 is the heavy chain CDR1 of the 4-1BB agonist monoclonal antibody utomirumab (PF-05082566).

[0133]

[0271] Sequence ID 16 is the heavy chain CDR2 of the 4-1BB agonist monoclonal antibody utomirumab (PF-05082566).

[0134]

[0272] Sequence ID No. 17 is the heavy chain CDR3 of the 4-1BB agonist monoclonal antibody utomirumab (PF-05082566).

[0135]

[0273] Sequence ID No. 18 is the light chain CDR1 of the 4-1BB agonist monoclonal antibody utomirumab (PF-05082566).

[0136]

[0274] Sequence ID 19 is the light chain CDR2 of the 4-1BB agonist monoclonal antibody utomirumab (PF-05082566).

[0137]

[0275] Sequence ID No. 20 is the light chain CDR3 of the 4-1BB agonist monoclonal antibody utomirumab (PF-05082566).

[0138]

[0276] Sequence ID 21 is the heavy chain of the 4-1BB agonist monoclonal antibody urelumab (BMS-663513).

[0139]

[0277] Sequence ID 22 is the light chain of the 4-1BB agonist monoclonal antibody urelumab (BMS-663513).

[0140]

[0278] Sequence ID 23 is the heavy chain variable region (V) of the 4-1BB agonist monoclonal antibody urelumab (BMS-663513). H )

[0141]

[0279] Sequence ID 24 is the light chain variable region (V) of the 4-1BB agonist monoclonal antibody urelumab (BMS-663513). L )

[0142]

[0280] Sequence ID No. 25 is the heavy chain CDR1 of the 4-1BB agonist monoclonal antibody urelumab (BMS-663513).

[0143]

[0281] Sequence ID No. 26 is the heavy chain CDR2 of the 4-1BB agonist monoclonal antibody urelumab (BMS-663513).

[0144]

[0282] Sequence ID No. 27 is the heavy chain CDR3 of the 4-1BB agonist monoclonal antibody urelumab (BMS-663513).

[0145]

[0283] Sequence ID No. 28 is the light chain CDR1 of the 4-1BB agonist monoclonal antibody urelumab (BMS-663513).

[0146]

[0284] Sequence ID 29 is the light chain CDR2 of the 4-1BB agonist monoclonal antibody urelumab (BMS-663513).

[0147]

[0285] Sequence ID No. 30 is the light chain CDR3 of the 4-1BB agonist monoclonal antibody urelumab (BMS-663513).

[0148]

[0286] Sequence ID 46 is the amino acid sequence of the 4-1BB ligand (4-1BBL).

[0149]

[0287] Sequence ID No. 47 is the soluble portion of the 4-1BBL polypeptide.

[0150]

[0288] SEQ ID NO: 48 is the heavy chain variable region (V) of the 4-1BB agonist antibody 4B4-1-1 version 1. H )

[0151]

[0289] SEQ ID NO: 49 is the light chain variable region (V) of the 4-1BB agonist antibody 4B4-1-1 version 1. L )

[0152]

[0290] SEQ ID NO: 50 is the heavy chain variable region (V) of the 4-1BB agonist antibody 4B4-1-1 version 2. H )

[0153]

[0291] Sequence ID 51 is the light chain variable region (V) of the 4-1BB agonist antibody 4B4-1-1 version 2. L )

[0154]

[0292] Sequence ID 52 is the heavy chain variable region (V) of the 4-1BB agonist antibody H39E3-2. H )

[0155]

[0293] Sequence ID 53 is the light chain variable region (V) of the 4-1BB agonist antibody H39E3-2. L )

[0156] Detailed description of the invention I. Introduction

[0294] Adoptive cell therapy using TILs cultured ex vivo via the Rapid Expansion Culture Protocol (REP) has demonstrated success in melanoma patients after host immunosuppression. Current injection suitability parameters rely on readings of TIL composition (e.g., CD28, CD8, or CD4 positivity) as well as numerical values ​​of the expansion factor and viability of the REP product.

[0157]

[0295] The current REP protocol provides little insight into the health of the TILs that will be injected into the patient. T cells undergo a significant metabolic shift during their maturation from naive T cells to effector T cells (see Chang, et al., Nat. Immunol. 2016, 17, 364 (expressly incorporated herein as a whole) and especially for considerations 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 are highly glycolytic and rely on aerobic glycolysis to supply the bioenergetic substrates they need for proliferation, migration, activation, and antitumor efficacy.

[0158]

[0296] Previous studies have shown that cells heavily reliant on glycolysis suffer nutrient depletion during adoptive migration, resulting in the death of most of the transplanted cells. Therefore, it is desirable to restrict TIL glycolysis and promote mitochondrial metabolism before migration. Consequently, in this field, it has been suggested that promoting mitochondrial metabolism may extend in vivo lifespan, and in fact, the use of glycolysis inhibitors before inducing an immune response has been proposed. See Chang et al. (Chang, et al., Nat. Immunol. 2016, 17(364)).

[0159]

[0297] The present invention further relates, in some embodiments, to a method for determining and quantifying this increase in metabolic health. Accordingly, the present invention provides a method for assaying the relative health of a TIL population using one or more common metabolic assessments, including, but not limited to, the rate and amount of glycolysis, oxidative phosphorylation, respiratory reserve (SRC), and glycolytic reserve.

[0160]

[0298] Furthermore, the present invention relates, in some embodiments, to a method for determining and quantifying this increase in metabolic health. Accordingly, the present invention provides a method for assaying the relative health of a TIL population using one or more common metabolic assessments, including, but not limited to, the rate and amount of glycolysis, oxidative phosphorylation, respiratory reserve (SRC), and glycolytic reserve.

[0161]

[0299] In addition, optional additional criteria may include, but are not limited to, ATP production, mitochondrial mass, and glucose uptake.

[0162] II. Definition

[0300] Unless otherwise defined, all technical and scientific terms used herein have the same meaning as those generally understood by those skilled in the art to which the present invention pertains. All patents and publications referenced herein are incorporated herein by reference in their entirety.

[0163]

[0301] The term "in vivo" refers to events that occur within the body of a subject.

[0164]

[0302] The term "in vitro" refers to events that occur outside the body of the subject. In vitro assays encompass cell-based assays using living or dead cells, and may also include cell-free assays that do not use intact cells.

[0165]

[0303] The term "exovivo" refers to an event involving the treatment or administration of cells, tissues, and / or organs extracted from a subject's body. Appropriately, the cells, tissues, and / or organs are returned to the subject's body by surgical or other means.

[0166]

[0304] The term "rapid expansion culture" refers to an increase in the number of antigen-specific TILs of at least approximately 3 times (or 4 times, 5 times, 6 times, 7 times, 8 times, or 9 times) over a one-week period, more preferably at least approximately 10 times (or 20 times, 30 times, 40 times, 50 times, 60 times, 70 times, 80 times, or 90 times) over a one-week period, or most preferably at least approximately 100 times over a one-week period. Several rapid expansion culture protocols are described below.

[0167]

[0305] In this specification, "tumor-infiltrating lymphocytes" or "TILs" refers to a group of cells that have migrated from the bloodstream into the tumor and were originally obtained as leukocytes. TILs are not limited to CD8 + Cytotoxic T cells (lymphocytes), Th1 and Th17 CD4 + Examples include T cells, natural killer cells, dendritic cells, and M1 macrophages. TILs include both primary and secondary TILs. "Primary TILs" are obtained from patient tissue samples as outlined herein (sometimes referred to as "freshly collected"), and "secondary TILs" are any TIL cell populations that have been cultured or grown as discussed herein, including but not limited to bulk TILs and expanded cultured TILs ("REP TILs" or "post-REP TILs"). TIL cell populations may include genetically modified TILs.

[0168]

[0306] In this specification, “cell population” (including TIL) means a large number of cells that share common traits. Generally, a population is roughly 1 × 10⁶ in number. 6 ~1 × 10 10 This is a range of individuals, and different TIL populations contain different numbers. For example, the initial growth of primary TILs in the presence of IL-2 is approximately 1 × 10⁻⁶. 8This yields a bulk TIL population of individual cells. REP expansion cultures generally yield 1.5 × 10⁶ cells. 9 ~1.5×10 10 This is done so that individual cell populations for injection are provided.

[0169]

[0307] In this specification, “cryopreserved TILs” means primary, bulk, or expanded culture (REP TILs) TILs that are processed and stored at a temperature range of approximately -150°C to -60°C. General cryopreservation methods are described in other parts of this specification, including in the examples. For clarity, “cryopreserved TILs” are distinct from frozen tissue samples that may be used as a source of primary TILs.

[0170]

[0308] In this specification, “thawed cryopreserved TILs” means a population of TILs that were previously cryopreserved but have been subsequently returned to room temperature or above, including, but not limited to, cell culture temperatures or temperatures at which TILs can be administered to patients, through processing.

[0171]

[0309] TILs can generally be defined biochemically using cell surface markers, or functionally by their ability to invade 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. In addition, and instead, TILs can be functionally defined by their ability to invade solid tumors upon reintroduction into a patient.

[0172]

[0310] The term "cryopreservation media" refers to any medium that can be used for the cryopreservation of cells. Such media may include media containing 7% to 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 may be referred to by the trade name "CryoStor® CS10". CS10 medium is a serum-free, animal-component-free medium containing DMSO.

[0173]

[0311] The term "central memory T cell" in humans is CD45R0+ and also CCR7(CCR7) hi ) and CD62L (CD62 hi This refers to a subset of T cells that constitutively express IL-2. The surface phenotype of central memory T cells also includes TCR, CD3, CD127 (IL-7R), and IL-15R. The transcription factors of central memory T cells include BCL-6, BCL-6B, MBD2, and BMI1. After TCR induction, central memory T cells mainly secrete IL-2 and CD40L as effector molecules. Central memory T cells are dominant in the CD4 compartment of the blood and are proportionally more concentrated in lymph nodes and tonsils in humans.

[0174]

[0312] The term "effector memory T cell" is similar to central memory T cells in that it is CD45R0+, but lacks constitutive expression of CCR7 (CCR7 lo ), and CD62L expression is heterogeneous or low (CD62L loCentral memory T cells refer to 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. The transcription factor of central memory T cells includes BLIMP1. Effector memory T cells rapidly secrete high levels of inflammatory cytokines, including interferon-γ, IL-4, and IL-5, after antigen stimulation. Effector memory T cells are dominant in the CD8 compartment of the blood and are proportionally more abundant in the lungs, liver, and intestines in humans. CD8+ effector memory T cells have a large amount of perforin.

[0175]

[0313] The term "closed system" refers to a system that is closed off from the external environment. In the method of the present invention, any closed system suitable for cell culture can be used. Examples of closed systems include, but are not limited to, closed G containers. After the tumor segment is added to the closed system, the system is not opened to the external environment until immediately before administration to a patient with TIL.

[0176]

[0314] The terms “fragmentation,” “fragment,” and “fragmented” as used herein to describe the process of tumor destruction include mechanical fragmentation methods such as crushing, slicing, dividing, and shredding tumor tissue, as well as any other method of destroying the physical structure of tumor tissue.

[0177]

[0315] The terms "peripheral blood mononuclear cells" and "PBMCs" refer to peripheral blood cells with a round nucleus, including lymphocytes (T cells, B cells, NK cells) and monocytes. Preferably, peripheral blood mononuclear cells are irradiated allogeneic peripheral blood mononuclear cells. PBMCs are a type of antigen-presenting cell.

[0178]

[0316] The term "anti-CD3 antibody" refers to an antibody or its variant, such as a monoclonal antibody, which includes human, humanized, chimeric, or mouse antibodies against the CD3 receptor on 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, teprizumab, and vizilizumab.

[0179]

[0317] The term "OKT-3" (also referred to herein as "OKT3") refers to monoclonal antibodies, including human, humanized, chimeric, or mouse antibodies, or their biosimilars or variants, against the CD3 receptor on the T cell antigen receptor of mature T cells, and includes commercially available forms such as OKT-3 (30 ng / mL, MACS GMP CD3 pure, Miltenyi Biotech, Inc., San Diego, CA, USA) and muromonab or its variants, conserved 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). Hybridomas capable of producing OKT-3 are deposited with the U.S. Cell Culture Line Preservation Service and have been assigned ATCC accession number CRL 8001. Hybridomas capable of producing OKT-3 are also deposited with the European Certified Cell Culture Line Preservation Service (ECACC) and have been assigned catalog number 86022706.

[0180] [Table 1]

[0181]

[0318] The term "IL-2" (also referred to herein as "IL2") refers to the T cell growth factor known as interleukin-2, and includes all forms of IL-2, including its human and mammalian forms, conserved 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 (these disclosures are incorporated herein by reference). Table 2 provides an amino acid sequence of recombinant human IL-2 suitable for use in the present invention (SEQ ID NO: 3). For example, the term IL-2 includes human recombinant forms of IL-2, such as aldesleukin (PROLEUKIN, commercially available from multiple suppliers at 22 million IU per disposable vial), 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 number CYT-209-b), and other commercially available equivalents from other distributors. 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. Table 2 provides the amino acid sequence of aldesleukin suitable for use in this invention (SEQ ID NO: 4). The term IL-2 also includes pegylated forms of IL-2 as described herein, including pegylated IL-2 prodrug NKTR-214, available from Nektar Therapeutics, South San Francisco, CA, USA. NKTR-214 and pegylated IL-2 suitable for use in the present invention are described in U.S. Patent Publication No. 2014 / 0328791 A1 and International Publication No. 2012 / 065086 A1 (these disclosures are incorporated herein by reference).Another form of conjugated IL-2 suitable for use in the present invention is described in U.S. Patents 4,766,106, 5,206,344, 5,089,261 and 4902,502 (these disclosures are incorporated herein by reference). A formulation of IL-2 suitable for use in the present invention is described in U.S. Patent 6,706,289 (this disclosure is incorporated herein by reference).

[0182] [Table 2]

[0183]

[0319] The term "IL-4" (also referred to herein as "IL4") refers to the cytokine known as interleukin-4, which is produced by Th2 T cells and also 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. Upon activation by IL-4, Th2 T cells subsequently produce further 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 this invention is commercially available from several suppliers, including 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 Gibco CTP0043). The amino acid sequences of recombinant human IL-4 suitable for use in this invention are provided in Table 2 (SEQ ID NO: 5).

[0184]

[0320] The term "IL-7" (also referred to herein as "IL7") refers to a glycosylated tissue-derived cytokine known as interleukin-7, which can be obtained 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 heterodimer IL-7 receptor, which consists of IL-7 receptor α and common gamma chain receptor, and this is 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 this invention is commercially available from several suppliers, including ProSpec-Tany TechnoGene Ltd., East Brunswick, NJ, USA (catalog no. CYT-254) and ThermoFisher Scientific, Inc., Waltham, MA, USA (human IL-15 recombinant protein, catalog no. Gibco PHC0071). Table 2 provides the amino acid sequence of recombinant human IL-7 suitable for use in the present invention (SEQ ID NO: 6).

[0185]

[0321] The term “IL-15” (also referred to herein as “IL15”) refers to the T cell growth factor known as interleukin-15, and includes all forms of IL-2, including its human and mammalian forms, conserved amino acid substitutions, glycoforms, biosimilars, and variants. IL-15 is described, for example, in Fehniger and Caligiuri, Blood 2001, 97, 14-32 (this disclosure is incorporated herein by reference). IL-15 shares β and γ signaling receptor subunits with IL-2. Recombinant human IL-15 is a single non-glycosylated polypeptide chain with a molecular weight of 12.8 kDa and containing 114 amino acids (and N-terminal methionine). 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 ThermoFisher Scientific, Inc., Waltham, MA, USA (recombinant human IL-15 protein, catalog number 34-8159-82). Table 2 provides amino acid sequences of recombinant human IL-15 suitable for use in this invention (SEQ ID NO: 7).

[0186]

[0322] The term “IL-21” (also referred to herein as “IL21”) refers to the pleiotropic cytokine protein known as interleukin-21, and includes all forms of IL-21, including its human and mammalian forms, conserved amino acid substitutions, glycoforms, biosimilars, and variants. For example, IL-21 is described in Spolski and Leonard, Nat. Rev. Drug. Disc. 2014, 13, 379-95 (this disclosure is incorporated herein by reference). IL-21 is primarily used in 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 weight of 15.4 kDa. Recombinant human IL-21 is commercially available from several suppliers, including ProSpec-Tany TechnoGene Ltd., East Brunswick, NJ, USA (catalog no. CYT-408-b) and ThermoFisher Scientific, Inc., Waltham, MA, USA (recombinant human IL-21 protein, catalog no. 14-8219-80). Table 2 provides amino acid sequences of recombinant human IL-21 suitable for use in this invention (SEQ ID NO: 8).

[0187]

[0323] When an "antitumor effective dose," "tumor inhibitory effective dose," or "therapeutic dose" is indicated, the precise dosage of the composition 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 the patient's (subject's) condition. Generally, the pharmaceutical compositions comprising tumor-infiltrating lymphocytes (e.g., secondary TILs or genetically modified cytotoxic lymphocytes) described herein are 10 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 10It can be said that the drug can be administered in doses of cells / kg body weight (including all integer values ​​within these ranges). Tumor-infiltrating lymphocytes (including, in some cases, genetically modified cytotoxic lymphocytes) compositions can also be administered multiple times in these doses. Tumor-infiltrating lymphocytes (including, in some cases, genetically modified ones) can be administered by infusion techniques commonly known in immunotherapy (see, for example, Rosenberg et al., New Eng. J. of Med. 319: 1676, 1988). The optimal dosage and treatment regimen for a particular patient can be easily determined by a person skilled in the medical field by monitoring the patient for signs of the disease and adjusting the treatment accordingly.

[0188]

[0324] The term "hematological malignancies" refers to cancers and tumors of hematopoietic and lymphoid tissues in mammals, including, but not limited to, blood, bone marrow, lymph nodes, and lymphoid tissues. Hematological malignancies are also called "humoral tumors." Examples of hematological malignancies, but not limited to, include acute lymphoblastic leukemia (ALL), chronic lymphocytic lymphoma (CLL), small lymphocytic lymphoma (SLL), acute myeloid leukemia (AML), chronic myeloid leukemia (CML), acute monocytic leukemia (AMoL), Hodgkin lymphoma, and non-Hodgkin lymphoma. The term "B-cell hematological malignancies" refers to hematological malignancies that affect B cells.

[0189]

[0325] The term "solid tumor" refers to an abnormal mass of tissue that does not typically contain cysts or fluid. Solid tumors can be benign or malignant. The term "solid tumor carcinoma" refers to a malignant, neoplastic, or cancerous solid tumor. Examples of solid tumor carcinomas include, but are not limited to, sarcomas, carcinomas, and lymphomas, such as lung cancer, breast cancer, prostate cancer, colon cancer, rectal cancer, and bladder cancer. The histological structure of a solid tumor includes interdependent tissue compartments containing parenchyma (cancer cells) and supporting stromal cells where cancer cells are dispersed and which can provide a supporting microenvironment.

[0190]

[0326] The term "humoral tumor" refers to an abnormal mass of cells that is essentially fluid. Humoral tumor cancers include, but are not limited to, leukemia, myeloma and lymphoma, and other hematological malignancies. TILs obtained from humoral tumors may also be referred to herein as bone marrow-infiltrating lymphocytes (MILs).

[0191]

[0327] The term “microenvironment,” as used herein, may refer to the entire microenvironment of a solid tumor or hematological tumor, or to individual cell subsets within the microenvironment. The tumor microenvironment, as used herein, 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 the tumor from host immunity, encourage treatment resistance, and provide a niche for the development of overt metastases, as described in Swartz, et al., Cancer Res., 2012, 72, 2473. Tumors express antigens that should be recognized by T cells, but due to immunosuppression by the microenvironment, immune system-mediated tumor clearance is rare.

[0192]

[0328] In one embodiment, the present invention includes a method for treating cancer with a TIL population, where the patient is pre-treated with non-myeloablative chemotherapy before infusion of the TIL according to the present invention. In some embodiments, a TIL population may be provided, where the patient is pre-treated with non-myeloablative chemotherapy before infusion of the TIL according to the present invention. In some embodiments, the non-myeloablative chemotherapy consists of cyclophosphamide 60 mg / kg / day for 2 days (days 27 and 26 before TIL infusion) and fludarabine 25 mg / m2 / day for 5 days (days 27-23 before TIL infusion). In some embodiments, after non-myeloablative chemotherapy and TIL infusion according to the present invention (day 0), the patient receives intravenous infusions of IL-2 at a rate of 720,000 IU / kg every 8 hours until a physiologically tolerable dose is reached.

[0193]

[0329] Experimental findings suggest that lymphocyte depletion prior to adoptive transfer of tumor-specific T lymphocytes plays a crucial role in enhancing therapeutic efficacy by eliminating regulatory T cells and competing elements of the immune system ("cytokine sinks"). Therefore, some embodiments of the present invention utilize a lymphocyte depletion step (also referred to as "immunosuppressive conditioning") in the patient before introducing the rTIL of the present invention.

[0194]

[0330] The terms “co-administration,” “to co-administer,” “administered in combination with,” “administered in combination with,” “simultaneous,” and “parallel,” as used herein, encompass the administration of two or more pharmacoactive ingredients to a subject such that both pharmacoactive ingredients and / or their metabolites are present in the subject at the same time point (in preferred embodiments of the present invention, for example, at least one potassium channel agonist in combination with multiple TILs). Co-administration includes simultaneous administration in separate compositions, administration in separate compositions at different time points, or administration in a composition in which two or more pharmacoactive ingredients are present. Simultaneous administration in separate compositions and administration in a composition in which both drugs are present are preferred.

[0195]

[0331] The term “effective dose” or “therapeutic effective dose” refers to the amount of a compound or combination of compounds as described herein that is sufficient to achieve the intended application, including, but not limited to, disease treatment. The therapeutic effective dose may vary depending on the intended application (in vitro or in vivo) or the subject and disease state under treatment (e.g., the subject’s weight, age, and sex), the severity of the disease state, or the method of administration. The term also applies to the dose that will produce a specific response in target cells (e.g., reduced platelet adhesion and / or cell migration). The specific dose will vary depending on the specific compound selected, the administration regimen to be followed, whether the compound is administered in combination with other compounds, the timing of administration, the target tissue, and the physical delivery system carrying the compound.

[0196]

[0332] The terms “treatment,” “treating,” and “to treat” refer to achieving a desired pharmacological and / or physiological effect. The effect may be preventative in the sense of completely or partially preventing a disease or its symptoms, and / or therapeutic in the sense of partially or completely curing a disease and / or adverse effects resulting from the disease. “Treatment,” as used herein, encompasses any treatment of a disease in mammals, particularly humans, and includes (a) preventing the onset of the disease in a subject that may be predisposed to the disease but has not yet been diagnosed as having it; (b) inhibiting the disease, i.e., stopping its onset or progression; and (c) mitigating the disease, i.e., causing regression of the disease and / or reducing one or more symptoms of the disease. “Treatment” is also intended to encompass the delivery of a drug to provide a pharmacological effect even in the absence of a disease or pathological condition. For example, “treatment” includes the delivery of a composition that can induce an immune response or confer immunity in the absence of a disease state, such as a vaccine.

[0197]

[0333] The term "heterogeneous," when used in relation to a nucleic acid or protein, indicates that the nucleic acid or protein contains two or more subsequences that are not found in nature in the same relation to one another. For example, a nucleic acid typically has two or more sequences from unrelated genes that have been recombinantly produced and organized 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 heterogeneous protein indicates that a protein contains two or more subsequences (e.g., a fusion protein) that are not found in nature in the same relation to one another.

[0198]

[0334] In the context of two or more nucleic acids or polypeptides, the terms “sequence identity,” “percent identity,” and “sequence percent identity” (or their synonyms, e.g., “99% identity”) refer to two or more sequences or subsequences that are identical, or have only a specific proportion of identical nucleotide or amino acid residues, when compared and aligned (with gaps introduced as necessary) to maximize correspondence, without considering any conserved amino acid substitutions as part of sequence identity. Percent identity can be measured using sequence comparison software or algorithms, or by visual inspection. Various algorithms and software that can be used to achieve amino acid or nucleotide sequence alignment are known in the art. A suitable program for determining percent sequence identity is, for example, the BLAST program suite available from the BLAST website of the U.S. government's National Center for Biotechnology Information. Comparison between two sequences can be performed using either the BLASTN or BLASTP algorithm. BLASTN is used for comparing nucleic acid sequences, while BLASTP is used for comparing amino acid sequences. ALIGN, ALIGN-2 (Genentech, South San Francisco, California), or MegAlign, available from DNASTAR, are further publicly available software programs that can be used for sequence alignment. 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.

[0199]

[0335] As used herein, the term “variant” includes, but is not limited to, an antibody or fusion protein having an amino acid sequence that differs from the amino acid sequence of a reference antibody in terms of one or more substitutions, deletions, and / or additions at specific positions within or adjacent to the amino acid sequence of the reference antibody. A variant may have one or more conserved 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 to specifically bind to the antigen of the reference antibody. The term “variant” also includes pegylated antibodies or proteins.

[0200]

[0336] In this specification, "tumor-infiltrating lymphocytes" or "TILs" refers to a group of cells that have migrated from the bloodstream into the tumor and were originally obtained as leukocytes. TILs are not limited to CD8 + Cytotoxic T cells (lymphocytes), Th1 and Th17 CD4 + Examples include T cells, natural killer cells, dendritic cells, and M1 macrophages. TILs include both primary and secondary TILs. "Primary TILs" are obtained from patient tissue samples as outlined herein (sometimes referred to as "freshly collected"), and "secondary TILs" are any TIL cell populations that have been cultured or grown as discussed herein, including, but are not limited to, bulk TILs, expanded cultured TILs ("REP TILs"), and "reREP TILs" as discussed herein. reREP TILs may include, for example, a second expanded cultured TIL or a second additional expanded cultured TIL (e.g., one containing a TIL referred to as a reREP TIL, as described in step D of Figure 27).

[0201]

[0337] TILs can generally be defined biochemically using cell surface markers, or functionally 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. In addition, and instead, TILs can be functionally defined by their ability to infiltrate solid tumors upon reintroduction into a patient. TILs can further be characterized by potency; for example, a TIL may be considered potent when interferon (IFN) release is higher than approximately 50 pg / mL, approximately 100 pg / mL, approximately 150 pg / mL, or approximately 200 pg / mL.

[0202]

[0338] The terms “pharmaceutically acceptable carrier” or “pharmaceutically acceptable excipient” include all solvents, dispersants, coatings, antimicrobial and antifungal agents, isotonic and absorption retardants, and inert components. The use of such pharmaceutically acceptable carriers or excipients for active pharmaceutical ingredients is well known in the art. Unless 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 intended. Additional active pharmaceutical ingredients, such as other drugs, may also be incorporated into the compositions and methods described.

[0203]

[0339] The terms “about” and “approximately” refer to a range of statistically meaningful values. Such a range 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 permissible variation included in the terms “about” or “approximately” depends on the specific system under study and is readily understandable to those skilled 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 greater or less, reflecting tolerances, conversion factors, rounding, measurement errors, and other factors known to those skilled in the art, as they may be. In general, dimensions, sizes, formulations, parameters, shapes, or other quantities or characteristics are “about” or “approximately,” whether or not they are explicitly stated to be so. Note that embodiments of significantly different sizes, shapes, and dimensions may adopt the terms described herein.

[0204]

[0340] When used in the attached claims, the transitional terms “including,” “essentially consisting of,” and “consisting of” define the claims in their original and modified forms, with respect to the exclusion of any additional claim elements or steps not described herein from the claims. The term “including” is intended to be inclusive or unrestricted and does not exclude any additional uncited elements, methods, steps, or materials. The term “consisting of” excludes any elements, steps, or materials other than those specified in the claims, and in the latter case, excludes any common impurities associated with the specified materials. The term “essentially consisting of” limits the claims to such an extent that it does not substantially affect the basic and novel characteristics of a particular element, step, or material and 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 “including,” “essentially consisting of,” and “consisting of.”

[0205] 4-1BB (CD137) Agonist

[0001] In one embodiment, the TNFRSF agonist is a 4-1BB (CD137) agonist. The 4-1BB agonist may be any 4-1BB binding molecule known in the art. The 4-1BB binding molecule may be a monoclonal antibody or fusion protein capable of binding to human or mammalian 4-1BB. The 4-1BB agonist or 4-1BB binding molecule may contain an immunoglobulin heavy chain of any isotype (e.g., IgG, IgE, IgM, IgD, IgA, and IgY), class (e.g., IgG1, IgG2, IgG3, IgG4, IgA1, and IgA2), or subclass of an immunoglobulin molecule. The 4-1BB agonist or 4-1BB binding molecule may have both a heavy chain and a light chain. As used herein, the term "binding molecule" includes antibodies (including full-length antibodies), monoclonal antibodies (including full-length monoclonal antibodies), polyclonal antibodies, multispecific antibodies (e.g., bispecific antibodies), human, humanized or chimeric antibodies, and antibody fragments, e.g., Fab fragments, F(ab') fragments, fragments produced by Fab expression libraries, any of the above epitope-binding fragments, and engineered forms of antibodies that bind to 4-1BB, e.g., scFv molecules. In one embodiment, the 4-1BB agonist is an antigen-binding protein that is a fully human antibody. In one embodiment, the 4-1BB agonist is an antigen-binding protein that is a humanized antibody. In some embodiments, 4-1BB agonists for use in the methods and compositions of this disclosure include anti-4-1BB antibodies, human anti-4-1BB antibodies, mouse anti-4-1BB antibodies, mammalian anti-4-1BB antibodies, monoclonal anti-4-1BB antibodies, polyclonal anti-4-1BB antibodies, chimeric anti-4-1BB antibodies, anti-4-1BB adnectin, anti-4-1BB domain antibodies, single-chain anti-4-1BB fragments, heavy-chain anti-4-1BB fragments, light-chain anti-4-1BB fragments, anti-4-1BB fusion proteins and their fragments, derivatives, conjugates, variants or biosimilars. Agonist anti-4-1BB antibodies are known to induce a strong immune response. Lee, et al., PLOS One 2013, 8, e69677.In a preferred embodiment, the 4-1BB agonist is an agonist, anti-4-1BB humanized, or fully human monoclonal antibody (i.e., an antibody derived from a single cell line). In one embodiment, the 4-1BB agonist is EU-101 (Eutilex Co. Ltd.), utomirumab, or urerumab, or fragments, derivatives, conjugates, variants, or biosimilars thereof. In a preferred embodiment, the 4-1BB agonist is utomirumab, or urerumab, or fragments, derivatives, conjugates, variants, or biosimilars thereof.

[0206]

[0002] In preferred embodiments, the 4-1BB agonist or 4-1BB binding molecule may also be a fusion protein. In preferred embodiments, multimeric 4-1BB agonists, such as trimer or hexameric 4-1BB agonists (having three or six ligand-binding domains), can induce superior receptor (4-1BBL) clustering and internal cell signaling complex formation compared to agonist monoclonal antibodies typically possessing two ligand-binding domains. Trimeric (trivalent), hexameric (or hexavalent), or larger fusion proteins comprising three TNFRSF-binding domains and IgG1-Fc, and optionally further linking two or more of these fusion proteins, are described, for example, in Gieffers, et al., Mol. Cancer Therapeutics 2013, 12, 2735-47.

[0207]

[0003] Agonist 4-1BB antibodies and fusion proteins are known to induce a strong immune response. In preferred embodiments, the 4-1BB agonist is a monoclonal antibody or fusion protein that specifically binds to the 4-1BB antigen in a manner sufficient to reduce toxicity. In some embodiments, the 4-1BB agonist is an agonist 4-1BB monoclonal antibody or fusion protein that suppresses antibody-dependent cytotoxicity (ADCC), such as NK cell cytotoxicity. In some embodiments, the 4-1BB agonist is an agonist 4-1BB monoclonal antibody or fusion protein that suppresses antibody-dependent phagocytosis (ADCP). In some embodiments, the 4-1BB agonist is an agonist 4-1BB monoclonal antibody or fusion protein that suppresses complement-dependent cytotoxicity (CDC). In some embodiments, the 4-1BB agonist is an agonist 4-1BB monoclonal antibody or fusion protein that suppresses Fc domain functionality.

[0208]

[0004] In some embodiments, the 4-1BB agonist is characterized by binding to human 4-1BB (SEQ ID NO: 9) with high affinity and agonist activity. In one embodiment, the 4-1BB agonist is a binding molecule that binds to human 4-1BB (SEQ ID NO: 9). In one embodiment, the 4-1BB agonist is a binding molecule that binds to mouse 4-1BB (SEQ ID NO: 10). The amino acid sequences of the 4-1BB antigen to which the 4-1BB agonist or binding molecule binds are summarized in Table 3.

[0209] [Table 3]

[0210]

[0005] In some embodiments, the compositions, processes and methods described are K at about 100 pM or less. D It binds to human or mouse 4-1BB, or K at approximately 90 pM or less. D It binds to human or mouse 4-1BB, or K at approximately 80 pM or less. D It binds to human or mouse 4-1BB, or K at approximately 70 pM or less.D that binds to human or mouse 4-1BB with a K of about 60 pM or less D that binds to human or mouse 4-1BB with a K of about 50 pM or less D that binds to human or mouse 4-1BB with a K of about 40 pM or less D that binds to human or mouse 4-1BB with a K of about 30 pM or less, or D that binds to human or mouse 4-1BB, and comprises a 4-1BB agonist.

[0211]

[0006] In some embodiments, the compositions, processes, and methods described herein are about 7.5 × 10 5 k of 1 / M·s or more assoc that binds to human or mouse 4-1BB with a k of about 7.5 × 10 5 1 / M·s or more assoc that binds to human or mouse 4-1BB with a k of about 8 × 10 5 1 / M·s or more assoc that binds to human or mouse 4-1BB with a k of about 8.5 × 10 5 1 / M·s or more assoc that binds to human or mouse 4-1BB with a k of about 9 × 10 5 1 / M·s or more assoc that binds to human or mouse 4-1BB with a k of about 9.5 × 10 5 1 / M·s or more assoc that binds to human or mouse 4-1BB with a k of about 1 × 10 6 1 / M·s or more assoc that binds to human or mouse 4-1BB, and comprises a 4-1BB agonist.

[0212]

[0007] In some embodiments, the compositions, processes, and methods described herein are about 2 × 10 -5 k of 1 / s or less dissoc that binds to human or mouse 4-1BB with a k of about 2.1 × 10 -5 1 / s or less dissoc that binds to human or mouse 4-1BB with a k of about 2.2 × 10 -5 1 / s or less dissoc that binds to human or mouse 4-1BB with a k of about 2.3 × 10 -5k less than 1 / s dissoc It binds to human or mouse 4-1BB, or approximately 2.4 × 10⁻⁶ -5 k less than 1 / s dissoc It binds to human or mouse 4-1BB, or approximately 2.5 × 10⁻⁶ -5 k less than 1 / s dissoc It binds to human or mouse 4-1BB, or approximately 2.6 × 10⁻⁶ -5 k less than 1 / s dissoc It binds to human or mouse 4-1BB, or approximately 2.7 × 10⁻⁶ -5 k less than 1 / s dissoc It binds to human or mouse 4-1BB, or approximately 2.8 × 10⁻⁶ -5 k less than 1 / s dissoc It binds to human or mouse 4-1BB, or approximately 2.9 × 10⁻⁶ -5 k less than 1 / s dissoc It binds to human or mouse 4-1BB, or approximately 3 × 10⁻⁶ -5 k less than 1 / s dissoc It contains a 4-1BB agonist that binds to human or mouse 4-1BB.

[0213]

[0008] In some embodiments, the compositions, processes and methods described are ICs of about 10 nM or less. 50 It binds to human or mouse 4-1BB, or ICs of approximately 9 nM or less. 50 It binds to human or mouse 4-1BB, or ICs with a minimum intensity of approximately 8 nM. 50 It binds to human or mouse 4-1BB, or ICs with a minimum pulse intensity of approximately 7 nM. 50 It binds to human or mouse 4-1BB, or ICs with a minimum intensity of approximately 6 nM. 50 It binds to human or mouse 4-1BB, or ICs with a minimum intensity of approximately 5 nM. 50 It binds to human or mouse 4-1BB, or ICs with a minimum pulse intensity of approximately 4 nM. 50 It binds to human or mouse 4-1BB, or ICs with a minimum pulse intensity of approximately 3 nM. 50 It binds to human or mouse 4-1BB, or ICs of approximately 2 nM or less. 50 It binds to human or mouse 4-1BB, or ICs of approximately 1 nM or less. 50 It contains a 4-1BB agonist that binds to human or mouse 4-1BB.

[0214]

[0009] In preferred embodiments, the 4-1BB agonist is utomirumab or a fragment, derivative, variant, or biosimilar thereof, also known as PF-05082566 or MOR-7480. Utomilumab is available from Pfizer, Inc. Utomilumab is an immunoglobulin G2-lambda, anti-[human (Homo sapiens) TNFRSF9 (tumor necrosis factor receptor (TNFR) superfamily member 9, 4-1BB, T cell antigen ILA, CD137)], human (Homo sapiens) (fully human) monoclonal antibody. The amino acid sequence of utomirumab is shown in Table 4. Utomilumab has glycosylation sites at Asn59 and Asn292; positions 22-96 (V H -V L ), 143-199(C H 1-C L ), 256-316(C H 2) and 362-420(C H 3) Disulfide bridges within the heavy chain; position 22'-87'(V H -V L ) and 136'-195'(C H 1-C LIncludes intrachain disulfide crosslinks of IgG2A isoform positions 218-218, 219-219, 222-222 and 225-225, IgG2A / B isoform positions 218-130, 219-219, 222-222 and 225-225 and IgG2B isoform positions 219-130(2), 222-222 and 225-225; and intrachain heavy-chain light-chain disulfide crosslinks of IgG2A isoform positions 130-213'(2), IgG2A / B isoform positions 218-213' and 130-213' and IgG2B isoform position 218-213'(2). Preparations and characterizations of utomirumab and its variants and fragments are described in U.S. Patents 8,821,867; 8,337,850; and 9,468,678 and International Publication 2012 / 032433 A1, the respective disclosures of which are incorporated herein by reference. Preclinical characteristics of utomirumab are described in Fisher, et al., Cancer Immunolog. & Immunother. 2012, 61, 1721-33. Current clinical trials of utomirumab in various hematological and solid tumor indications include the National Institutes of Health Clinicaltrials.gov identifiers NCT02444793, NCT01307267, NCT02315066, and NCT02554812.

[0215]

[0010] In one embodiment, the 4-1BB agonist includes a heavy chain shown in SEQ ID NO: 11 and a light chain shown in SEQ ID NO: 12. In one embodiment, the 4-1BB agonist includes a heavy chain and a light chain having the sequences shown in SEQ ID NO: 11 and SEQ ID NO: 12, respectively, or an antigen-binding fragment, a Fab fragment, a single-chain variable fragment (scFv), a variant or conjugate thereof. In one embodiment, the 4-1BB agonist includes a heavy chain and a light chain that are at least 99% identical to the sequences shown in SEQ ID NO: 11 and SEQ ID NO: 12, respectively. In one embodiment, the 4-1BB agonist includes a heavy chain and a light chain that are at least 98% identical to the sequences shown in SEQ ID NO: 11 and SEQ ID NO: 12, respectively. In one embodiment, the 4-1BB agonist includes a heavy chain and a light chain that are at least 97% identical to the sequences shown in SEQ ID NO: 11 and SEQ ID NO: 12, respectively. In one embodiment, the 4-1BB agonist includes a heavy chain and a light chain that are at least 96% identical to the sequences shown in SEQ ID NO: 11 and SEQ ID NO: 12, respectively. In one embodiment, the 4-1BB agonist includes a heavy chain and a light chain that are at least 95% identical to the sequences shown in SEQ ID NO: 11 and SEQ ID NO: 12, respectively.

[0216]

[0011] In one embodiment, the 4-1BB agonist includes the heavy chain and light chain CDR or variable region (VR) of utomirumab. In one embodiment, the 4-1BB agonist heavy chain variable region (V H ) contains the sequence shown in Sequence ID No. 13, and the 4-1BB agonist light chain variable region (V L ) comprises the sequence shown in SEQ ID NO: 14 and its conserved amino acid substitutions. In one embodiment, the 4-1BB agonist is V, which is at least 99% identical to the sequences shown in SEQ ID NO: 13 and SEQ ID NO: 14, respectively. H and V L Includes a region. In one embodiment, the 4-1BB agonist is V, which is at least 98% identical to the sequences shown in SEQ ID NO: 13 and SEQ ID NO: 14, respectively. H and V LIncludes a region. In one embodiment, the 4-1BB agonist is at least 97% identical to the sequences shown in SEQ ID NO: 13 and SEQ ID NO: 14, respectively. H and V L Includes a region. In one embodiment, the 4-1BB agonist is at least 96% identical to the sequences shown in SEQ ID NO: 13 and SEQ ID NO: 14, respectively. H and V L Includes a region. In one embodiment, the 4-1BB agonist is at least 95% identical to the sequences shown in SEQ ID NO: 13 and SEQ ID NO: 14, respectively. H and V L Includes a region. In one embodiment, the 4-1BB agonist is V, which is at least 99% identical to the sequences shown in SEQ ID NO: 13 and SEQ ID NO: 14, respectively. H and V L Contains scFv antibody including the region.

[0217]

[0012] In one embodiment, the 4-1BB agonist comprises heavy chain CDR1, CDR2, and CDR3 domains having the sequences described in SEQ ID NO: 15, SEQ ID NO: 16, and SEQ ID NO: 17, and their conserved amino acid substitutions, and light chain CDR1, CDR2, and CDR3 domains having the sequences described in SEQ ID NO: 18, SEQ ID NO: 19, and SEQ ID NO: 20, and their conserved amino acid substitutions.

[0218]

[0013] In one embodiment, the 4-1BB agonist is a 4-1BB agonist biosimilar monoclonal antibody approved by a drug regulatory authority with respect to utomirumab. In one embodiment, the biosimilar monoclonal antibody comprises a 4-1BB antibody comprising an amino acid sequence having at least 97% sequence identity, e.g., 97%, 98%, 99%, or 100% sequence identity with respect to the amino acid sequence of a reference drug or reference biological product, and comprising one or more post-translational modifications compared to the reference drug or reference biological product, where the reference drug or reference biological product is utomirumab. In some embodiments, the one or more post-translational modifications are selected from one or more of glycosylation, oxidation, deamidation, and cleavage. In some embodiments, the biosimilar is a 4-1BB agonist antibody that has been approved or submitted for approval, and the 4-1BB agonist antibody is provided in a formulation different from the formulation of the reference drug or reference biological product, where the reference drug or reference biological product is utomirumab. 4-1BB agonist antibodies may be approved by drug regulatory authorities such as the US FDA and / or the European Union's EMA. In some embodiments, the biosimilar is provided as a composition further comprising one or more excipients, wherein one or more excipients are identical or different from those contained in a reference drug or reference biological product, the reference drug or reference biological product being utomirumab. In some embodiments, the biosimilar is provided as a composition further comprising one or more excipients, wherein one or more excipients are identical or different from those contained in a reference drug or reference biological product, the reference drug or reference biological product being utomirumab.

[0219] [Table 4]

[0220]

[0014] In preferred embodiments, the 4-1BB agonist is the monoclonal antibody urelumab, also known as BMS-663513 and 20H4.9.h4a, or a fragment, derivative, variant, or biosimilar thereof. Urelumab is available from Bristol-Myers Squibb, Inc. and Creative Biolabs, Inc. Urelumab is an immunoglobulin G4-kappa, anti-[human (Homo sapiens) TNFRSF9 (tumor necrosis factor receptor superfamily member 9, 4-1BB, T cell antigen ILA, CD137)], human (Homo sapiens) (fully human) monoclonal antibody. The amino acid sequence of urelumab is shown in Table 5. Urelumab has an N-glycosylation site at position 298 (and 298''); position 22-95 (V H -V L ), 148-204(C H 1-C L ), 262-322(C H 2) and 368-426(C H 3) Heavy chain disulfide bridges at positions 22''-95'', 148''-204'', 262''-322'' and 368''-426''; position 23''-88''(V H -V L ) and 136'-196'(C H 1-C LThe compound includes light chain intra-disulfide bridges at positions 23'''-88'' and 136'''-196'''; intra-chain heavy chain-heavy chain disulfide bridges at positions 227-227'' and 230-230''; and intra-chain heavy chain-light chain disulfide bridges at positions 135-216'' and 135''-216''''. Preparations and characterization of urelumab and its variants and fragments are described in U.S. Patents No. 7,288,638 and No. 8,962,804, the disclosures of which are incorporated herein by reference. The preclinical and clinical characteristics of urelumab are described in Segal, et al., Clin. Cancer Res. 2016, available at http: / dx.doi.org / 10.1158 / 1078-0432.CCR-16-1272. Current clinical trials of urelumab for various hematological and solid tumor indications include the National Institutes of Health Clinicaltrials.gov identifiers NCT01775631, NCT02110082, NCT02253992, and NCT01471210.

[0221]

[0015] In one embodiment, the 4-1BB agonist includes a heavy chain shown in SEQ ID NO: 21 and a light chain shown in SEQ ID NO: 22. In one embodiment, the 4-1BB agonist includes a heavy chain and a light chain having the sequences shown in SEQ ID NO: 21 and SEQ ID NO: 22, respectively, or an antigen-binding fragment, a Fab fragment, a single-chain variable fragment (scFv), a variant or conjugate thereof. In one embodiment, the 4-1BB agonist includes a heavy chain and a light chain that are at least 99% identical to the sequences shown in SEQ ID NO: 21 and SEQ ID NO: 22, respectively. In one embodiment, the 4-1BB agonist includes a heavy chain and a light chain that are at least 98% identical to the sequences shown in SEQ ID NO: 21 and SEQ ID NO: 22, respectively. In one embodiment, the 4-1BB agonist includes a heavy chain and a light chain that are at least 97% identical to the sequences shown in SEQ ID NO: 21 and SEQ ID NO: 22, respectively. In one embodiment, the 4-1BB agonist includes a heavy chain and a light chain that are at least 96% identical to the sequences shown in SEQ ID NO: 21 and SEQ ID NO: 22, respectively. In one embodiment, the 4-1BB agonist includes a heavy chain and a light chain that are at least 95% identical to the sequences shown in SEQ ID NO: 21 and SEQ ID NO: 22, respectively.

[0222]

[0016] In one embodiment, the 4-1BB agonist includes the heavy chain and light chain CDR or variable region (VR) of urelumab. In one embodiment, the 4-1BB agonist heavy chain variable region (V H ) contains the sequence shown in Sequence ID No. 23, and the 4-1BB agonist light chain variable region (V L ) comprises the sequence shown in SEQ ID NO: 24 and its conserved amino acid substitutions. In one embodiment, the 4-1BB agonist is V, which is at least 99% identical to the sequences shown in SEQ ID NO: 23 and SEQ ID NO: 24, respectively. H and V L Includes a region. In one embodiment, the 4-1BB agonist is V, which is at least 98% identical to the sequences shown in SEQ ID NO: 23 and SEQ ID NO: 24, respectively. H and V LIncludes a region. In one embodiment, the 4-1BB agonist is V, which is at least 97% identical to the sequences shown in SEQ ID NO: 23 and SEQ ID NO: 24, respectively. H and V L Includes a region. In one embodiment, the 4-1BB agonist is at least 96% identical to the sequences shown in SEQ ID NO: 23 and SEQ ID NO: 24, respectively. H and V L Includes a region. In one embodiment, the 4-1BB agonist is V, which is at least 95% identical to the sequences shown in SEQ ID NO: 23 and SEQ ID NO: 24, respectively. H and V L Includes a region. In one embodiment, the 4-1BB agonist is V, which is at least 99% identical to the sequences shown in SEQ ID NO: 23 and SEQ ID NO: 24, respectively. H and V L Contains scFv antibody including the region.

[0223]

[0017] In one embodiment, the 4-1BB agonist comprises heavy chain CDR1, CDR2, and CDR3 domains having the sequences described in SEQ ID NO: 25, SEQ ID NO: 26, and SEQ ID NO: 27, and their conserved amino acid substitutions, and light chain CDR1, CDR2, and CDR3 domains having the sequences described in SEQ ID NO: 28, SEQ ID NO: 29, and SEQ ID NO: 30, and their conserved amino acid substitutions.

[0224]

[0018] In one embodiment, the 4-1BB agonist is a 4-1BB agonist biosimilar monoclonal antibody approved by a drug regulatory authority with respect to urelumab. In one embodiment, the biosimilar monoclonal antibody comprises a 4-1BB antibody comprising an amino acid sequence having at least 97% sequence identity, e.g., 97%, 98%, 99%, or 100% sequence identity with respect to the amino acid sequence of a reference drug or reference biological product, and comprising one or more post-translational modifications compared to the reference drug or reference biological product, where the reference drug or reference biological product is urelumab. In some embodiments, the one or more post-translational modifications are selected from one or more of glycosylation, oxidation, deamidation, and cleavage. In some embodiments, the biosimilar is a 4-1BB agonist antibody that has been approved or submitted for approval, and the 4-1BB agonist antibody is provided in a formulation different from the formulation of the reference drug or reference biological product, where the reference drug or reference biological product is urelumab. 4-1BB agonist antibodies may be approved by drug regulatory authorities such as the U.S. FDA and / or the European Union's EMA. In some embodiments, the biosimilar is provided as a composition further comprising one or more excipients, wherein one or more excipients are identical or different from those contained in a reference drug or reference biological product, and the reference drug or reference biological product is urelumab. In some embodiments, the biosimilar is provided as a composition further comprising one or more excipients, wherein one or more excipients are identical or different from those contained in a reference drug or reference biological product, and the reference drug or reference biological product is urelumab.

[0225] [Table 5]

[0226]

[0019] In one embodiment, the 4-1BB agonist is an antibody produced by the cell line disclosed in U.S. Patent No. 6,974,863, deposited as 1D8, 3Elor, 4B4 (BioLegend 309809), H4-1BB-M127 (BD Pharmingen 552532), BBK2 (Thermo Fisher MS621PABX), 145501 (Leinco Technologies B591), ATCC number HB-11248, 5F4 (BioLegend 31 1503), C65-485 (BD Pharmingen Antibodies disclosed in U.S. Patent Application Publication No. 2005 / 0095244 (559446), antibodies disclosed in U.S. Patent No. 7,288,638 (such as 20H4.9-IgGl (BMS-663031)), antibodies disclosed in U.S. Patent No. 6,887,673 (such as 4E9 or BMS-554271), antibodies disclosed in U.S. Patent No. 7,214,493, antibodies disclosed in U.S. Patent No. 6,303,121, antibodies disclosed in U.S. Patent No. 6,569,997, antibodies disclosed in U.S. Patent No. 6,905,685 (such as 4E9 or BMS-554271), antibodies disclosed in U.S. Patent No. 6,362,325 (1D8 or BMS-469492; 3H3 or BMS-4694 A selection from the group consisting of antibodies such as 97; or 3El (etc.), antibodies disclosed in U.S. Patent No. 6,974,863 (such as 53A2); antibodies disclosed in U.S. Patent No. 6,210,669 (such as 1D8, 3B8, or 3El); antibodies described in U.S. Patent No. 5,928,893; antibodies disclosed in U.S. Patent No. 6,303,121; antibodies disclosed in U.S. Patent No. 6,569,997; antibodies disclosed in International Publication No. 2012 / 177788, International Publication No. 2015 / 119923, and International Publication No. 2010 / 042433, and their fragments, derivatives, conjugates, variants, or biosimilars, wherein each of the aforementioned patents or patent application publications is incorporated herein by reference.

[0227]

[0020] In one embodiment, the 4-1BB agonist is as described in International Publication Nos. 2008 / 025516 A1, 2009 / 007120 A1, 2010 / 003766 A1, 2010 / 010051 A1 and 2010 / 078966 A1; U.S. Patent Application Publication Nos. 2011 / 0027218 A1, 2015 / 0126709 A1, 2011 / 0111494 A1, 2015 / 0110734 A1 and 2015 / 0126710 A1; and the 4-1BB agonist fusion proteins described in U.S. Patents 9,359,420, 9,340,599, 8,921,519 and 8,450,460, the disclosures thereof are incorporated herein by reference.

[0228]

[0021] In one embodiment, the 4-1BB agonist is a 4-1BB agonist fusion protein represented by structure IA (C-terminal Fc antibody fragment fusion protein) or structure IB (N-terminal Fc antibody fragment fusion protein), or a fragment, derivative, conjugate, variant, or biosimilar thereof: In structures IA and IB, the cylinders refer to individual polypeptide-binding domains (see Figure 140). Structures IA and IB contain three linearly linked TNFRSF-binding domains derived, for example, from an antibody that binds to 4-1BBL or 4-1BB, which fold to form a trivalent protein, and then IgG1-Fc(C) H 3 and C H The TNFRSF-binding domain, shown as a cylinder, is linked to a second trivalent protein via a V-linker, which is then used to link the two trivalent proteins via a disulfide bond (small, elongated oval), stabilizing the structure and providing an agonist capable of combining the intracellular signaling domains and signaling proteins of the six receptors to form a signaling complex. The TNFRSF-binding domain is linked by a linker that may, for example, contain hydrophilic residues as well as Gly and Ser sequences for flexibility and Glu and Lys for solubility. H and V LIt may be an scFv domain including a chain. Any scFv domain design may be used, such as those described in de Marco, Microbial Cell Factories, 2011, 10, 44; Ahmad, et al., Clin. & Dev.Immunol. 2012, 980250; Monnier, et al., Antibodies, 2013, 2, 193-208; or references incorporated elsewhere in this specification. Fusion protein structures in this form are described in U.S. Patents 9,359,420, 9,340,599, 8,921,519 and 8,450,460, the disclosures of which are incorporated herein by reference.

[0229]

[0022] The amino acid sequences of other polypeptide domains of structure IA are shown in Table 6. The Fc domain preferably comprises a complete constant domain (amino acids 17-230 of SEQ ID NO: 31), a complete hinge domain (amino acids 1-16 of SEQ ID NO: 31), or a portion of a hinge domain (e.g., amino acids 4-16 of SEQ ID NO: 31). A preferred linker for affixing the C-terminal Fc antibody may be selected from the embodiments shown in SEQ ID NOs. 32-41, which include a linker suitable for fusion of additional polypeptides.

[0230] [Table 6]

[0231]

[0023] Table 7 shows the amino acid sequences of other polypeptide domains of structure IB. When the Fc antibody fragment is fused to the N-terminus of the TNRFSF fusion protein as in structure IB, the sequence of the Fc module is preferably the one shown in SEQ ID NO: 42, and the linker sequence is preferably selected from the embodiments shown in SEQ ID NOs: 43 to 45.

[0232] [Table 7]

[0233]

[0024] In one embodiment, the 4-1BB agonist fusion protein according to structure IA or IB comprises a variable heavy chain and a variable light chain of utomirumab, a variable heavy chain and a variable light chain of urerumab, a variable heavy chain and a variable light chain of utomirumab, a variable heavy chain and a variable light chain selected from the variable heavy chains and variable light chains listed in Table 8, any combination of the above variable heavy chains and variable light chains, and one or more 4-1BB binding domains selected from the group consisting of fragments, derivatives, conjugates, variants and biosimilars thereof.

[0234]

[0025] In one embodiment, the 4-1BB agonist fusion protein according to structure IA or IB includes one or more 4-1BB binding domains containing a 4-1BBL sequence. In one embodiment, the 4-1BB agonist fusion protein according to structure IA or IB includes one or more 4-1BB binding domains containing the sequence according to SEQ ID NO: 46. In one embodiment, the 4-1BB agonist fusion protein according to structure IA or IB includes one or more 4-1BB binding domains containing a soluble 4-1BBL sequence. In one embodiment, the 4-1BB agonist fusion protein according to structure IA or IB includes one or more 4-1BB binding domains containing the sequence according to SEQ ID NO: 47.

[0235]

[0026] In one embodiment, the 4-1BB agonist fusion protein with structure IA or IB is at least 95% identical to the sequences shown in SEQ ID NO: 13 and SEQ ID NO: 14, respectively. H and V L It contains one or more 4-1BB binding domains which are scFv domains containing the region, where V H and V L The domains are linked by a linker. In one embodiment, the 4-1BB agonist fusion protein with structure IA or IB is at least 95% identical to the sequences shown in SEQ ID NO: 23 and SEQ ID NO: 24, respectively. H and V L It contains one or more 4-1BB binding domains which are scFv domains containing the region, where V H and V LThe domains are connected by a linker. In one embodiment, the 4-1BB agonist fusion protein with structure IA or IB is shown in Table 8. H and V L V is at least 95% identical to the sequence. H and V L It contains one or more 4-1BB binding domains which are scFv domains containing the region, where V H and V L Domains are connected by linkers.

[0236] [Table 8]

[0237]

[0027] In one embodiment, the 4-1BB agonist is a 4-1BB agonist single-chain fusion polypeptide comprising (i) a first soluble 4-1BB binding domain, (ii) a first peptide linker, (iii) a second soluble 4-1BB binding domain, (iv) a second peptide linker, and (v) a third soluble 4-1BB binding domain, further comprising an additional domain at the N-terminus and / or C-terminus, wherein the additional domain is a Fab or Fc fragment domain. In one embodiment, the 4-1BB agonist is a 4-1BB agonist single-chain fusion polypeptide comprising (i) a first soluble 4-1BB binding domain, (ii) a first peptide linker, (iii) a second soluble 4-1BB binding domain, (iv) a second peptide linker, and (v) a third soluble 4-1BB binding domain, further comprising additional domains at the N-terminus and / or C-terminus, wherein the additional domains are Fab or Fc fragment domains, each of the soluble 4-1BB domains lacking a stalk region (which contributes to trimerization and provides a certain distance to the cell membrane but is not part of the 4-1BB binding domain), and the first and second peptide linkers independently having a length of 3 to 8 amino acids.

[0238]

[0028] In one embodiment, the 4-1BB agonist is a 4-1BB agonist single-chain fusion polypeptide comprising (i) a first soluble tumor necrosis factor (TNF) superfamily cytokine domain, (ii) a first peptide linker, (iii) a second soluble TNF superfamily cytokine domain, (iv) a second peptide linker, and (v) a third soluble TNF superfamily cytokine domain, where each soluble TNF superfamily cytokine domain lacks a stalk region, the first and second peptide linkers independently have a length of 3 to 8 amino acids, and each TNF superfamily cytokine domain is a 4-1BB binding domain.

[0239]

[0029] In one embodiment, the 4-1BB agonist is the V L The aforementioned V linked to any of the domains H It is a 4-1BB agonist scFv antibody containing one of the domains.

[0240]

[0030] In one embodiment, the 4-1BB agonist is BPS Bioscience 4-1BB agonist antibody catalog number 79097-2, which is commercially available from BPS Bioscience, San Diego, CA, USA. In one embodiment, the 4-1BB agonist is Creative Biolabs 4-1BB agonist antibody catalog number MOM-18179, which is commercially available from Creative Biolabs, Shirley, NY, USA.

[0241] Manufacturing method of Sch.TIL

[0341] An example of a TIL process known as Process 2A, which includes some of these features, is shown in Figure 1, and some of the advantages of this embodiment of the present invention over Process 1C are shown in Figure 2, as well as in Figure 84. Process 1C is shown in Figure 3 for comparison. Two alternative timelines for TIL treatment based on Process 2A are shown in Figure 4 (high cell count) and Figure 5 (low cell count). Embodiments of Process 2A are shown in Figures 6 and 27. Figures 83 and 84 further provide an exemplary 2A process compared to an exemplary 1C process.

[0242]

[0342] As discussed herein, the present invention may include steps relating to increasing the metabolic activity and, consequently, the relative health of cryopreserved TILs by restimulating them before transplantation into a patient, and methods for testing said metabolic health. Generally as outlined herein, TILs are generally collected from patient samples and manipulated to increase their number before transplantation into a patient. In some embodiments, TILs may optionally be genetically engineered as discussed below.

[0243]

[0343] In some embodiments, TILs may be cryopreserved. After thawing, TILs may be restimulated to increase their metabolism before injection into the patient.

[0244]

[0344] In some embodiments, as will be discussed in detail below and in the examples and figures, the first expansion culture (including a process referred to as pre-REP and the process shown as step A in Figure 27) is shortened to 3 to 14 days, and the second expansion culture (including a process referred to as REP and the process shown as step B in Figure 27) is shortened to 7 to 14 days. In some embodiments, as will be discussed in the examples and shown in Figures 4, 5 and 27, the first expansion culture (e.g., the expansion culture described as step B in Figure 27) is shortened to 11 days, and the second expansion culture (e.g., the expansion culture described as step D in Figure 27) is shortened to 11 days. In some embodiments, as will be discussed in detail below and in the examples and figures, the combination of the first and second expansion cultures (e.g., the expansion cultures described as steps B and D in Figure 27) is shortened to 22 days.

[0245]

[0345] The following "Step" names A, B, C, etc., refer to Figure 27 and the embodiments described herein. The order of steps below and in Figure 27 is illustrative, and any combination or order of steps, as well as additional steps, repetition of steps and / or omission of steps, are intended by the methods disclosed herein and herein.

[0246] A. Step A: Obtain patient tumor samples.

[0346] Generally, TILs are first obtained from patient tumor samples ("primary TILs"), then cultured in larger populations for further manipulation as described herein, optionally cryopreserved, restimulated as outlined herein, and optionally assessed for phenotypic and metabolic parameters as indicators of TIL health.

[0247]

[0347] Patient tumor samples can generally be obtained by surgical resection, needle biopsy, or other means for obtaining a sample containing a mixture of tumor and TIL cells, using methods known in the art. Generally, tumor samples may be from any solid tumor, including primary tumors, invasive tumors, or metastatic tumors. Tumor samples may also be humoral tumors, such as tumors obtained from hematological malignancies. Solid tumors may be from any type of cancer, including, but are not limited to, breast cancer, pancreatic cancer, prostate cancer, colorectal cancer, lung cancer, brain cancer, kidney 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, particularly due to reports of high levels of TILs.

[0248]

[0348] The term "solid tumor" refers to an abnormal tissue mass that does not typically contain cysts or fluid areas. Solid tumors can be benign or malignant. The term "solid tumor carcinoma" refers to a malignant, neoplastic, or cancerous solid tumor. Examples of solid tumor carcinomas include, but are not limited to, lung cancer, breast cancer, triple-negative breast cancer, prostate cancer, colon cancer, rectal cancer, and bladder cancer, as well as sarcomas, carcinomas, and lymphomas. In some embodiments, 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 tissue structure of a solid tumor includes interdependent tissue compartments comprising parenchyma (cancer cells) and supporting stromal cells where cancer cells are dispersed and which can provide a supporting microenvironment.

[0249]

[0349] The term "hematological malignancies" refers to cancers and tumors of hematopoietic and lymphoid tissues in mammals, including, but not limited to, blood, bone marrow, lymph nodes, and lymphoid tissues. Hematological malignancies are also called "humoral tumors." Examples of hematological malignancies, but not limited to, include acute lymphoblastic leukemia (ALL), chronic lymphocytic lymphoma (CLL), small lymphocytic lymphoma (SLL), acute myeloid leukemia (AML), chronic myeloid leukemia (CML), acute monocytic leukemia (AMoL), Hodgkin lymphoma, and non-Hodgkin lymphoma. The term "B-cell hematological malignancies" refers to hematological malignancies that affect B cells.

[0250]

[0350] After acquisition, tumor specimens are generally dissected using sharp dissection to a depth of 1 to approximately 8 mm. 3 It is fragmented into small pieces, about 2-3 mm in size. 3 This is particularly useful. TILs are cultured from these fragments using enzymatic tumor digests. Such tumor digests can be prepared by incubation in an enzymatic medium (e.g., Roswell Park Memorial Laboratory (RPMI) 1640 buffer, 2 mM glutamate, 10 mcg / mL gentamicin, 30 units / mL DNase, and 1.0 mg / mL collagenase) followed by mechanical dissociation (e.g., using a tissue dissociation device). Tumor digests can be prepared by placing the tumor in the enzymatic medium, mechanically dissociating the tumor for about 1 minute, then incubating at 37°C under 5% CO2 for 30 minutes, and then repeating the cycle of mechanical dissociation and incubation under the above conditions until only very small tissue fragments remain. If the cell suspension contains a large number of erythrocytes or dead cells at the end of this process, these cells can be removed by density gradient separation using FICOLL branched hydrophilic polysaccharides. Alternative methods known in the art may be used, such as those described in U.S. Patent Application Publication No. 2012 / 0244133 A1 (this disclosure is incorporated herein by reference). Any of the aforementioned methods can be used in any embodiment described herein relating to methods for expanding TIL culture or treating cancer.

[0251]

[0351] Generally, a collected cell suspension is called a "primary cell population" or a "freshly collected" cell population.

[0252]

[0352] In some embodiments, fragmentation includes physical fragmentation, such as detachment and digestion. In some embodiments, fragmentation is physical fragmentation. In some embodiments, fragmentation is detachment. In some embodiments, fragmentation is by digestion. In some embodiments, TILs can be cultured initially from enzymatic tumor digests and tumor fragments obtained from a patient. In one embodiment, TILs can be cultured initially from enzymatic tumor digests and tumor fragments obtained from a patient.

[0253]

[0353] In some embodiments, if the tumor is a solid tumor, after obtaining the tumor sample in step A (provided in Figure 27), for example, the tumor undergoes physical fragmentation. In some embodiments, fragmentation is performed before cryopreservation. In some embodiments, fragmentation is performed after cryopreservation. In some embodiments, fragmentation is performed 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 the first expansion culture. In some embodiments, the tumor is fragmented and 30 or 40 fragments or pieces are placed in each container for the first expansion culture. In some embodiments, the tumor is fragmented and 40 fragments or pieces are placed in each container for the first expansion culture. In some embodiments, the fragments consist of about 4 to about 50 fragments, each fragment being about 27 mm. 3 It has a volume of approximately 1300 mm. In some embodiments, the multiple fragments are approximately 1300 mm 3 ~approx. 1500mm 3 It contains approximately 30 to 60 fragments with a total volume of approximately 1350 mm. In some embodiments, the fragments are approximately 1350 mm 3 It contains about 50 fragments having a total volume of . In some embodiments, the multiple fragments consist of about 50 fragments having a total mass of about 1 g to about 1.5 g. In some embodiments, the multiple fragments consist of about 4 fragments.

[0254]

[0354] In some embodiments, the TIL is obtained from a tumor fragment. In some embodiments, the tumor fragment is obtained by sharp dissection. In some embodiments, the tumor fragment is approximately 1 mm in size. 3 ~10mm 3 In some embodiments, the tumor fragment is approximately 1 mm. 3 ~8mm 3 In some embodiments, the tumor fragment is approximately 1 mm. 3 In some embodiments, the tumor fragment is approximately 2 mm in size. 3 In some embodiments, the tumor fragment is approximately 3 mm. 3 In some embodiments, the tumor fragment is approximately 4 mm in size. 3 In some embodiments, the tumor fragment is approximately 5 mm. 3 In some embodiments, the tumor fragment is approximately 6 mm. 3 In some embodiments, the tumor fragment is approximately 7 mm. 3 In some embodiments, the tumor fragment is approximately 8 mm. 3 In some embodiments, the tumor fragment is approximately 9 mm. 3 In some embodiments, the tumor fragment is approximately 10 mm. 3 In some embodiments, the tumor is 1-4 mm × 1-4 mm × 1-4 mm. In some embodiments, the tumor is 1 mm × 1 mm × 1 mm. In some embodiments, the tumor is 2 mm × 2 mm × 2 mm. In some embodiments, the tumor is 3 mm × 3 mm × 3 mm. In some embodiments, the tumor is 4 mm × 4 mm × 4 mm.

[0255]

[0355] In some embodiments, the tumor is excised to minimize the amount of bleeding, necrosis, and / or fatty tissue on each fragment. In some embodiments, the tumor is excised to minimize the amount of bleeding tissue on each fragment. In some embodiments, the tumor is excised to minimize the amount of necrotic tissue on each fragment. In some embodiments, the tumor is excised to minimize the amount of fatty tissue on each fragment.

[0256]

[0356] In some embodiments, tumor fragmentation is performed to preserve the internal structure of the tumor. In some embodiments, tumor fragmentation is performed without performing a sawing motion with a scalpel. In some embodiments, TIL is obtained from tumor digestate. In some embodiments, tumor digestate is prepared by incubation in an enzyme medium, for example, 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). After placing the tumor in the enzyme medium, 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 mechanically dissociated again for about 1 minute. After incubation again at 37°C under 5% CO2 for 30 minutes, the tumor may be mechanically dissociated a third time for about 1 minute. In some embodiments, if large tissue fragments were present after the third mechanical disruption, one or two further mechanical dissociations were applied to the sample, with or without a further 30-minute incubation at 37°C under 5% CO2. In some embodiments, if the cell suspension contained a large number of erythrocytes or dead cells at the end of the final incubation, such cells could be removed by density gradient separation using Ficoll.

[0257]

[0357] In some embodiments, the cell suspension collected before the first expansion culture step is referred to as the “primary cell population” or “freshly collected” cell population.

[0258]

[0358] In some embodiments, the cells may be optionally frozen after sampling and stored frozen before proceeding to the expansion culture described in step B, similarly illustrated in Figure 27, which is described in more detail below.

[0259] B. Step B: First Expansion Culture 1.Young TIL

[0359] In some embodiments, this method provides the acquisition of immature TILs that can increase the replication cycle upon administration to the subject / patient, and thus may provide additional therapeutic benefits beyond mature TILs (i.e., TILs that have undergone more replication cycles before administration to the subject / patient). The characteristics of immature TILs are described in the literature. For example, Donia, at al., Scandinavian Journal of Immunology, 75:157-167 (2012), Dudley et al., Clin Cancer Res, 16:6122-6131 (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 are incorporated herein by reference in their entirety.

[0260]

[0360] Diverse antigen receptors for T and B lymphocytes are produced by somatic recombination of a limited but numerous gene segment. These gene segments V (variable), D (diversity), J (binding), and C (constant) determine the binding specificity and downstream application of immunoglobulins and T cell receptors (TCRs). The present invention provides a method for producing TILs that exhibit and increase T cell repertoire diversity. In some embodiments, TILs obtained by the present method exhibit increased T cell repertoire diversity. In some embodiments, TILs obtained by the present method 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 Figure 27. In some embodiments, as illustrated in Figure 83, TILs obtained by the present method exhibit increased T cell repertoire diversity compared to freshly harvested TILs and / or TILs prepared using a method referred to as process 1C. In some embodiments, TILs obtained in a 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 lies in the immunoglobulin heavy chain. In some embodiments, the immunoglobulin diversity lies in the immunoglobulin light chain. In some embodiments, the diversity lies in the T cell receptor. In some embodiments, the diversity lies in one of the T cell receptors selected from the group consisting of alpha, beta, gamma, and delta receptors. In some embodiments, the expression of T cell receptor (TCR) alpha and / or beta is increased. In some embodiments, the expression of T cell receptor (TCR) alpha is increased. In some embodiments, the expression of T cell receptor (TCR) beta is increased. In some embodiments, the expression of TCRab (i.e., TCRα / β) is increased.

[0261]

[0361] After detachment or digestion of the tumor fragments, the resulting cells are cultured in serum-containing IL-2 under conditions more favorable to TIL growth than tumor and other cells, for example, as described in step A of Figure 27. In some embodiments, the tumor digest is incubated in 2 mL wells in a medium containing inactivated human AB serum with 6000 IU / mL of IL-2. This primary cell population is cultured for several days, generally 3 to 14 days, thereby growing a bulk TIL population, generally about 1 × 10⁶. 8 A bulk TIL population of approximately 1 × 10¹⁶ cells is obtained. In some embodiments, this primary cell population is cultured for a period of 7 to 14 days, thereby yielding a bulk TIL population, generally about 1 × 10¹⁶ cells. 8 A bulk TIL population of approximately 1 × 10¹⁶ cells is obtained. In some embodiments, this primary cell population is cultured for a period of 10–14 days, thereby yielding a bulk TIL population, generally about 1 × 10¹⁶ cells. 8 A bulk TIL population of approximately 1 × 10¹⁶ cells is obtained. In some embodiments, this primary cell population is cultured for a period of approximately 11 days, thereby yielding a bulk TIL population, generally about 1 × 10¹⁶ cells. 8 Individual bulk TIL cells are obtained.

[0262]

[0362] In a preferred embodiment, TIL expansion culture can be carried out using an initial bulk TIL expansion culture step (e.g., as described in step B of Figure 27, which may include a process referred to as pre-REP), followed by a second expansion culture (including a process referred to as step D, the Rapid Expansion Protocol (REP) step), as described below under step D and herein, followed by optional cryopreservation and a subsequent second step D (including a process referred to as the restimulation REP step). The TILs obtained by this process may optionally be characterized with phenotypic features and metabolic parameters as described herein.

[0263]

[0363] In an embodiment, if TIL culture is initiated in a 24-well plate, for example using a Costar 24-well cell culture cluster, flat-bottom (Corning Incorporated, Corning, NY), each well contains 1 × 10¹⁶ IL-2 (6000 IU / mL; Chiron Corp., Emeryville, CA) in 2 mL of complete medium (CM). 6 Individual tumor digestive cells or a single tumor fragment can be seeded. In some embodiments, the tumor fragment is approximately 1 mm in size. 3 ~10mm 3 That is the case.

[0264]

[0364] In some embodiments, the first expanded culture medium is referred to as "CM," an abbreviation for culture medium. In some embodiments, the CM in step B consists of GlutaMAX-containing RPMI 1640 supplemented with 10% human AB serum, 25 mM hepes, and 10 mg / mL gentamicin. The culture is in a 40 mL volume and 10 cm³. 2 In embodiments initiated in a gas-permeable silicon-bottomed gas-permeable flask (e.g., G-Rex10; Wilson Wolf Manufacturing, New Brighton, MN) (Figure 1), each flask contains 10-40 mL of IL-2-containing CM, with 10-40 × 10 6 Each cell was loaded with either 10 tumor digestive cells or 5-30 tumor fragments. Both G-Rex10 and 24-well plates were incubated in a humidified incubator at 37°C under 5% CO2. Five days after the start of culture, half of the medium was removed and replenished with fresh CM and IL-2. From day five onward, half of the medium was replaced every 2-3 days.

[0265]

[0365] Following the preparation of tumor fragments, the resulting cells (i.e., fragments) are cultured in serum-containing IL-2 under conditions more favorable to TIL growth than tumor and other cells. In some embodiments, tumor digests are incubated in 2 mL wells in a medium containing inactivated human AB serum with 6000 IU / mL of IL-2 (or, if applicable, 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 growing a bulk TIL population, generally about 1 × 10⁶ 8 A bulk TIL cell count is obtained. In some embodiments, the growth medium during the first expansion culture contains IL-2 or a variant thereof. In some embodiments, IL is recombinant human IL-2 (rhIL-2). In some embodiments, the IL-2 stock solution contains 20-30 × 10⁶ cells per 1 mg vial. 6 It has a specific activity of IU / mg. In some embodiments, the IL-2 stock solution is 20 to 10 times per 1 mg vial. 6 It has a specific activity of IU / mg. In some embodiments, the IL-2 stock solution is 25 × 10⁶ per 1 mg vial. 6 It has a specific activity of IU / mg. In some embodiments, the IL-2 stock solution is 30 × 10⁶ per 1 mg vial. 6 It has a specific activity of IU / mg. In some embodiments, the IL-2 stock solution is 4-8 × 10⁻⁶. 6 The final concentration of IL-2 is IU / mg. In some embodiments, the IL-2 stock solution is 5-7 × 10⁻⁶. 6 It has a final concentration of IL-2 IU / mg. In some embodiments, the IL-2 stock solution is 6 × 10⁻⁶ 6The final concentration of IL-2 is IU / mg. In some embodiments, the IL-2 stock solution is prepared as described in Example 4. In some embodiments, the first expanded culture medium contains about 10,000 IU / mL of IL-2, about 9,000 IU / mL of IL-2, about 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 expanded 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 expanded 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 expanded 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 expanded culture medium contains about 6,000 IU / mL of IL-2. In some 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 contains IL-2. In some embodiments, the cell culture medium contains about 3,000 IU / mL of IL-2. In one embodiment, the cell culture medium contains IL-2 at approximately 1000 IU / mL, 1500 IU / mL, 2000 IU / mL, 2500 IU / mL, 3000 IU / mL, 3500 IU / mL, 4000 IU / mL, 4500 IU / mL, 5000 IU / mL, 5500 IU / mL, 6000 IU / mL, 6500 IU / mL, 7000 IU / mL, 7500 IU / mL, or 8000 IU / mL. In one embodiment, the cell culture medium contains IL-2 in concentrations of 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 approximately 8000 IU / mL.

[0266]

[0366] In some embodiments, the first expanded culture medium contains approximately 500 IU / mL of IL-15, approximately 400 IU / mL of IL-15, approximately 300 IU / mL of IL-15, approximately 200 IU / mL of IL-15, approximately 180 IU / mL of IL-15, approximately 160 IU / mL of IL-15, approximately 140 IU / mL of IL-15, approximately 120 IU / mL of IL-15, or approximately 100 IU / mL of IL-15. In some embodiments, the first expanded culture medium contains approximately 500 IU / mL of IL-15 to approximately 100 IU / mL of IL-15. In some embodiments, the first expanded culture medium contains approximately 400 IU / mL of IL-15 to approximately 100 IU / mL of IL-15. In some embodiments, the first expanded culture medium contains approximately 300 IU / mL of IL-15 to approximately 100 IU / mL of IL-15. In some embodiments, the first expanded culture medium contains about 200 IU / mL of IL-15. In some embodiments, the cell culture medium contains about 180 IU / mL of IL-15. In some embodiments, the cell culture medium further contains IL-15. In a preferred embodiment, the cell culture medium contains about 180 IU / mL of IL-15.

[0267]

[0367] In some embodiments, the first expanded culture medium contains approximately 20 IU / mL of IL-21, approximately 15 IU / mL of IL-21, approximately 12 IU / mL of IL-21, approximately 10 IU / mL of IL-21, approximately 5 IU / mL of IL-21, approximately 4 IU / mL of IL-21, approximately 3 IU / mL of IL-21, approximately 2 IU / mL of IL-21, approximately 1 IU / mL of IL-21, or approximately 0.5 IU / mL of IL-21. In some embodiments, the first expanded culture medium contains approximately 20 IU / mL of IL-21 to approximately 0.5 IU / mL of IL-21. In some embodiments, the first expanded culture medium contains approximately 15 IU / mL of IL-21 to approximately 0.5 IU / mL of IL-21. In some embodiments, the first expanded culture medium contains approximately 12 IU / mL of IL-21 to approximately 0.5 IU / mL of IL-21. In some embodiments, the first expanded culture medium contains approximately 10 IU / mL of IL-21 to approximately 0.5 IU / mL of IL-21. In some embodiments, the first expanded culture medium contains approximately 5 IU / mL of IL-21 to approximately 1 IU / mL of IL-21. In some embodiments, the first expanded culture medium contains approximately 2 IU / mL of IL-21. In some embodiments, the cell culture medium contains approximately 1 IU / mL of IL-21. In some embodiments, the cell culture medium contains approximately 0.5 IU / mL of IL-21. In one embodiment, the cell culture medium further contains IL-21. In a preferred embodiment, the cell culture medium contains approximately 1 IU / mL of IL-21.

[0268]

[0368] In one embodiment, the cell culture medium contains OKT-3 antibody. In some embodiments, the cell culture medium contains approximately 30 ng / mL of OKT-3 antibody. In one embodiment, the cell culture medium contains approximately 0.1 ng / mL, approximately 0.5 ng / mL, approximately 1 ng / mL, approximately 2.5 ng / mL, approximately 5 ng / mL, approximately 7.5 ng / mL, approximately 10 ng / mL, approximately 15 ng / mL, approximately 20 ng / mL, approximately 25 ng / mL, approximately 30 ng / mL, approximately 35 ng / mL, approximately 40 ng / mL, approximately 50 ng / mL, approximately 60 ng / mL, approximately 70 ng / mL, approximately 80 ng / mL, approximately 90 ng / mL, approximately 100 ng / mL, approximately 200 ng / mL, approximately 500 ng / mL, and approximately 1 μg / mL of OKT-3 antibody. In one embodiment, the cell culture medium contains OKT-3 antibodies in concentrations of 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. In some embodiments, the cell culture medium does not contain OKT-3 antibodies.

[0269]

[0369] In some embodiments, the cell culture medium contains one or more TNFRSF agonists. In some embodiments, the TNFRSF agonist contains a 4-1BB agonist. In some embodiments, the TNFRSF agonist is a 4-1BB agonist, which is selected from the group consisting of urelumab, utomirumab, EU-101, fusion proteins and their fragments, derivatives, variants, biosimilars and combinations. In some embodiments, the TNFRSF agonist is added to the cell culture medium at a concentration sufficient to achieve a concentration of 0.1 μg / mL to 100 μg / mL. In some embodiments, the TNFRSF agonist is added to the cell culture medium at a concentration sufficient to achieve a concentration of 20 μg / mL to 40 μg / mL.

[0270]

[0370] In some embodiments, in addition to one or more TNFRSFs, the cell culture medium further contains IL-2 at an initial concentration of approximately 3000 IU / mL and OKT-3 antibody at an initial concentration of approximately 30 ng / mL, and one or more TNFRSF agonists contain 4-1BB agonists.

[0271]

[0371] In some embodiments, the first expanded culture medium is referred to as "CM," an abbreviation for culture medium. In some embodiments, this is referred to as CM1 (Culture Medium 1). In some embodiments, CM consists of GlutaMAX-containing RPMI 1640 supplemented with 10% human AB serum, 25 mM hepes, and 10 mg / mL gentamicin. The culture is in a 40 mL volume and 10 cm³. 2 In embodiments initiated in a gas-permeable silicon-bottomed gas-permeable flask (e.g., G-Rex10; Wilson Wolf Manufacturing, New Brighton, MN) (Figure 1), each flask contains 10-40 mL of IL-2-containing CM with 10-40 × 10 6 Each cell was loaded with tumor digestive live cells or 5 to 30 tumor fragments. Both G-Rex10 and 24-well plates were incubated in a humidified incubator at 37°C under 5% CO2. Five days after the start of culture, half of the medium was removed and replenished with fresh CM and IL-2. From day five onward, half of the medium was replaced every 2 to 3 days. In some embodiments, CM is CM1 as described in the examples; see Example 5. In some embodiments, the first expansion culture is performed in initial cell culture medium or first cell culture medium. In some embodiments, the initial cell culture medium or first cell culture medium contains IL-2.

[0272]

[0372] In some embodiments, as discussed in the examples and figures, the first expansion culture process (including the process described in step B of Figure 27, which may also be referred to as pre-REP) is shortened to 3 to 14 days. In some embodiments, the first expansion culture (including the process described in step B of Figure 27, which may also be referred to as pre-REP) is shortened to 7 to 14 days, as discussed in the examples and including the expansion culture described in Figures 4 and 5 and, for example, step B of Figure 27. In some embodiments, as discussed in the examples and shown in Figures 4 and 5, the first expansion culture in step B is shortened to 10 to 14 days. In some embodiments, as discussed in the examples and including the expansion culture described in Figures 4 and 5 and, for example, step B of Figure 27, the first expansion culture is shortened to 11 days.

[0273]

[0373] In some embodiments, the first TIL expansion culture may be continued for 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, or 14 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 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.

[0274]

[0374] In some embodiments, a combination of IL-2, IL-7, IL-15, and / or IL-21 is used as the combination during the first expansion culture. In some embodiments, IL-2, IL-7, IL-15, and / or IL-21, and any optional combination thereof, may be included in the first expansion culture, including, for example, the Step B process as shown in Figure 27 and described herein. In some embodiments, a combination of IL-2, IL-15, and IL-21 is used as the combination during the first expansion culture. In some embodiments, IL-2, IL-15, and IL-21, and any optional combination thereof, may be included in the Step B process, for example, as shown in Figure 27 and described herein.

[0275]

[0375] In some embodiments, as discussed in the examples and figures, the first expansion culture process (including, for example, the process described in step B of Figure 27, referred to as pre-REP) is shortened to 3 to 14 days. In some embodiments, as discussed in the examples and shown in Figures 4 and 5, the first expansion culture in step B is shortened to 7 to 14 days. In some embodiments, as discussed in the examples and shown in Figures 4, 5 and 27, the first expansion culture in step B is shortened to 10 to 14 days. In some embodiments, as discussed in the examples and shown in Figures 4, 5 and 27, the first expansion culture is shortened to 11 days.

[0276]

[0376] In some embodiments, the first expansion culture, for example, step B according to Figure 27, is carried out in a closed-system bioreactor. In some embodiments, a closed system is used for TIL expansion culture 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 a G-REX-100. In some embodiments, the closed-system bioreactor is a single bioreactor.

[0277] C. Step C: Transition from the first expansion culture to the second expansion culture.

[0377] In some cases, for example, as shown in Figure 27, the bulk TIL population obtained from the first expansion culture, including the TIL population obtained from step B, can be immediately cryopreserved using the protocol discussed below herein. Alternatively, the TIL population obtained from the first expansion culture, referred to as the second TIL population, can be subjected to a second expansion culture (which may include an expansion culture sometimes referred to as REP) and then cryopreserved as discussed below. Similarly, when genetically modified TILs are used therapeutically, the first TIL population (sometimes referred to as the bulk TIL population) or the second TIL population (which in some embodiments may include a population referred to as the REP TIL population) can be subjected to genetic modification for appropriate treatment before expansion culture or after the first expansion culture and before the second expansion culture.

[0278]

[0378] In some embodiments, TILs obtained from the first expansion culture (e.g., from step B as shown in Figure 27) are stored until phenotyping for selection. In some embodiments, TILs obtained from the first expansion culture (e.g., from step B as shown in Figure 27) are not stored and proceed directly to the second expansion culture. In some embodiments, TILs obtained from the first expansion culture are not cryopreserved after the first expansion culture but before the second expansion culture. In some embodiments, the transition from the first expansion culture to the second expansion culture occurs at approximately 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, or 14 days after fragmentation. In some embodiments, the transition from the first expansion culture to the second expansion culture occurs at approximately 3 to 14 days after fragmentation. In some embodiments, the transition from the first expansion culture to the second expansion culture occurs at approximately 4 to 14 days after fragmentation. In some embodiments, the transition from the first expansion culture to the second expansion culture occurs approximately 4 to 10 days after fragmentation. In some embodiments, the transition from the first expansion culture to the second expansion culture occurs approximately 7 to 14 days after fragmentation. In some embodiments, the transition from the first expansion culture to the second expansion culture occurs approximately 14 days after fragmentation.

[0279]

[0379] In some embodiments, the transition from the first expansion culture to the second expansion culture takes place on days 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, or 14 days after fragmentation. In some embodiments, the transition from the first expansion culture to the second expansion culture takes place between 1 and 14 days after fragmentation. In some embodiments, the first TIL expansion culture may be continued for 2 to 14 days. In some embodiments, the transition from the first expansion culture to the second expansion culture takes place between 3 and 14 days after fragmentation. In some embodiments, the transition from the first expansion culture to the second expansion culture takes place between 4 and 14 days after fragmentation. In some embodiments, the transition from the first expansion culture to the second expansion culture takes place between 5 and 14 days after fragmentation. In some embodiments, the transition from the first expansion culture to the second expansion culture takes place between 6 and 14 days after fragmentation. In some embodiments, the transition from the first expansion culture to the second expansion culture takes place 7 to 14 days after fragmentation. In some embodiments, the transition from the first expansion culture to the second expansion culture takes place 8 to 14 days after fragmentation. In some embodiments, the transition from the first expansion culture to the second expansion culture takes place 9 to 14 days after fragmentation. In some embodiments, the transition from the first expansion culture to the second expansion culture takes place 10 to 14 days after fragmentation. In some embodiments, the transition from the first expansion culture to the second expansion culture takes place 11 to 14 days after fragmentation. In some embodiments, the transition from the first expansion culture to the second expansion culture takes place 12 to 14 days after fragmentation. In some embodiments, the transition from the first expansion culture to the second expansion culture takes place 13 to 14 days after fragmentation. In some embodiments, the transition from the first expansion culture to the second expansion culture takes place 14 days after fragmentation. In some embodiments, the transition from the first expansion culture to the second expansion culture takes place 1 to 11 days after fragmentation has occurred.In some embodiments, the transition from the first expansion culture to the second expansion culture takes place 2 to 11 days after fragmentation. In some embodiments, the transition from the first expansion culture to the second expansion culture takes place 3 to 11 days after fragmentation. In some embodiments, the transition from the first expansion culture to the second expansion culture takes place 4 to 11 days after fragmentation. In some embodiments, the transition from the first expansion culture to the second expansion culture takes place 5 to 11 days after fragmentation. In some embodiments, the transition from the first expansion culture to the second expansion culture takes place 6 to 11 days after fragmentation. In some embodiments, the transition from the first expansion culture to the second expansion culture takes place 7 to 11 days after fragmentation. In some embodiments, the transition from the first expansion culture to the second expansion culture takes place 8 to 11 days after fragmentation. In some embodiments, the transition from the first expansion culture to the second expansion culture takes place 9 to 11 days after fragmentation. In some embodiments, the transition from the first expansion culture to the second expansion culture takes place 10 to 11 days after fragmentation.

[0280]

[0380] In some embodiments, TILs are not stored after the first expansion culture but before the second expansion culture, and the TILs proceed directly to the second expansion culture (for example, in some embodiments, no storage occurs during the transition from step B to step D, as shown in Figure 27). In some embodiments, the transition is carried out in a closed system, as described herein. In some embodiments, TILs from the first expansion culture are TILs from the second TIL population and proceed directly to the second expansion culture without a transition period.

[0281]

[0381] In some embodiments, the transition from the first expansion culture to the second expansion culture, for example, step C shown in Figure 27, is carried out in a closed bioreactor. In some embodiments, a closed system is used for TIL expansion culture 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, the closed bioreactor is a single bioreactor.

[0282] D. Step D: Second expansion culture

[0382] In some embodiments, the TIL cell population increases in number after transitions referred to as steps A and B and step C, as shown in Figure 27, followed by harvesting and initial bulk processing. This further increase is referred to herein as a second expansion culture, which may include an expansion culture process (REP and the process shown in step D of Figure 27) commonly referred to in the art as a rapid expansion culture process. The second expansion culture can generally be achieved using a culture medium in a gas-permeable vessel containing several components, including feeder cells, cytokine sources, and anti-CD3 antibodies.

[0283]

[0383] In some embodiments, the second TIL expansion culture or second TIL expansion culture (which may also be referred to as REP; and may include the process shown in step D of Figure 27) can be carried out using any TIL flask or container known to those skilled in the art. In some embodiments, the second TIL expansion culture may be continued for 7, 8, 9, 10, 11, 12, 13, or 14 days. In some embodiments, the second TIL expansion culture may be continued for about 7 to about 14 days. In some embodiments, the second TIL expansion culture may be continued for about 8 to about 14 days. In some embodiments, the second TIL expansion culture may be continued for about 9 to about 14 days. In some embodiments, the second TIL expansion culture may be continued for about 10 to about 14 days. In some embodiments, the second TIL expansion culture may be continued for about 11 to about 14 days. In some embodiments, the second TIL expansion culture may be continued for about 12 to about 14 days. In some embodiments, the second TIL expansion culture may be continued for approximately 13 to 14 days.

[0284]

[0384] In one embodiment, a second expansion culture can be carried out in a gas-permeable vessel using the methods of the present disclosure (e.g., expansion culture called REP and the process shown in step D of Figure 27). For example, TILs can be rapidly expanded using nonspecific T cell receptor stimulation in the presence of interleukin-2 (IL-2) or interleukin-15 (IL-15). Examples of nonspecific T cell receptor stimulation include anti-CD3 antibodies such as OKT3 at approximately 30 ng / ml, mouse monoclonal anti-CD3 antibodies (commercially available from Ortho-McNeil, Raritan, NJ or Miltenyi Biotech, Auburn, CA) or UHCT-1 (commercially available from BioLegend, San Diego, CA, USA). TILs can be expanded in vitro to induce further stimulation of TILs by including one or more cancer antigens, including their antigenic moieties, in a second expansion culture, in the presence of a T cell growth factor, optionally such as 300 IU / mL IL-2 or IL-15, and optionally one or more epitopes that can be expressed from a vector, such as 0.3 μM MART-1:26-35 (27L) or gpl 00:209-217 (210M). Other suitable antigens include, for example, NY-ESO-1, TRP-1, TRP-2, tyrosinase cancer antigen, MAGE-A3, SSX-2, and VEGFR2 or their antigenic moieties. TILs can also be rapidly expanded by restimulation with the same one or more cancer antigens pulsed on HLA-A2 expressing antigen-presenting cells. Alternatively, TILs can be further restimulated, for example, by irradiated autologous lymphocytes or by 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.

[0285]

[0385] In some embodiments, the cell culture medium further contains IL-2. In some embodiments, the cell culture medium contains about 3000 IU / mL of IL-2. In some embodiments, the cell culture medium further contains IL-2. In some embodiments, the cell culture medium contains about 3000 IU / mL of IL-2. In some embodiments, the cell culture medium contains 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 one embodiment, the cell culture medium contains IL-2 at concentrations of 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.

[0286]

[0386] In one embodiment, the cell culture medium contains OKT-3 antibody. In some embodiments, the cell culture medium contains approximately 30 ng / mL of OKT-3 antibody. In some embodiments, the cell culture medium contains approximately 0.1 ng / mL, approximately 0.5 ng / mL, approximately 1 ng / mL, approximately 2.5 ng / mL, approximately 5 ng / mL, approximately 7.5 ng / mL, approximately 10 ng / mL, approximately 15 ng / mL, approximately 20 ng / mL, approximately 25 ng / mL, approximately 30 ng / mL, approximately 35 ng / mL, approximately 40 ng / mL, approximately 50 ng / mL, approximately 60 ng / mL, approximately 70 ng / mL, approximately 80 ng / mL, approximately 90 ng / mL, approximately 100 ng / mL, approximately 200 ng / mL, approximately 500 ng / mL, and approximately 1 μg / mL of OKT-3 antibody. In one embodiment, the cell culture medium contains OKT-3 antibodies in concentrations of 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. In some embodiments, the cell culture medium does not contain OKT-3 antibodies.

[0287]

[0387] In some embodiments, the cell culture medium contains one or more TNFRSF agonists. In some embodiments, the TNFRSF agonist contains a 4-1BB agonist. In some embodiments, the TNFRSF agonist is a 4-1BB agonist, which is selected from the group consisting of urelumab, utomirumab, EU-101, fusion proteins and their fragments, derivatives, variants, biosimilars and combinations. In some embodiments, the TNFRSF agonist is added to the cell culture medium at a concentration sufficient to achieve a concentration of 0.1 μg / mL to 100 μg / mL. In some embodiments, the TNFRSF agonist is added to the cell culture medium at a concentration sufficient to achieve a concentration of 20 μg / mL to 40 μg / mL.

[0288]

[0388] In some embodiments, in addition to one or more TNFRSFs, the cell culture medium further contains IL-2 at an initial concentration of approximately 3000 IU / mL and OKT-3 antibody at an initial concentration of approximately 30 ng / mL, and one or more TNFRSF agonists contain 4-1BB agonists.

[0289]

[0389] In some embodiments, a combination of IL-2, IL-7, IL-15, and / or IL-21 is used as the combination during the second expansion culture. In some embodiments, IL-2, IL-7, IL-15, and / or IL-21, and any combination thereof, may be included in the second expansion culture, including, for example, the process in step D, as shown in Figure 27 and described herein. In some embodiments, a combination of IL-2, IL-15, and IL-21 is used as the combination during the second expansion culture. In some embodiments, IL-2, IL-15, and IL-21, and any combination thereof, may be included in the process in step D, as shown in Figure 27 and described herein.

[0290]

[0390] In some embodiments, the second expansion culture may be carried out in a supplemented cell culture medium containing IL-2, OKT-3, antigen-presenting feeder cells, and optionally a TNFRSF agonist. In some embodiments, the second expansion culture is carried out 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 cells (APCs; also referred to as antigen-presenting feeder cells). In some embodiments, the second expansion culture occurs in a cell culture medium containing IL-2, OKT-3, and antigen-presenting feeder cells (i.e., antigen-presenting cells).

[0291]

[0391] In some embodiments, the second expanded culture medium contains approximately 500 IU / mL of IL-15, approximately 400 IU / mL of IL-15, approximately 300 IU / mL of IL-15, approximately 200 IU / mL of IL-15, approximately 180 IU / mL of IL-15, approximately 160 IU / mL of IL-15, approximately 140 IU / mL of IL-15, approximately 120 IU / mL of IL-15, or approximately 100 IU / mL of IL-15. In some embodiments, the second expanded culture medium contains approximately 500 IU / mL of IL-15 to approximately 100 IU / mL of IL-15. In some embodiments, the second expanded culture medium contains approximately 400 IU / mL of IL-15 to approximately 100 IU / mL of IL-15. In some embodiments, the second expanded culture medium contains approximately 300 IU / mL of IL-15 to approximately 100 IU / mL of IL-15. In some embodiments, the second expanded culture medium contains approximately 200 IU / mL of IL-15. In some embodiments, the cell culture medium contains approximately 180 IU / mL of IL-15. In some embodiments, the cell culture medium further contains IL-15. In a preferred embodiment, the cell culture medium contains approximately 180 IU / mL of IL-15.

[0292]

[0392] In some embodiments, the second expanded culture medium contains approximately 20 IU / mL of IL-21, approximately 15 IU / mL of IL-21, approximately 12 IU / mL of IL-21, approximately 10 IU / mL of IL-21, approximately 5 IU / mL of IL-21, approximately 4 IU / mL of IL-21, approximately 3 IU / mL of IL-21, approximately 2 IU / mL of IL-21, approximately 1 IU / mL of IL-21, or approximately 0.5 IU / mL of IL-21. In some embodiments, the second expanded culture medium contains approximately 20 IU / mL of IL-21 to approximately 0.5 IU / mL of IL-21. In some embodiments, the second expanded culture medium contains approximately 15 IU / mL of IL-21 to approximately 0.5 IU / mL of IL-21. In some embodiments, the second expanded culture medium contains approximately 12 IU / mL of IL-21 to approximately 0.5 IU / mL of IL-21. In some embodiments, the second expanded culture medium contains approximately 10 IU / mL of IL-21 to approximately 0.5 IU / mL of IL-21. In some embodiments, the second expanded culture medium contains approximately 5 IU / mL of IL-21 to approximately 1 IU / mL of IL-21. In some embodiments, the second expanded culture medium contains approximately 2 IU / mL of IL-21. In some embodiments, the cell culture medium contains approximately 1 IU / mL of IL-21. In some embodiments, the cell culture medium contains approximately 0.5 IU / mL of IL-21. In one embodiment, the cell culture medium further contains IL-21. In a preferred embodiment, the cell culture medium contains approximately 1 IU / mL of IL-21.

[0293]

[0393] In some embodiments, antigen-presenting feeder cells (APCs) are PBMCs. In some embodiments, the ratio of TILs to PBMCs and / or antigen-presenting cells in rapid expansion culture and / or second expansion culture is approximately 1:25, 1:50, 1:100, 1:125, 1:150, 1:175, 1:200, 1:225, 1:250, 1:275, 1:300, 1:325, 1:350, 1:375, 1:400, or 1:500. In some embodiments, the ratio of TILs to PBMCs in rapid expansion culture and / or second expansion culture is 1:50 to 1:300. In some embodiments, the ratio of TILs to PBMCs in rapid expansion culture and / or second expansion culture is 1:100 to 1:200.

[0294]

[0394] In one embodiment, REP and / or a second expansion culture is carried out in a flask by mixing bulk TIL with 100- or 200-fold excess inactivated feeder cells in 150 ml of medium, 30 mg / mL OKT3 anti-CD3 antibody, and 3000 IU / mL IL-2. Medium is replenished until the cells are transferred to another growth chamber (generally two-thirds medium replenishment via respiration with fresh medium). The other growth chamber contains a G-REX flask and a gas-permeable container, as will be discussed in more detail below.

[0295]

[0395] In some embodiments, the second expansion culture (which may include a process referred to as the REP process) is shortened to 7 to 14 days, as discussed in the examples and figures. In some embodiments, the second expansion culture is shortened to 11 days.

[0296]

[0396] In some embodiments, REP and / or the second expansion culture may be carried out using a T-175 flask and a gas-permeable bag or gas-permeable culture apparatus (G-Rex flask) as described above (Tran, et al., J. Immunother. 2008, 31, 742-51; Dudley, et al., J. Immunother. 2003, 26, 332-42). In some embodiments, the second expansion culture (including expansion cultures referred to as rapid expansion cultures) is carried out in a T-175 flask and approximately 1 × 10⁶ of culture medium suspended in 150 mL of medium. 6 One TIL can be added to each T-175 flask. The TIL can 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 can be incubated at 37°C under 5% CO2. Half of the medium can be replaced on day 5 with 50 / 50 medium containing 3000 IU / mL of IL-2. In some embodiments, on day 7, cells from two T-175 flasks can be combined into a 3L bag, to which 300 mL of TIL suspension was added 300 mL of AIM V containing 5% human AB serum and 3000 IU / mL of IL-2. The number of cells in each bag was counted daily or every two days, and fresh medium was added to reduce the cell count to 0.5–2.0 × 10⁶. 6 Maintained at cells / mL

[0297]

[0397] In one embodiment, a second expansion culture (which may include the expansion culture referred to as REP; and the one referred to as step D in Figure 27) may be carried out in a 500 mL gas-permeable flask with a 100 cm gas-permeable silicone bottom (G-Rex 100, commercially available from Wilson Wolf Manufacturing Corporation, New Brighton, MN, USA), with 5 × 10¹⁶ cells in 400 mL of 50 / 50 medium supplemented with 5% human AB serum, 3000 IU / mL of IL-2, and 30 ng / mL of anti-CD3 (OKT3). 6 or 10 x 10 6The TILs can be cultured with PBMCs. The G-Rex 100 flask can be incubated at 37°C under 5% CO2. On day 5, 250 mL of supernatant can be taken out and placed in a centrifuge bottle and centrifuged at 1500 rpm (491 × g) for 10 minutes. The TIL pellet can be resuspended in 150 mL of fresh medium containing 5% human AB serum and 3000 IU / mL of IL-2 and returned to the original G-Rex 100 flask. When continuously expanding the TIL culture in the G-Rex 100 flask, on day 7, the TILs from each G-Rex 100 can be suspended in 300 mL of medium present in each flask, and the cell suspension can be divided into three 100 mL aliquots, which can be used for seeding the three G-Rex 100 flasks. Next, 150 mL of AIM-V containing 5% human AB serum and 3000 IU / mL of IL-2 can be added to each flask. G-Rex 100 flasks can be incubated at 37°C under 5% CO2, and after 4 days, 150 mL of AIM-V containing 3000 IU / mL of IL-2 can be added to each G-Rex 100 flask. Cells can be harvested on day 14 of culture.

[0298]

[0398] In one embodiment, a second expansion culture (including an expansion culture referred to as REP) is carried out in a flask by mixing bulk TIL with 100- or 200-fold excess inactivated feeder cells in 150 ml of medium, 30 mg / mL OKT3 anti-CD3 antibody, and 3000 IU / mL IL-2. In some embodiments, the medium is changed until the cells are transferred to another growth chamber. In some embodiments, two-thirds of the medium is replaced with fresh medium respiration. In some embodiments, the other growth chamber includes a G-REX flask and a gas-permeable container, as will be discussed in more detail below.

[0299]

[0399] In one embodiment, a second expansion culture (including an expansion culture referred to as REP) is performed, which further includes the step of selecting TILs for superior tumor responsiveness. Any selection method known in the art can be used. For example, the method described in U.S. Patent Application Publication 2016 / 0010058 A1 (this disclosure is incorporated herein by reference) may be used for selecting TILs for superior tumor responsiveness.

[0300]

[0400] Optionally, after a second expansion culture (including an expansion culture referred to as REP expansion culture), a cell viability assay can be performed using a standard assay known in the art. For example, a trypan blue exclusion assay can be performed on a bulk TIL sample, which selectively labels dead cells and allows for viability evaluation. In some embodiments, a Cellometer K2 automated cell counter (Nexcelom Bioscience, Lawrence, MA) can be used to count the TIL sample and determine viability. In some embodiments, viability is determined according to the Cellometer K2 Image Cytometer automated cell counter protocol, for example, as described in Example 15.

[0301]

[0401] In some embodiments, the second expansion culture of TIL (including the expansion culture referred to as REP) can be carried out using a T-175 flask and a gas-permeable bag (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 a gas-permeable G-Rex flask, as described above. In some embodiments, the second expansion culture is carried out using a flask. In some embodiments, the second expansion culture is carried out using a gas-permeable G-Rex flask. In some embodiments, the second expansion culture is carried out using a T-175 flask and approximately 1 × 10⁻⁶ 6Each TIL is suspended in approximately 150 mL of culture medium and added to each T-175 flask. The TILs are cultured in a 1:100 ratio with irradiated (50 Gy) allogeneic PBMCs as "feeder" cells, and the cells are cultured in a 1:1 mixture (50 / 50 medium) 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 are incubated at 37°C under 5% CO2. In some embodiments, half of the medium is replaced on day 5 with 50 / 50 medium containing 3000 IU / mL of IL-2. In some embodiments, on day 7, cells from two T-175 flasks are combined in a 3 L bag, and 300 mL of AIM-V containing 5% human AB serum and 3000 IU / mL of IL-2 is added to its 300 mL TIL suspension. The number of cells in each bag can be counted daily or every two days, and the number of cells can be increased to approximately 0.5 to 2.0 × 10⁻⁶ by adding fresh culture medium. 6 Can be maintained at cells / mL.

[0302]

[0402] In some embodiments, the second expansion culture (including the expansion culture referred to as REP) is 100 cm². 2 The procedure was performed in a 500 mL volume flask with a gas-permeable silicon bottom (G-Rex 100, Wilson Wolf) (Figure 1), and approximately 5 × 10¹⁶ units were obtained 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 6The TILs are cultured with irradiated allogeneic PBMCs in a 1:100 ratio. The G-Rex100 flasks are incubated at 37°C under 5% CO2. In some embodiments, on day 5, 250 mL of supernatant is taken out and 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 returned to the original G-Rex100 flask. In embodiments where the TILs are continuously expanded and cultured in the 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 for seeding in 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 are incubated at 37°C under 5% CO2, and after 4 days, 150 mL of AIM-V containing 3000 IU / mL of IL-2 is added to each G-Rex100 flask. Cells are harvested on day 14 of culture.

[0303]

[0403] Diverse antigen receptors for T and B lymphocytes are produced by somatic recombination of a limited but numerous gene segments. These gene segments, V (variable), D (diversity), J (binding), and C (constant), determine the binding specificity and downstream application of immunoglobulins and T cell receptors (TCRs). The present invention provides a method for generating TILs that exhibit and increase T cell repertoire diversity. In some embodiments, TILs obtained by the present method exhibit increased T cell repertoire diversity. In some embodiments, TILs obtained in a second 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, immunoglobulin diversity lies in the immunoglobulin heavy chain. In some embodiments, immunoglobulin diversity lies in the immunoglobulin light chain. In some embodiments, diversity lies in the T cell receptor. In some embodiments, diversity lies in one of the T cell receptors selected from the group consisting of alpha, beta, gamma, and delta receptors. In some embodiments, the expression of T cell receptor (TCR) alpha and / or beta is increased. In some embodiments, the expression of T cell receptor (TCR) alpha is increased. In some embodiments, the expression of T cell receptor (TCR) beta is increased. In some embodiments, the expression of TCRab (i.e., TCRα / β) is increased.

[0304]

[0404] In some embodiments, the second expanded culture medium (sometimes referred to as CM2 or the second cell culture medium) contains IL-2, OKT-3, and antigen-presenting feeder cells (APCs), as will be discussed in more detail below.

[0305]

[0405] In some embodiments, a second expansion culture, for example, step D according to Figure 27, is carried out in a closed-system bioreactor. In some embodiments, a closed system is used for TIL expansion culture 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 a G-REX-100. In some embodiments, the closed-system bioreactor is a single bioreactor.

[0306] 1. Feeder cells and antigen-presenting cells

[0406] In one embodiment, the second expansion culture procedure described herein (including, for example, the expansion culture described in step D of Figure 27 and the expansion culture referred to as REP) requires an excess amount of feeder cells during the REP TIL expansion culture and / or the second expansion 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.

[0307]

[0407] Generally, allogeneic PBMCs are inactivated by either irradiation or heat treatment and used in REP procedures, as described in the Examples, particularly Example 14 which provides an exemplary protocol for evaluating the non-replication properties of irradiated allogeneic PBMCs.

[0308]

[0408] In some embodiments, if the total number of viable cells on day 14 is less than the initial number of viable cells that transitioned to 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 non-reproducible and are permitted for use in the TIL expansion culture procedure described herein. See, for example, Example 14.

[0309]

[0409] In some embodiments, if the total number of viable cells cultured in the presence of OKT3 and IL-2 has not increased by day 7 and day 14 from the initial number of viable cells that entered 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 non-replicable and are permitted for use in the TIL expansion culture procedure described herein. In some embodiments, the PBMCs are cultured in the presence of 30 ng / ml of OKT3 antibody and 3000 IU / ml of IL-2. See, for example, Example 13.

[0310]

[0410] In some embodiments, if the total number of viable cells cultured in the presence of OKT3 and IL-2 has not increased on days 7 and 14 from the initial number of viable cells that entered 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 non-replicable and are permitted for use in the TIL expansion culture procedure described herein. In some embodiments, PBMCs are cultured in the presence of 5-60 ng / ml of OKT3 antibody and 1000-6000 IU / ml of IL-2. In some embodiments, PBMCs are cultured in the presence of 10-50 ng / ml of OKT3 antibody and 2000-5000 IU / ml of IL-2. In some embodiments, PBMCs are cultured in the presence of 20-40 ng / ml of OKT3 antibody and 2000-4000 IU / ml of IL-2. In some embodiments, PBMCs are cultured in the presence of 25-35 ng / ml of OKT3 antibody and 2500-3500 IU / ml of IL-2.

[0311]

[0411] 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 approximately 1:25, 1:50, 1:100, 1:125, 1:150, 1:175, 1:200, 1:225, 1:250, 1:275, 1:300, 1:325, 1:350, 1:375, 1:400, or 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.

[0312]

[0412] In one embodiment, the second expansion culture procedure described herein involves approximately 2.5 × 10⁻⁶ cells. 9 A feeder cell and approximately 100 x 10 6 A ratio of TILs is required. In another embodiment, the second expansion culture procedure described herein is approximately 2.5 × 10 9 A feeder cell and approximately 50 x 10 6 A ratio of TILs is required. In yet another embodiment, the second expansion culture procedure described herein is approximately 2.5 × 10 9 A feeder cell and approximately 25 × 10⁶ cells 6 It requires one TIL.

[0313]

[0413] In some embodiments, the second expansion culture procedure described herein requires an excess volume of feeder cells during the second expansion 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.

[0314]

[0414] Generally, allogeneic PBMCs are inactivated by either irradiation or heat treatment and used in the TIL expansion culture procedures described herein, including the exemplary procedures shown in Figures 4, 5, and 27.

[0315]

[0415] In one embodiment, artificial antigen-presenting cells are used in a second expansion culture, either as a substitute for or in combination with PBMCs.

[0316] 2. Cytokines

[0416] The expansion culture method described herein generally uses a culture medium containing high doses of cytokines, particularly IL-2, as is known in the art.

[0317]

[0417] Alternatively, it is possible to use cytokine combinations for rapid expansion culture or second expansion culture of TILs, and combinations of two or more IL-2, IL-15, and IL-21 are generally outlined in International Publications 2015 / 189356 and 2015 / 189357 (expressly incorporated herein by reference in whole). Possible combinations include IL-2 and IL-15, IL-2 and IL-21, IL-15 and IL-21 and IL-2, and IL-15 and IL-21, the latter of which have specific uses in many embodiments. As described in these specifications, the use of cytokine combinations is advantageous in particular for the generation of lymphocytes, and more specifically, T cells.

[0318] 3. Anti-CD3 antibody

[0418] In some embodiments, the culture medium used in the expansion culture method described herein (including the one referred to as REP; see, for example, Figure 27) also includes an anti-CD3 antibody. When the anti-CD3 antibody is used in combination with IL-2, T cell activation and cell division are induced in the TIL population. This effect can be observed with the full-length antibody as well as the Fab and F(ab')2 fragments, with the former being generally preferred; see, for example, Tsoukas et al., J.Immunol.1985, 135, 1719 (referred to herein by reference in its entirety).

[0319]

[0419] As those skilled in the art will understand, there are several suitable anti-human CD3 antibodies found for use in the present invention, but are not limited to, anti-human CD3 polyclonal and monoclonal antibodies from various mammals, including those from murids, humans, primates, rats, and canines. In detailed embodiments, the OKT3 anti-CD3 antibody is used (commercially available from Ortho-McNeil, Raritan, NJ or Miltenyi Biotech, Auburn, CA).

[0320] E. Step E: Recover TIL

[0420] After the second expansion culture step, the cells can be harvested. In some embodiments, the TILs are harvested after one, two, three, four or more expansion culture steps, as provided, for example, in Figure 27. In some embodiments, the TILs are harvested after two expansion culture steps, as provided, for example, in Figure 27.

[0321]

[0421] TIL can be recovered by any suitable and sterile method, including, for example, centrifugation. TIL recovery methods are well known in the art, and any such known method can be used in conjunction with this process. In some embodiments, TIL is recovered using an automated system.

[0322]

[0422] Cell harvesters and / or cell processing systems are commercially available from various sources, including, for example, Fresenius Kabi, Tomtec Life Science, Perkin Elmer, and Inotech Biosystems International, Inc. Any cell-based harvester can be used in this method. In some embodiments, the cell harvester and / or cell processing system is a membrane-based cell harvester. In some embodiments, cell recovery is performed via a cell processing system such as a LOVO system (manufactured by Fresenius Kabi). The term “LOVO cell processing system” also refers to any equipment or apparatus manufactured by any vendor that can deliver a solution containing cells through a membrane or filter, such as a rotating membrane or rotating filter, in a sterile and / or closed system environment, enabling continuous flow and cell processing with removal of supernatant or cell culture without pelletizing. 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.

[0323]

[0423] In some embodiments, recovery, for example, step E shown in Figure 27, is carried out from a closed-system bioreactor. In some embodiments, a closed system is used for TIL expansion culture 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, the closed-system bioreactor is a single bioreactor.

[0324]

[0424] In some embodiments, step E shown in Figure 27 is carried out according to the process described in Example 30. In some embodiments, to maintain the sterility and closure of the system, the closed system is accessed via a syringe under sterile conditions. In some embodiments, a closed system such as that described in Example 30 is used.

[0325]

[0425] In some embodiments, TIL is recovered according to the method described in Example 30. In some embodiments, TIL from day 1 to day 11 is recovered using a method such as the one described in Section 8.5 (referred to as TIL recovery on day 11 in Example 30). In some embodiments, TIL from day 12 to day 22 is recovered using a method such as the one described in Section 8.12 (referred to as TIL recovery on day 22 in Example 30).

[0326] F. Step F: Final formulation and / or transfer to infusion bag

[0426] After steps A to E are completed, as provided in the illustrative order in Figure 27 and outlined in detail above and herein, 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 culture method described above, the TILs are transferred to a container for use in administration to a patient.

[0327]

[0427] In one embodiment, TILs cultured using the APC of this disclosure are administered to a patient as a pharmaceutical composition. In one embodiment, the pharmaceutical composition is a suspension of TILs in sterile buffer. TILs cultured using the PBMC of this disclosure may be administered by any preferred route known in the art. In some embodiments, T cells are administered as a single intra-arterial or intravenous infusion, which is preferably continued for about 30 to 60 minutes. Other preferred routes of administration include intraperitoneal, intrathecal, and intralymphatic.

[0328] 1. Pharmaceutical composition, dosage, and administration regimen

[0428] In one embodiment, TILs cultured using the method of the Disclosure are administered to a patient as a pharmaceutical composition. In one embodiment, the pharmaceutical composition is a suspension of TILs in sterile buffer. TILs cultured using the PBMCs of the Disclosure may be administered by any preferred route known in the Art. In some embodiments, T cells are administered as a single intra-arterial or intravenous infusion, which is preferably continued for about 30 to 60 minutes. Other preferred routes of administration include intraperitoneal, intrathecal, and intralymphatic administration.

[0329]

[0429] Any suitable dose of TIL can be administered. In some embodiments, approximately 2.3 × 10⁻⁶ 10 ~Approx. 13.7×10 10 Individual TILs were administered, and especially in cases of melanoma, the average was approximately 7.8 × 10⁶ 10 This is the TIL of a certain number. In one embodiment, it is approximately 1.2 × 10⁻⁶. 10 ~Approx. 4.3×10 10 Individual TILs are administered. In some embodiments, approximately 3 × 10 10 ~Approx. 12×10 10 Individual TILs are administered. In some embodiments, approximately 4 × 10 10 ~About 10×10 10 A TIL is administered. In some embodiments, approximately 5 × 10 10 ~Approx. 8×10 10 Individual TILs are administered. In some embodiments, approximately 6 × 10 10 ~Approx. 8×10 10 Individual TILs are administered. In some embodiments, approximately 7 × 10 10 ~Approx. 8×10 10 A TIL is administered. In some embodiments, the therapeutically effective dose is approximately 2.3 × 10⁶. 10 ~Approx. 13.7×10 10 It is one. In some embodiments, the therapeutically effective dose is about 7.8 × 10⁻¹⁴, especially when the cancer is melanoma. 10 This is a TIL of one unit. In some embodiments, the therapeutically effective dosage is approximately 1.2 × 10⁻⁶. 10 ~Approx. 4.3×10 10This is a TIL of one unit. In some embodiments, the therapeutically effective dosage is approximately 3 × 10⁻⁶. 10 ~Approx. 12×10 10 This is a TIL of one unit. In some embodiments, the therapeutically effective dosage is approximately 4 × 10⁻⁶. 10 ~About 10×10 10 This is a TIL of one unit. In some embodiments, the therapeutically effective dosage is approximately 5 × 10⁻⁶. 10 ~Approx. 8×10 10 This is a TIL of one unit. In some embodiments, the therapeutically effective dosage is approximately 6 × 10⁶ units. 10 ~Approx. 8×10 10 This is a TIL of one unit. In some embodiments, the therapeutically effective dosage is approximately 7 × 10⁻⁶. 10 ~Approx. 8×10 10 This is the TIL for each individual.

[0330]

[0430] In some embodiments, the number of TILs provided in the pharmaceutical composition of the present invention is about 1 × 10⁶ 6 , 2×10 6 , 3 x 10 6 , 4×10 6 , 5×10 6 , 6×10 6 , 7×10 6 , 8×10 6 , 9×10 6 , 1 x 10 7 , 2×10 7 , 3 x 10 7 , 4×10 7 , 5×10 7 , 6×10 7 , 7×10 7 , 8×10 7 , 9×10 7 , 1 x 10 8 , 2×10 8 , 3 x 10 8 , 4×10 8 , 5×10 8 , 6×10 8 , 7×10 8 , 8×10 8 , 9×10 8 , 1 x 10 9 , 2×10 9 , 3 x 10 9 , 4×10 9 , 5×109 , 6×10 9 , 7×10 9 , 8×10 9 , 9×10 9 , 1 x 10 10 , 2×10 10 , 3 x 10 10 , 4×10 10 , 5×10 10 , 6×10 10 , 7×10 10 , 8×10 10 , 9×10 10 , 1 x 10 11 , 2×10 11 , 3 x 10 11 , 4×10 11 , 5×10 11 , 6×10 11 , 7×10 11 , 8×10 11 , 9×10 11 , 1 x 10 12 , 2×10 12 , 3 x 10 12 , 4×10 12 , 5×10 12 , 6×10 12 , 7×10 12 , 8×10 12 , 9×10 12 , 1 x 10 13 , 2×10 13 , 3 x 10 13 , 4×10 13 , 5×10 13 , 6×10 13 , 7×10 13 , 8×10 13 and 9×10 13 There are 1. In one embodiment, the number of TILs provided in the pharmaceutical composition of the present invention is 1 × 10⁶. 6 ~5×10 6 , 5×10 6 ~1 × 10 7 , 1 x 10 7 ~5×10 7 , 5×10 7 ~1 × 10 8 , 1 x 10 8 ~5×10 8 , 5×10 8 ~1 × 10 9 , 1 x 109 ~5×10 9 , 5×10 9 ~1 × 10 10 , 1 x 10 10 ~5×10 10 , 5×10 10 ~1 × 10 11 , 5×10 11 ~1 × 10 12 , 1 x 10 12 ~5×10 12 and 5×10 12 ~1 × 10 13 It is within the range of an individual.

[0331]

[0431] In some embodiments, the concentration of TIL 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%, and 0.07% of the pharmaceutical composition. The percentages are less than %, 0.06%, 0.05%, 0.04%, 0.03%, 0.02%, 0.01%, 0.009%, 0.008%, 0.007%, 0.006%, 0.005%, 0.004%, 0.003%, 0.002%, 0.001%, 0.0009%, 0.0008%, 0.0007%, 0.0006%, 0.0005%, 0.0004%, 0.0003%, 0.0002%, or 0.0001% w / w, w / v, or v / v.

[0332]

[0432] In some embodiments, the concentrations of TIL provided in the pharmaceutical composition of the present invention are 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%, and 15.5% of the pharmaceutical composition. 0%, 15.25%, 15%, 14.75%, 14.50%, 14.25%, 14%, 13.75%, 13.50%, 13.25%, 13%, 12.75%, 12.50%, 12.25%, 12%, 11.75%, 11.50%, 11.25%, 11%, 10.75%, 10.50%, 10.25%, 10%, 9.75%, 9.50%, 9.25%, 9%, 8.75%, 8.50%, 8.25%, 8%, 7.75% , 7.50%, 7.25%, 7%, 6.75%, 6.50%, 6.25%, 6%, 5.75%, 5.50%, 5.25%, 5%, 4.75%, 4.50%, 4.25%, 4%, 3.75%, 3.50%, 3.25%, 3%, 2.75%, 2.50%, 2.25%, 2%, 1.75%, 1.50%, 125%, 1%, 0.5%, 0.4%, 0.3%, 0.2%, 0.1%, 0.09%, 0.08%, 0.07%, 0. The percentages are 0.6%, 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.

[0333]

[0433] In some embodiments, the concentration of TIL provided in the pharmaceutical composition of the present invention is approximately 0.0001% to approximately 50%, approximately 0.001% to approximately 40%, approximately 0.01% to approximately 30%, approximately 0.02% to approximately 29%, approximately 0.03% to approximately 28%, approximately 0.04% to approximately 27%, approximately 0.05% to approximately 26%, approximately 0.06% to approximately 25%, and approximately 0.07% to approximately 24% of the pharmaceutical composition. The ranges are approximately 0.08% to 23%, 0.09% to 22%, 0.1% to 21%, 0.2% to 20%, 0.3% to 19%, 0.4% to 18%, 0.5% to 17%, 0.6% to 16%, 0.7% to 15%, 0.8% to 14%, 0.9% to 12%, or 1% to 10% in the range of w / w, w / v, or v / v.

[0334]

[0434] In some embodiments, the concentration of TIL 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%, about 0.1% to about 0.9% w / w, w / v, or v / v of the pharmaceutical composition.

[0335]

[0435] In some embodiments, the amount of TIL 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, 0.09g, 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 or less.

[0336]

[0436] In some embodiments, the amount of TIL provided in the pharmaceutical composition of the present invention is 0.0001g, 0.0002g, 0.0003g, 0.0004g, 0.0005g, 0.0006g, 0.0007g, 0.0008g, 0.0009g, 0.001g, 0.0015g, 0.002g, 0.002g 5g, 0.003g, 0.0035g, 0.004g, 0.0045g, 0.005g, 0.0055g, 0.006g, 0.0065g, 0.00 7g, 0.0075g, 0.008g, 0.0085g, 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, 0.09g, 0.095g, 0.1g, 0.15g, 0.2g, 0.25g, 0.3g, 0.35g, 0.4g, 0.45g , 0.5g, 0.55g, 0.6g, 0.65g, 0.7g, 0.75g, 0.8g, 0.85g, 0.9g, 0.95g, 1g, 1.5g, 2g, 2.5, 3g, 3.5, 4g, 4.5g, 5g, 5.5g, 6g, 6.5g, 7g, 7.5g, 8g, 8.5g, 9g, 9.5g or more than 10g.

[0337]

[0437] The TILs provided in the pharmaceutical compositions of the present invention are effective over a wide range of dosages. The exact dosage will depend on the route of administration, the form of compound administration, the sex and age of the patient, the patient's weight, and the attending physician's preference and experience. Clinically established TIL dosages may also be used as appropriate. The amount of the pharmaceutical composition administered using the method 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 properties of the active pharmaceutical ingredient, and the discretion of the prescribing physician.

[0338]

[0438] In some embodiments, TIL may be administered as a single dose. Such administration may be by injection, for example, intravenous injection. In some embodiments, TIL may be administered in multiple doses. Administration may be once, twice, three times, four times, five times, six times, or more than six times per year. Administration may be once a month, once every two weeks, once a week, or once every other day. Administration of TIL may be continued for as long as necessary.

[0339]

[0439] In some embodiments, the effective dose of TIL is approximately 1 × 10⁻⁶ 6 , 2×10 6 , 3 x 10 6 , 4×10 6 , 5×10 6 , 6×10 6 , 7×10 6 , 8×10 6 , 9×10 6 , 1 x 10 7 , 2×10 7 , 3 x 10 7 , 4×10 7 , 5×10 7 , 6×10 7 , 7×10 7 , 8×10 7 , 9×10 7 , 1 x 10 8 , 2×10 8 , 3 x 10 8 , 4×10 8 , 5×10 8 , 6×10 8 , 7×10 8 , 8×10 8 , 9×10 8 , 1 x 10 9 , 2×10 9 , 3 x 10 9 , 4×10 9 , 5×10 9 , 6×10 9 , 7×10 9 , 8×10 9 , 9×10 9 , 1 x 10 10 , 2×10 10 , 3 x 10 10 , 4×10 10 , 5×10 10 , 6×10 10 , 7×1010 , 8×10 10 , 9×10 10 , 1 x 10 11 , 2×10 11 , 3 x 10 11 , 4×10 11 , 5×10 11 , 6×10 11 , 7×10 11 , 8×10 11 , 9×10 11 , 1 x 10 12 , 2×10 12 , 3 x 10 12 , 4×10 12 , 5×10 12 , 6×10 12 , 7×10 12 , 8×10 12 , 9×10 12 , 1 x 10 13 , 2×10 13 , 3 x 10 13 , 4×10 13 , 5×10 13 , 6×10 13 , 7×10 13 , 8×10 13 and 9×10 13 It is one unit. In some embodiments, the effective dose of TIL is 1 × 10⁻⁶. 6 ~5×10 6 , 5×10 6 ~1 × 10 7 , 1 x 10 7 ~5×10 7 , 5×10 7 ~1 × 10 8 , 1 x 10 8 ~5×10 8 , 5×10 8 ~1 × 10 9 , 1 x 10 9 ~5×10 9 , 5×10 9 ~1 × 10 10 , 1 x 10 10 ~5×10 10 , 5×10 10 ~1 × 10 11 , 5×10 11 ~1 × 10 12 , 1 x 10 12 ~5×10 12 and 5×1012 ~1 × 10 13 It is within the range of an individual.

[0340]

[0440] In some embodiments, the effective dosage of TIL is approximately 0.01 mg / kg to 4.3 mg / kg, approximately 0.15 mg / kg to 3.6 mg / kg, approximately 0.3 mg / kg to 3.2 mg / kg, approximately 0.35 mg / kg to 2.85 mg / kg, approximately 0.15 mg / kg to 2.85 mg / kg, approximately 0.3 mg to 2.15 mg / kg, approximately 0.45 mg / kg to 1.7 mg / kg, approximately 0.15 mg / kg to 1.3 mg / kg, approximately 0.3 mg / kg to 1.15 mg / kg, approximately 0.45 mg / kg to 1 mg / kg, approximately 0.55 mg / kg to 0.85 mg / kg, approximately 0.65 mg / kg to 0.8 mg / kg, and approximately 0.7 mg / kg. The ranges are approximately 0.75 mg / kg, 0.7 mg / kg to 2.15 mg / kg, 0.85 mg / kg to 2 mg / kg, 1 mg / kg to 1.85 mg / kg, 1.15 mg / kg to 1.7 mg / kg, 1.3 mg / kg to 1.6 mg / kg, 1.35 mg / kg to 1.5 mg / kg, 2.15 mg / kg to 3.6 mg / kg, 2.3 mg / kg to 3.4 mg / kg, 2.4 mg / kg to 3.3 mg / kg, 2.6 mg / kg to 3.15 mg / kg, 2.7 mg / kg to 3 mg / kg, 2.8 mg / kg to 3 mg / kg, or 2.85 mg / kg to 2.95 mg / kg.

[0341]

[0441] In some embodiments, the effective dosage of TIL is approximately 1 mg to 500 mg, 10 mg to 300 mg, 20 mg to 250 mg, 25 mg to 200 mg, 1 mg to 50 mg, 5 mg to 45 mg, 10 mg to 40 mg, 15 mg to 35 mg, 20 mg to 30 mg, 23 mg to 28 mg, 50 mg to 150 mg, and 60 mg to 140 mg. The ranges are approximately 70mg to 130mg, 80mg to 120mg, 90mg to 110mg, 95mg to 105mg, 98mg to 102mg, 150mg to 250mg, 160mg to 240mg, 170mg to 230mg, 180mg to 220mg, 190mg to 210mg, 195mg to 205mg, or 198mg to 207mg.

[0342]

[0442] An effective dose of TIL may be administered as a single dose or in multiple doses by any method of administration generally accepted for drugs of similar utility, including intranasal and transdermal routes, intra-arterial injection, intravenous, intraperitoneal, parenteral, intramuscular, subcutaneous, topical, implantation, or inhalation.

[0343] G. Analysis of randomly selected cell viability

[0443] Optionally, after the first expansion culture in Step B, cell viability assays can be performed using standard assays known in the art. For example, a trypan blue exclusion assay can be performed on a bulk TIL sample, which selectively labels dead cells and allows for viability evaluation. Other assays used for viability testing include, but are not limited to, the Alamer blue assay and the MTT assay.

[0344] 1. Cell count, viability, flow cytometry

[0444] In some embodiments, cell number and / or viability are measured. Expression of markers such as CD3, CD4, CD8, and CD56, and any other markers disclosed or described herein, can be measured by antibody-assisted flow cytometry, for example, using a FACSCanto® flow cytometer (BD Biosciences), commercially available from BD Bio-sciences (BD Biosciences, San Jose, CA), but not limited to such devices. Cells can be manually counted 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.

[0345]

[0445] In some embodiments, TIL is analyzed in terms of the percentage of the CD3+ cell population. In some embodiments, TIL for use in treatment is analyzed in terms of the percentage of the CD3+ cell population. In some embodiments, TIL is CD3+ / CD45+ TIL. In some embodiments, the percentage of CD3+ is approximately 70% to approximately 99.9%. In some embodiments, the percentage of CD3+ is approximately 74% to approximately 99.9%. In some embodiments, the percentage of CD3+ is approximately 74% to approximately 99.9%. In some embodiments, the percentage of CD3+ is approximately 74% to approximately 97.1%. In some embodiments, the percentage of CD3+ is approximately 80% to approximately 99.9%. In some embodiments, the percentage of CD3+ is approximately 85% to approximately 99.9%. In some embodiments, the percentage of CD3+ is approximately 90% to approximately 99.9%. In some embodiments, the percentage of CD3+ is approximately 85% to 95%. In some embodiments, the percentage of CD3+ is approximately 80% to 95%. In some embodiments, the percentage of CD3+ is approximately 95% to 99.9%. In some embodiments, the percentage of CD3+ / CD45+ is approximately 70% to 99.9%. In some embodiments, the percentage of CD3+ / CD45+ is approximately 74% to 99.9%. In some embodiments, the percentage of CD3+ / CD45+ is approximately 74% to 99.9%. In some embodiments, the percentage of CD3+ / CD45+ is approximately 74% to 97.1%. In some embodiments, the percentage of CD3+ / CD45+ is approximately 80% to 99.9%. In some embodiments, the percentage of CD3+ / CD45+ is approximately 85% to 99.9%. In some embodiments, the percentage of CD3+ / CD45+ is approximately 90% to approximately 99.9%. In some embodiments, the percentage of CD3+ / CD45+ is approximately 85% to approximately 95%. In some embodiments, the percentage of CD3+ / CD45+ is approximately 80% to approximately 95%. In some embodiments, the percentage of CD3+ / CD45+ is approximately 95% to approximately 99.9%.

[0346]

[0446] In some cases, the bulk TIL population can be immediately cryopreserved using the protocol discussed below. Alternatively, the bulk TIL population may be subjected to REP (recommended gene modification) and then cryopreserved, as discussed below. Similarly, if genetically modified TILs are to be used in therapy, the bulk or REP TIL population may be subjected to gene modification for suitable therapeutic purposes.

[0347] 2.Cell culture

[0447] In one embodiment, the method for expanding TILs may include using about 5,000 mL to about 25,000 mL of cell medium, about 5,000 mL to about 10,000 mL of cell medium, or about 5,800 mL to about 8,700 mL of cell medium. In one embodiment, one or fewer types of cell culture medium are used to expand the number of TILs. Any suitable cell culture medium, for example, AIM-V cell medium (L-glutamine, 50 μM streptomycin sulfate, and 10 μM gentamicin sulfate) or cell culture medium (Invitrogen, Carlsbad CA) may be used. In this regard, the method of the present invention is advantageous in that the amount of medium and the number of types of medium required to expand the number of TILs are reduced. In one embodiment, expanding the number of TILs may include adding fresh cell culture medium to the cells (also referred to as feeding cells) at a frequency of every two or three days or less. Expanding the number of cells in a gas-permeable container simplifies the procedure required to expand the number of cells by reducing the feeding frequency required for cell expansion culture.

[0348]

[0448] In one embodiment, the cell culture medium in the first and / or second gas-permeable vessel is not filtered. Using an unfiltered cell culture medium may simplify the procedure required for increasing the number of cells. In one embodiment, the cell culture medium in the first and / or second gas-permeable vessel does not contain β-mercaptoethanol (BME).

[0349]

[0449] In one embodiment, the required duration of the method includes: obtaining a tumor tissue sample from a mammal; culturing the tumor tissue sample in a first gas-permeable container containing cell culture medium; obtaining TILs from the tumor tissue sample; and expanding the number of TILs for approximately 14 to approximately 42 days, for example, approximately 28 days, in a second gas-permeable container containing cell culture medium using aAPC.

[0350]

[0450] In one embodiment, TILs are cultured in a gas-permeable container. Gas-permeable containers are used for the expansion culture of TILs using PBMCs in methods, compositions, and apparatus known in the art, including those described in U.S. Patent Application Publication 2005 / 0106717 A1 (this disclosure is incorporated herein by reference). In one embodiment, TILs are cultured in a gas-permeable bag. In one embodiment, TILs are cultured using a cell expansion culture system that expands TILs in a gas-permeable bag, such as the Xuri Cell Expansion System W25 (GE Healthcare). In one embodiment, TILs are cultured using a cell expansion culture system that expands 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 culture system includes a gas-permeable cell bag having a volume selected from the group consisting of approximately 100 mL, 200 mL, 300 mL, 400 mL, 500 mL, 600 mL, 700 mL, 800 mL, 900 mL, 1 L, 2 L, 3 L, 4 L, 5 L, 6 L, 7 L, 8 L, 9 L, and 10 L. In one embodiment, the TIL can be expanded and cultured in a G-Rex flask (commercially available from Wilson Wolf Manufacturing). In such embodiments, the cell population is expanded to approximately 5 × 10⁶ 5 cells / cm 2 From 10x10 6 ~30×10 6 cells / cm 2This allows for expansion culture. In one embodiment, this expansion culture is performed without adding fresh cell culture medium to the cells (also referred to as feeding the cells). In one embodiment, this is no feeding as long as there is about 10 cm of medium in the GRex flask. In one embodiment, there is no feeding, but one or more cytokines are added. In one embodiment, the cytokines can be added as a bolus without any need to mix the cytokines with the medium. Such containers, apparatus and methods are known in the art and have been used for the expansion of TILs, as published in U.S. Patent Application Publication No. 2014 / 0377739A1, International Publication No. 2014 / 210036A1, U.S. Patent Application Publication No. 2013 / 0115617A1, International Publication No. 2013 / 188427A1, U.S. Patent Application Publication No. 2011 / 0136228A1, U.S. Patent No. 8,809,050 B2, International Publication No. 2011 / 072088A2, U.S. Patent Application Publication No. 2016 / 0208216A1, U.S. Patent Application Publication No. 2012 / 0244133A1, International Publication No. 2012 / 129201A1, U.S. Patent Application Publication No. 2013 / 0102075A1, U.S. Patent No. 8,956,860 This includes the information contained in Patent No. B2, International Publication No. 2013 / 173835 A1, and U.S. Patent Application Publication No. 2015 / 0175966 A1 (these disclosures are incorporated herein by reference). Such processes are also described in Jin et al., J. Immunotherapy, 2012, 35:283-292.

[0351] Selective TIL gene modification

[0451] In some embodiments, the TIL may be optionally, but not limited to, genetically modified to include additional functionality, such as high-affinity T cell receptors (TCRs), e.g., TCRs targeting 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-specific cell surface molecules (e.g., CD19).

[0352] H. Optional TIL cryopreservation

[0452] As discussed above and illustrated in steps A-E provided in Figure 27, cryopreservation can be performed at various points throughout the TIL expansion culture process. In some embodiments (for example, as per step D in Figure 27), the expanded cultured TIL population after the second expansion culture can be cryopreserved. Cryopreservation can generally be achieved by placing the TIL population in a cryopreservation solution, such as 85% complement-inactivated AB serum and 15% dimethyl sulfoxide (DMSO). The cells in this solution are placed in a cryogenic vial and stored at -80°C for 24 hours, and optionally transferred to a gaseous nitrogen freezer for cryopreservation. See Sadeghi, et al., Acta Oncologica 2013, 52, 978-986. In some embodiments, TILs are cryopreserved in 5% DMSO. In some embodiments, TILs are cryopreserved in cell culture medium + 5% DMSO. In some embodiments, TILs are cryopreserved according to the methods provided in Examples 8 and 9.

[0353]

[0453] Where appropriate, the cells are removed from the freezer and thawed in a 37°C water bath until approximately four-fifths of the solution has thawed. The cells are generally resuspended in complete medium and optionally washed one or more times. In some embodiments, the thawed TIL can be counted as known in the art and evaluated in relation to viability.

[0354] I. Phenotypic characteristics of cultured TILs

[0454] In some embodiments, TILs are analyzed for the expression of numerous phenotypic markers after expansion culture, including those described herein and in the examples. In some embodiments, the expression of one or more phenotypic markers is examined. In some embodiments, the phenotypic features of TILs are analyzed after the first expansion culture in step B. In some embodiments, the phenotypic features of TILs are analyzed during the transition in step C. In some embodiments, the phenotypic features of TILs are analyzed during the transition in step C and after cryopreservation. In some embodiments, the phenotypic features of TILs are analyzed after the second expansion culture in step D. In some embodiments, the phenotypic features of TILs are analyzed after two or more expansion cultures 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 marker is selected from the group consisting of TCRab (i.e., TCRα / β), CD57, CD28, CD4, CD27, CD56, and CD8a. In some embodiments, the marker is selected from the group consisting of CD45RA, CD8a, CCR7, CD4, CD3, CD38, and HLA-DR. In some embodiments, the expression of 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, or 14 markers is examined. In some embodiments, the expression of one or more markers from each group is examined. In some embodiments, one or more HLA-DR, CD38, and CD69 expressions are maintained in fresh TILs compared to thawed TILs (i.e., there is no statistically significant difference). In some embodiments, the activated state of TILs is maintained in thawed TILs.

[0355]

[0455] In some embodiments, the expression of one or more regulatory markers is measured. In some embodiments, the regulatory markers are 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 markers are selected from the group consisting of CD137, CD8a, Lag3, CD4, CD3, PD-1, and TIM-3. In some embodiments, the regulatory markers are selected from the group consisting of CD69, CD8a, TIGIT, CD4, CD3, KLRG1, and CD154. In some embodiments, the expression of regulatory molecules is reduced in thawed TILs compared to fresh TILs. In some embodiments, the expression of regulatory molecules LAG-3 and TIM-3 is reduced in thawed TILs compared to fresh TILs. In some embodiments, there is no significant difference in the expression of CD4, CD8, NK, and TCRαβ. In some embodiments, there is no significant difference in CD4, CD8, NK, TCRαβ expression and / or memory markers in fresh TILs compared to thawed TILs. In some embodiments, there is no significant difference in CD4, CD8, NK, TCRαβ expression between TILs prepared by the methods provided herein and / or TILs prepared by other methods not provided herein, including, for example, methods not embodied in Figure 27.

[0356]

[0456] In some embodiments, the selection of a first TIL population, a second TIL population, a third TIL population, a recovered TIL population, and / or a therapeutic TIL population based on CD4, CD8, and / or NK, TCRαβ expression is not performed during any of the steps discussed above or including the example in Figure 27. In some embodiments, the selection of a first TIL population based on CD4, CD8, and / or NK, TCRαβ is not performed. In some embodiments, the selection of a second TIL population based on CD4, CD8, and / or NK, TCRαβ expression is not performed. In some embodiments, the selection of a third TIL population based on CD4, CD8, and / or NK, TCRαβ expression is not performed. In some embodiments, the selection of a recovered TIL population based on CD4, CD8, and / or NK, TCRαβ expression is not performed. In some embodiments, the selection of a therapeutic TIL population based on CD4, CD8, and / or NK, TCRαβ expression is not performed.

[0357]

[0457] In one embodiment, the selection of a first TIL population, a second TIL population, a third TIL population, or recovered TILs based on CD4, CD8 and / or NK, TCRαβ expression is not performed during any of steps (a) to (f) of the method for expanding the culture of tumor-infiltrating lymphocytes (TILs) into a therapeutic TIL population, and this step is (a) Obtaining a first TIL population from tumors excised from patients by processing tumor samples obtained from patients into multiple tumor fragments; (b) Adding tumor fragments to a closed system; (c) Performing a first expansion culture to produce a second TIL population by culturing a first TIL population in a cell culture medium containing IL-2, wherein the first expansion culture is performed in a closed container providing a first gas-permeable surface area, and the first expansion culture is performed for approximately 3 to 14 days to obtain a second TIL population, the second TIL population being at least 50 times larger in number 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 of the second TIL population by adding additional IL-2, OKT-3 and antigen-presenting cells (APCs) to the cell culture medium of the second TIL population to produce a third TIL population, the second expansion culture being performed over approximately 7–14 days to obtain the third TIL population, the third TIL population being a therapeutic TIL population including an increased subpopulation of effector T cells and / or central memory T cells compared to the second TIL population, the second expansion culture being performed in a closed vessel 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 opening the system; and (f) Transferring the TIL population recovered from step (e) to an infusion bag, wherein the transition from step (e) to (f) occurs without opening the system. Includes.

[0358]

[0458] In one embodiment, the selection of recovered TILs based on a first TIL population, a second TIL population, a third TIL 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 comprising administering the expanded cultured tumor-infiltrating lymphocytes (TILs) is (a) Obtaining a first TIL population from tumors resected from patients by processing tumor samples obtained from subjects into multiple tumor fragments; (b) Adding tumor fragments to a closed system; (c) Performing a first expansion culture to produce a second TIL population by culturing a first TIL population in a cell culture medium containing IL-2, wherein the first expansion culture is performed in a closed container providing a first gas-permeable surface area, and the first expansion culture is performed for approximately 3 to 14 days to obtain a second TIL population, the second TIL population being at least 50 times larger in number 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 of the second TIL population by adding additional IL-2, OKT-3 and antigen-presenting cells (APCs) to the cell culture medium of the second TIL population to produce a third TIL population, the second expansion culture being performed over approximately 7–14 days to obtain the third TIL population, the third TIL population being a therapeutic TIL population including an increased subpopulation of effector T cells and / or central memory T cells compared to the second TIL population, the second expansion culture being performed in a closed vessel 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 opening the system; and (f) Transferring the TIL population recovered from step (e) to an infusion bag, wherein the transition from step (e) to (f) occurs without opening the system; (g) optionally cryopreserve the infusion bag containing the TIL population recovered from step (f) using a cryopreservation process; and (h) Administer a third TIL population of a therapeutically effective dose to the patient from the infusion bag in step (g). Includes.

[0359]

[0459] In some embodiments, the memory marker is selected from the group consisting of CCR7 and CD62L.

[0360]

[0460] In some embodiments, the survival rate of fresh TIL compared to thawed TIL 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 survival rates of both fresh and thawed TIL are 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 survival rates of both fresh and thawed products are 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 survival rate of both fresh and thawed products is greater than 86%.

[0361]

[0461] In some embodiments, restimulated TILs can also be evaluated for cytokine release using a cytokine release assay. In some embodiments, TILs can be evaluated for interferon-7 (IFN-7) secretion in response to stimulation by either OKT3 or co-culture with autologous tumor digests. For example, in embodiments using OKT3 stimulation, the TILs are thoroughly washed and placed in a 96-well flat-bottom plate pre-coated with 0.1 or 1.0 μg / mL of OKT3 diluted in phosphate-buffered saline, with 1 × 10¹⁶ TILs per 0.2 mL CM. 5 Prepare duplicate wells with cells. After incubation overnight, collect the supernatant and measure IFN-7 in the supernatant by ELISA (Pierce / Endogen, Woburn, MA). For co-culture assays, 1 × 10⁶ 5 Individual TIL cells are placed in a 96-well plate together with autologous tumor cells (1:1 ratio). After 24 hours of incubation, the supernatant is collected, and IFN-7 release can be quantified, for example, by ELISA.

[0362]

[0462] Flow cytometry analysis of cell surface biomarkers: TIL samples were aliquoted for flow cytometry analysis of cell surface markers. See, for example, Examples 7, 8, and 9.

[0363]

[0463] In some embodiments, TIL is determined with respect to various regulatory markers. In some embodiments, the regulatory marker is selected from the group consisting of TCRα / β, CD56, 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 adjustment marker is KLRG1. In some embodiments, the adjustment marker is CD122.

[0364]

[0464] In one embodiment, expanded TILs are analyzed for the expression of numerous phenotypic markers, including those described herein and in the examples. In some embodiments, the 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, the expression of 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, or 14 markers is examined. In some embodiments, the expression of one or more markers from each group is examined. In some embodiments, one or more HLA-DR, CD38, and CD69 expressions are maintained in fresh TILs compared to thawed TILs (i.e., there is no statistically significant difference). In some embodiments, the activated state of TILs is maintained in thawed TILs.

[0365]

[0465] In some embodiments, the expression of one or more regulatory markers is measured. In some embodiments, the regulatory markers are 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 markers are selected from the group consisting of CD137, CD8a, Lag3, CD4, CD3, PD1, and TIM-3. In some embodiments, the regulatory markers are selected from the group consisting of CD69, CD8a, TIGIT, CD4, CD3, KLRG1, and CD154. In some embodiments, the expression of regulatory molecules is reduced in thawed TILs compared to fresh TILs. In some embodiments, the expression of regulatory molecules LAG-3 and TIM-3 is reduced in thawed TILs compared to fresh TILs. In some embodiments, there is no significant difference in the expression of CD4, CD8, NK, and TCRαβ. In some embodiments, there are no significant differences in CD4, CD8, NK, TCRαβ expression and / or memory markers in fresh TIL compared to thawed TIL.

[0366]

[0466] In some embodiments, the memory marker is selected from the group consisting of CCR7 and CD62L.

[0367]

[0467] In some embodiments, the survival rate of fresh TIL compared to thawed TIL 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 survival rates of both fresh and thawed TIL are 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 survival rates of both fresh and thawed products are 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 survival rate of both fresh and thawed products is greater than 86%.

[0368]

[0468] In some embodiments, restimulated TILs can also be evaluated for cytokine release using a cytokine release assay. In some embodiments, TILs can be evaluated for interferon-7 (IFN-7) secretion in response to stimulation by either OKT3 or co-culture with autologous tumor digests. For example, in embodiments using OKT3 stimulation, the TILs are thoroughly washed and placed in a 96-well flat-bottom plate pre-coated with 0.1 or 1.0 μg / mL of OKT3 diluted in phosphate-buffered saline, with 1 × 10¹⁶ TILs per 0.2 mL CM. 5 Prepare duplicate wells with cells. After incubation overnight, collect the supernatant and measure IFN-7 in the supernatant by ELISA (Pierce / Endogen, Woburn, MA). For co-culture assays, 1 × 10⁶ 5 Individual TIL cells are placed in a 96-well plate together with autologous tumor cells (1:1 ratio). After 24 hours of incubation, the supernatant is collected, and IFN-7 release can be quantified, for example, by ELISA.

[0369]

[0469] In some embodiments, phenotypic characterization is examined after cryopreservation.

[0370] J. Metabolic health of expanded cultured TILs

[0470] Restimulated TILs are characterized by a significant increase in basal glycolysis when compared to both freshly collected TILs and / or thawed TILs. In some embodiments, selection of a first TIL population, a second TIL population, a third TIL population, a recovered 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 example in Figure 27. In some embodiments, selection of a first TIL population based on CD8 expression is not performed. In some embodiments, selection of a second TIL population based on CD8 expression is not performed. In some embodiments, selection of a third TIL population based on CD8 expression is not performed. In some embodiments, selection of a recovered TIL population based on CD8 expression is not performed. In some embodiments, selection of a therapeutic TIL population based on CD8 expression is not performed.

[0371]

[0471] In one embodiment, the selection of a first TIL population, a second TIL population, a third TIL population, or recovered TILs based on CD8 expression is not performed during any of steps (a) to (f) of the method for expanding the culture of tumor-infiltrating lymphocytes (TILs) into a therapeutic TIL population, and this step is performed. (a) Obtaining a first TIL population from tumors excised from patients by processing tumor samples obtained from patients into multiple tumor fragments; (b) Adding tumor fragments to a closed system; (c) Performing a first expansion culture to produce a second TIL population by culturing a first TIL population in a cell culture medium containing IL-2, wherein the first expansion culture is performed in a closed container providing a first gas-permeable surface area, and the first expansion culture is performed for approximately 3 to 14 days to obtain a second TIL population, the second TIL population being at least 50 times larger in number 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 of the second TIL population by adding additional IL-2, OKT-3 and antigen-presenting cells (APCs) to the cell culture medium of the second TIL population to produce a third TIL population, the second expansion culture being performed over approximately 7–14 days to obtain the third TIL population, the third TIL population being a therapeutic TIL population including an increased subpopulation of effector T cells and / or central memory T cells compared to the second TIL population, the second expansion culture being performed in a closed vessel 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 opening the system; and (f) Transferring the TIL population recovered from step (e) to an infusion bag, wherein the transition from step (e) to (f) occurs without opening the system. Includes.

[0372]

[0472] In one embodiment, the selection of a first TIL population, a second TIL population, a third TIL population, or a recovered TIL based on CD8 expression is not performed during any of steps (a) to (h) of the method for treating a subject with cancer, and the method, which includes administering the expanded cultured tumor-infiltrating lymphocytes (TILs), (a) Obtaining a first TIL population from tumors resected from patients by processing tumor samples obtained from subjects into multiple tumor fragments; (b) Adding tumor fragments to a closed system; (c) Performing a first expansion culture to produce a second TIL population by culturing a first TIL population in a cell culture medium containing IL-2, wherein the first expansion culture is performed in a closed container providing a first gas-permeable surface area, and the first expansion culture is performed for approximately 3 to 14 days to obtain a second TIL population, the second TIL population being at least 50 times larger in number 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 of the second TIL population by adding additional IL-2, OKT-3, and antigen-presenting cells (APCs) to the cell culture medium of the second TIL population to produce a third TIL population, the second expansion culture being performed over approximately 7–14 days to obtain the third TIL population, the third TIL population being a T-cell therapeutic TIL population including an increased subpopulation of effector T cells and / or central memory T cells compared to the second TIL population, the second expansion culture being performed in a closed vessel 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 opening the system; and (f) Transferring the TIL population recovered from step (e) to an infusion bag, wherein the transition from step (e) to (f) occurs without opening the system; (g) optionally cryopreserve the infusion bag containing the TIL population recovered from step (f) using a cryopreservation process; and (h) Administer a third TIL population of a therapeutically effective dose to the patient from the infusion bag in step (g). Includes.

[0373]

[0473] TILs prepared by the methods described herein are characterized by a significant difference in basal glycolysis compared to TILs prepared using methods other than those provided herein, including, for example, freshly harvested TILs and / or methods other than those embodied in Figure 27. In some embodiments, selection of a first TIL population, a second TIL population, a third TIL population, a recovered 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 example in Figure 27. In some embodiments, selection of a first TIL population based on CD8 expression is not performed. In some embodiments, selection of a second TIL population based on CD8 expression is not performed. In some embodiments, selection of a third TIL population based on CD8 expression is not performed. In some embodiments, selection of a recovered TIL population based on CD8 expression is not performed. In some embodiments, selection of a therapeutic TIL population based on CD8 expression is not performed. In one embodiment, the selection of recovered TILs based on a first TIL population, a second TIL population, a third TIL population, or CD8 expression is not performed during any of steps (a) to (h).

[0374]

[0474] For TILs cultured using the different methods described herein, the reserve respiratory capacity (SRC) and glycolytic reserve can be determined. The Seahorse XF cell mitostress test measures mitochondrial function by directly measuring the 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 injected sequentially to measure ATP production, maximal respiration, and non-mitochondrial respiration, respectively. These parameters and basal respiration are then used to calculate proton leak and reserve 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 related to cellular ATP production. Carbonyl cyanide-4 (trifluoromethoxy)phenylhydrazone (FCCP) is an uncoupling agent that disrupts the proton gradient and destroys the mitochondrial membrane potential. As a result, electron flow through the electron transport chain is not suppressed, and oxygen is consumed to the maximum extent by complex IV. Subsequently, the reserve respiratory volume, defined as the difference between maximal and basal respiration, can be calculated using FCCP-stimulated OCR. Reserve respiratory volume (SRC) is a measure of a cell's ability 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 driven by processes outside the mitochondria. In some embodiments, comparisons are made, for example, with 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.

[0375]

[0475] In some embodiments, the metabolic assay is basal respiration. Generally, the second expanded cultured TIL has a basal respiration rate of 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 collected TIL and / or TIL prepared using methods other than those provided herein, including methods other than those embodied in Figure 27. In some embodiments, the basal respiration rate is about 50% to about 99% of the basal respiration rate of freshly collected TIL and / or TIL prepared using methods other than those provided herein, including methods other than those embodied in Figure 27. In some embodiments, the basal respiration rate is about 60% to about 99% of the basal respiration rate of freshly collected TIL and / or TIL prepared using methods other than those provided herein, including methods other than those embodied in Figure 27. In some embodiments, the basal respiratory rate is about 70% to about 99% of the basal respiratory rate of freshly collected TIL and / or TIL prepared using methods other than those provided herein, including methods other than those embodied in Figure 27. In some embodiments, the basal respiratory rate is about 80% to about 99% of the basal respiratory rate of freshly collected TIL and / or TIL prepared using methods other than those provided herein, including methods other than those embodied in Figure 27. In some embodiments, the basal respiratory rate is about 90% to about 99% of the basal respiratory rate of freshly collected TIL and / or TIL prepared using methods other than those provided herein, including methods other than those embodied in Figure 27. In some embodiments, the basal respiratory rate is about 95% to about 99% of the basal respiratory rate of freshly collected TIL and / or TIL prepared using methods other than those provided herein, including methods other than those embodied in Figure 27.In some embodiments, a second expanded culture TIL or a second additional expanded culture TIL (including, for example, a TIL referred to as a reREP TIL, as described in step D of Figure 27) has a basal respiratory rate that is not statistically significant with that of a freshly harvested TIL and / or a TIL prepared using methods other than those provided herein, including methods other than those embodied in Figure 27. In some embodiments, the comparison is against a freshly harvested TIL and / or a TIL prepared using methods other than those provided herein, including methods other than those embodied in Figure 27.

[0376]

[0476] In some embodiments, the metabolic assay is reserve respiratory volume. Generally, a second expanded culture TIL or a second additional expanded culture TIL (e.g., including a TIL referred to as a reREP TIL, as described in step D of Figure 27) has a reserve respiratory volume of 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 respiratory rate of a freshly collected TIL and / or a TIL prepared using other methods other than those provided herein, including methods other than those embodied in Figure 27. In some embodiments, the reserve respiratory volume is about 50% to about 99% of the basal respiratory rate of a freshly collected TIL. In some embodiments, the reserve respiratory volume is about 50% to about 99% of the basal respiratory rate of freshly collected TIL and / or TIL prepared using methods other than those provided herein, including methods other than those embodied in Figure 27. In some embodiments, the reserve respiratory volume is about 60% to about 99% of the basal respiratory rate of freshly collected TIL and / or TIL prepared using methods other than those provided herein, including methods other than those embodied in Figure 27. In some embodiments, the reserve respiratory volume is about 70% to about 99% of the basal respiratory rate of freshly collected TIL and / or TIL prepared using methods other than those provided herein, including methods other than those embodied in Figure 27. In some embodiments, the reserve respiratory volume is about 80% to about 99% of the basal respiratory rate of freshly collected TIL. In some embodiments, the reserve respiratory volume is about 90% to about 99% of the basal respiratory rate of freshly collected TIL and / or TIL prepared using methods other than those provided herein, including methods other than those embodied in Figure 27. In some embodiments, the reserve respiratory volume is about 95% to about 99% of the basal respiratory rate of freshly collected TIL and / or TIL prepared using methods other than those provided herein, including methods other than those embodied in Figure 27.In some embodiments, a second expanded cultured TIL or a second additional expanded cultured TIL (including, for example, a TIL referred to as a reREP TIL, as described in step D of Figure 27) has a reserve respiratory volume that does not show a statistically significant difference from the basal respiratory rate of a freshly harvested TIL and / or a TIL prepared using other methods other than those provided herein, including methods other than those embodied in, for example, Figure 27.

[0377]

[0477] Generally, a second expanded culture TIL or a second additional expanded culture TIL (including, for example, a TIL referred to as reREP TIL, as described in step D of Figure 27) has a reserve respiratory capacity of 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 respiratory rate of a freshly harvested TIL and / or a TIL prepared using methods other than those provided herein, including methods other than those embodied in Figure 27. In some embodiments, the metabolic assay measured is glycolytic reserve. In some embodiments, the metabolic assay is reserve respiratory capacity. To measure cellular (respiratory) metabolism, cells were treated with inhibitors of mitochondrial respiration and glycolysis to determine the metabolic profile of the TIL, consisting of the following measures: baseline oxidative phosphorylation (measured by OCR), reserve respiratory capacity, baseline glycolytic activity (measured by ECAR), and glycolytic reserve. Metabolic profiles were performed using the Seahorse combination mitochondrial / glycolytic stress test assay (including kits commercially available from Agilent®), which can determine the ability of cells to perform glycolysis when mitochondrial ATP production is blocked. In some embodiments, cells are placed in a glucose-starved state, then injected with glucose, followed by injection of a stress agonist. In some embodiments, the stress agonist 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 capacity and / or non-glycolytic acidification are measured.Generally, TIL has a glycolytic reserve of 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 respiratory rate of freshly collected TIL and / or TIL prepared using methods other than those provided herein, including methods other than those embodied in Figure 27. In some embodiments, the glycolytic reserve is about 50% to about 99% of the basal respiratory rate of freshly collected TIL and / or TIL prepared using methods other than those provided herein, including methods other than those embodied in Figure 27. In some embodiments, the glycolytic reserve is about 60% to about 99% of the basal respiratory rate of freshly collected TIL and / or TIL prepared using methods other than those provided herein, including methods other than those embodied in Figure 27. In some embodiments, glycolytic reserve is the basal respiratory rate of about 70% to about 99% of freshly collected TIL and / or TIL prepared using methods other than those provided herein, including methods other than those embodied in Figure 27. In some embodiments, glycolytic reserve is the basal respiratory rate of about 80% to about 99% of freshly collected TIL and / or TIL prepared using methods other than those provided herein, including methods other than those embodied in Figure 27. In some embodiments, glycolytic reserve is the basal respiratory rate of about 90% to about 99% of freshly collected TIL and / or TIL prepared using methods other than those provided herein, including methods other than those embodied in Figure 27. In some embodiments, glycolytic reserve is the basal respiratory rate of about 95% to about 99% of freshly collected TIL and / or TIL prepared using methods other than those provided herein, including methods other than those embodied in Figure 27.

[0378]

[0478] In some embodiments, the metabolic assay is basal glycolysis. In some embodiments, a second expanded culture TIL or a second additional expanded culture TIL (e.g., including a TIL referred to as a reREP TIL, as described in step D of Figure 27) has at least a 2-fold, 3-fold, 4-fold, 5-fold, 6-fold, 7-fold, 8-fold, 9-fold, or 10-fold increase in basal glycolysis compared to a freshly harvested TIL and / or a TIL prepared using other methods other than those provided herein, including methods other than those embodied in Figure 27. In some embodiments, a second expanded culture TIL or a second additional expanded culture TIL (e.g., including a TIL referred to as a reREP TIL, as described in step D of Figure 27) has about a 2-fold to about a 10-fold increase in basal glycolysis compared to a freshly harvested TIL and / or a TIL prepared using other methods other than those provided herein, including methods other than those embodied in Figure 27. In some embodiments, a second expanded culture TIL or a second additional expanded culture TIL (e.g., including a TIL referred to as reREP TIL, as described in step D of Figure 27) has about 2 to 8 times the increase in basal glycolysis compared to freshly harvested TIL and / or TIL prepared using other methods other than those provided herein, including methods other than those embodied in Figure 27. In some embodiments, a second expanded culture TIL or a second additional expanded culture TIL (e.g., including a TIL referred to as reREP TIL, as described in step D of Figure 27) has about 3 to 7 times the increase in basal glycolysis compared to freshly harvested TIL and / or TIL prepared using other methods other than those provided herein, including methods other than those embodied in Figure 27. In some embodiments, a second expanded culture TIL or a second additional expanded culture TIL (including, for example, a TIL referred to as reREP TIL, as described in step D of Figure 27) has about 2 to 4 times the increase in basal glycolysis compared to freshly harvested TIL and / or TIL prepared using other methods other than those provided herein, including methods other than those embodied in, for example, Figure 27.In some embodiments, a second expanded culture TIL or a second additional expanded culture TIL (including, for example, a TIL referred to as reREP TIL, as described in step D of Figure 27) has about 2 to 3 times the increase in basal glycolysis compared to freshly harvested TIL and / or TIL prepared using other methods other than those provided herein, including methods other than those embodied in, for example, Figure 27.

[0379]

[0479] Generally, a second expanded culture TIL or a second additional expanded culture TIL (including, for example, a TIL referred to as a reREP TIL, as described in step D of Figure 27) has 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 glycolytic reserve of a freshly harvested TIL and / or a TIL prepared using other methods not provided herein, including methods other than those embodied in, for example, Figure 27. In some embodiments, the glycolytic reserve is about 50% to about 99% of the basal respiratory rate of a freshly harvested TIL. In some embodiments, the glycolytic reserve is about 60% to about 99% of the basal respiratory rate of a freshly harvested TIL and / or a TIL prepared using other methods not provided herein, including methods other than those embodied in, for example, Figure 27. In some embodiments, glycolytic reserve is the basal respiratory rate of freshly collected TIL and / or TIL prepared using methods other than those provided herein, including methods other than those embodied in Figure 27. In some embodiments, glycolytic reserve is the basal respiratory rate of freshly collected TIL and / or TIL prepared using methods other than those provided herein, including methods other than those embodied in Figure 27. In some embodiments, glycolytic reserve is the basal respiratory rate of freshly collected TIL and / or TIL prepared using methods other than those provided herein, including methods other than those embodied in Figure 27. In some embodiments, glycolytic reserve is the basal respiratory rate of freshly collected TIL and / or TIL prepared using methods other than those provided herein, including methods other than those embodied in Figure 27. In some embodiments, glycolytic reserve is the basal respiratory rate of freshly collected TIL.

[0380]

[0480] Granzyme B Production: Granzyme B is another measure of the ability of a TIL to kill target cells. Granzyme B levels were also evaluated in the supernatant of culture media restimulated as described above using antibodies against CD3, CD28, and CD137 / 4-1BB, using the Human Granzyme B DuoSet ELISA Kit (R&D Systems, Minneapolis, MN) according to the manufacturer's instructions. In some embodiments, a second expanded culture TIL or a second additional expanded culture TIL (e.g., including a TIL referred to as a reREP TIL, as described in step D of Figure 27) increases granzyme B production. In some embodiments, a second expanded culture TIL or a second additional expanded culture TIL (e.g., including a TIL referred to as a reREP TIL, as described in step D of Figure 27) increases cytotoxic activity.

[0381]

[0481] In some embodiments, telomere length can be used as a measure of cell viability and / or cellular function. In some embodiments, telomeres were remarkably identical in length to those of TILs prepared by the present invention compared to TILs prepared using methods other than those provided herein, including methods other than those embodied in Figure 27. 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 ultimate criterion for measuring telomere length (de Lange et al., 1990). However, the main limitation of TRF is that it requires a large amount of DNA (1.5 g). Two widely used techniques for measuring telomere length, namely fluorescence in situ hybridization (FISH; Agilent Technologies, Santa Clara, CA) and quantitative PCR, can be used in the present invention. In some embodiments, there is no change in telomere length between TILs initially recovered in step A and TILs expanded and cultured from step D, for example, provided in Figure 27.

[0382]

[0482] In some embodiments, TIL health is measured by IFN-gamma (IFN-γ) secretion. In some embodiments, IFN-γ secretion is an indicator of active TILs. In some embodiments, an efficacy 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 culture medium of TILs stimulated with antibodies against CD3, CD28, and CD137 / 4-1BB. The level of IFN-γ in the culture medium from these stimulated TILs can be determined by measuring IFN-γ release. In some embodiments, for example, the TIL shown in Figure 27 shows increased IFN-γ production in step D compared to the TIL initially harvested in step A, as shown in Figure 27. This increase in IFN-γ production in step D is an indicator of increased cytotoxicity of the TIL in step D. In some embodiments, IFN-γ secretion increases 1-fold, 2-fold, 3-fold, 4-fold, or 5-fold or more. In some embodiments, IFN-γ secretion increases 1-fold. In some embodiments, IFN-γ secretion increases 2-fold. In some embodiments, IFN-γ secretion increases threefold. In some embodiments, IFN-γ secretion increases fourfold. In some embodiments, IFN-γ secretion increases fivefold. In some embodiments, IFN-γ is measured using...

Claims

1. A method for expanding the culture of tumor-infiltrating lymphocytes (TILs) into a therapeutic TIL population, (a) Add processed tumor fragments from tumors resected from patients to a closed system to obtain a first TIL population; (b) Performing a first expansion culture to produce a second TIL population by culturing the first TIL population in a cell culture medium containing IL-2, wherein the first expansion culture is performed in a closed container providing a first gas-permeable surface area, and the first expansion culture is performed for 7 to 11 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 to produce a third TIL population by adding additional IL-2 and antigen-presenting cells (APCs) to the cell culture medium of the second TIL population, wherein the second expansion culture is performed for 108 to 132 hours to obtain the third TIL population, the second expansion culture is performed in a closed container providing a second gas-permeable surface area, and the transition from step (b) to step (c) occurs without opening the system; (d) The third TIL population is a first of five or fewer TIL subpopulations, each having at least 1.0 × 10 ９ Dividing the TILs into subpopulations containing individual TILs, and then adding additional IL-2 to the cell culture medium of each of the TIL subpopulations to perform a third expansion culture of the first five or fewer TIL subpopulations to produce a second plurality of TIL subpopulations, wherein the second plurality of TIL subpopulations includes a therapeutic TIL population, the third expansion culture is performed over a period of 132 to 156 hours, the third expansion culture for each of the TIL subpopulations is performed in a closed container providing a third 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 opening the system; and (f) Transferring the recovered TIL mass from step (e) to a plurality of infusion bags, wherein the transition from step (e) to (f) occurs without opening the system. A method that includes this.

2. The method according to claim 1, wherein in step (b), (c), and / or (d), the cell culture medium further comprises OKT-3.

3. The method according to claim 1, wherein the recovered TIL mass from step (e) is transferred to 3 to 4 infusion bags.

4. The method according to any one of claims 1 to 3, wherein step (b) is carried out over a period of 10, 11, or 12 days.

5. The method according to any one of claims 1 to 3, wherein step (b) is carried out within a period of 11 days.

6. The method according to claim 1, wherein step (c) is performed for 108 hours, 5 days, or 132 hours.

7. The method according to claim 6, wherein step (c) is carried out for five days.

8. The method according to any one of claims 1 to 7, wherein step (d) is carried out for six days.

9. The method according to any one of claims 1 to 8, wherein steps (a) to (f) are carried out within a period of 10 to 22 days.

10. The method according to any one of claims 1 to 8, wherein steps (a) to (f) are carried out within a period of 10 to 20 days.

11. The method according to any one of claims 1 to 8, wherein steps (a) to (f) are carried out within a period of 10 to 15 days.

12. The method according to any one of claims 1 to 11, wherein steps (c) and (d) are carried out within a period of 10 to 12 days.

13. The method according to claim 12, wherein steps (c) and (d) are carried out within a period of 10, 11, or 12 days.

14. The method according to any one of claims 1 to 13, wherein the therapeutic TIL population comprises 1.5 × 10⁹ to 1.5 × 10¹⁰ TILs.

15. The therapeutic TIL population was 7.8 × 10 ９ pieces ~ 6.7×10 １０ The method according to any one of claims 1 to 13, comprising TILs.

16. The method according to any one of claims 1 to 15, wherein the second TIL group is at least 50 times larger in number than the first TIL group.

17. The method according to any one of claims 1 to 16, further comprising the step of freezing and preserving the infusion bag containing the recovered TIL population using a cryopreservation process.

18. The method according to claim 17, wherein the cryopreservation process is carried out using a 1:1 ratio of the recovered TIL population to the cryopreservation medium.

19. The method according to claim 18, wherein the frozen infusion bags are placed in a cassette.

20. The method according to claim 19, wherein the cassette is made of aluminum.

21. The method according to any one of claims 1 to 20, wherein the infusion bag in step (f) is a freezer bag.

22. The method according to any one of claims 1 to 21, wherein the tumor fragment is obtained from a tumor excised from a patient suffering from metastatic melanoma.

23. The method according to any one of claims 1 to 22, wherein each infusion bag contains a final volume of 100 mL to 600 mL.

24. The method according to any one of claims 1 to 23, wherein the closed system is accessible via syringe under sterile conditions, and / or the closed system includes a Luer lock and a heat seal system.

25. The method according to any one of claims 1 to 24, further comprising adding IL-2 to the recovered TIL.

26. The method according to claim 25, wherein IL-2 is added to a final concentration of 300 IU / mL.

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