Treatment of cancer patients with tumor-infiltrating lymphocyte therapy in combination with KRAS inhibitors

Abstract

The present invention provides improved and / or shortened processes and methods for preparing TILs in combination with KRAS inhibitors to prepare therapeutic TIL populations with increased therapeutic efficacy for the treatment of cancers harboring KRAS mutations with TILs as described herein.

Description

[Technical Field]

[0001] CROSS-REFERENCE TO RELATED APPLICATIONS This application claims priority to U.S. Provisional Application No. 63 / 226,751, filed July 28, 2021, the disclosure of which is incorporated herein in its entirety. [Background technology]

[0002] The treatment of bulky, refractory cancers using adoptive autologous transfer of tumor-infiltrating lymphocytes (TILs) offers a powerful approach to treating patients with poor prognosis. Gattinoni, et al., Nat. Rev. Immunol. 2006, 6, 383-393. TILs are dominated by T cells, and IL-2-based TIL expansion followed by the "rapid expansion process" (REP) has become the preferred method of TIL expansion due to its 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. Many approaches to improving response to TIL therapy in melanoma and extending TIL therapy to other tumor types have met with limited success, and the field remains challenging. Goff, et al., J. Clin. Oncol. 2016, 34, 2389-97; Dudley, et al., J. Clin. Oncol. 2008, 26, 5233-39; Rosenberg, et al., Clin. Cancer Res. 2011, 17, 4550-57. Combination studies with single immune checkpoint inhibitors have also been described, but further studies are ongoing and additional treatment options are needed (Kverneland, et al., Oncotarget, 2020, 11(22), 2092-2105).

[0003] Furthermore, current TIL production and treatment processes are limited by length, cost, sterility concerns, and other factors described herein, thus significantly limiting the ability to treat patients who are refractory to KRAS inhibitor treatment. There is an urgent need to provide TIL production processes and therapies based on such processes that are suitable for use in treating patients who have few or no remaining viable treatment options. The present invention fills this need by providing a manufacturing process for use in generating TILs that can be used in combination with KRAS inhibitors to treat patients. Summary of the Invention

[0004] Provided herein are methods of treating cancer in a patient using an expanded TIL population and at least one KRAS inhibitor, as well as methods of generating a therapeutic TIL population from a patient or subject pretreated with at least one KRAS inhibitor.

[0005] In some embodiments, the disclosure provides a method of treating cancer in a patient or subject in need thereof, comprising administering a population of tumor-infiltrating lymphocytes (TILs) and at least one KRAS inhibitor, optionally wherein the patient or subject has received at least one prior therapy, wherein the at least one prior therapy optionally includes an anti-PD1 antibody.

[0006] In some embodiments, the present disclosure provides a method of treating cancer in a patient or subject in need thereof, comprising administering a population of tumor-infiltrating lymphocytes (TILs) and at least one KRAS inhibitor, the method comprising: (a) obtaining and / or receiving a first population of TILs from a tumor excised from a subject or patient by processing a tumor sample obtained from the subject into a plurality of tumor fragments; (b) adding the first population of TILs to the closed system; (c) performing a first expansion by culturing the first TIL population in a cell culture medium containing IL-2 to generate a second TIL population, wherein the first expansion is performed in a closed container that provides a first gas permeable surface area, and the first expansion is performed for about 3 to 14 days to obtain a second TIL population, and the transition from step (b) to step (c) occurs without opening the system to generate the second TIL population; (d) performing a second expansion by supplementing the cell culture medium of the second TIL population with additional IL-2, OKT-3, and antigen-presenting cells (APCs) to generate a third TIL population, wherein the second expansion is performed for about 7 to 14 days to obtain a third TIL population, the third TIL population being a therapeutic TIL population, and wherein the second expansion is performed in a closed container that provides a second gas-permeable surface area, and wherein the transition from step (c) to step (d) occurs without opening the system. (e) harvesting the therapeutic TIL population obtained from step (d), wherein the transition from step (d) to step (e) occurs without opening the system; (f) transferring the harvested TIL population from step (e) to an infusion bag, wherein the transfer from step (e) to (f) occurs without opening the system; (g) cryopreserving the infusion bag containing the harvested TIL population from step (f) using a cryopreservation process; (h) administering a therapeutically effective dose of the third population of TILs from the infusion bag of step (g) to the subject; (i) administering at least one KRAS inhibitor to the subject.

[0007] In some embodiments, the present disclosure provides a method of treating cancer in a patient or subject in need thereof, comprising administering a population of tumor-infiltrating lymphocytes (TILs) and at least one KRAS inhibitor, the method comprising: (a) obtaining a first population of TILs from a tumor excised from a subject by processing a tumor sample obtained from the subject into a plurality of tumor fragments; (b) adding tumor fragments to the closed system; (c) performing a first expansion by culturing the first TIL population in a cell culture medium comprising IL-2 to generate a second TIL population, wherein the first expansion is performed in a closed container providing a first gas permeable surface area, and wherein the first expansion is performed for about 3-11 days to obtain a second TIL population, wherein the second TIL population is at least 50-fold more numerous than the first TIL population, and wherein the transition from step (b) to step (c) occurs without opening the system; (d) performing a second expansion by supplementing the cell culture medium of the second TIL population with additional IL-2, OKT-3, and antigen-presenting cells (APCs) to generate a third TIL population, wherein the second expansion is performed for about 7 to 11 days to obtain a third TIL population, and wherein the second expansion is performed in a closed container that provides a second gas-permeable surface area, and the transition from step (c) to step (d) occurs without opening the system to generate the third TIL population; (e) harvesting the third population of TILs obtained from step (d), wherein the transition from step (d) to step (e) occurs without opening the system; (f) transferring the harvested third population of TILs from step (e) to an infusion bag, wherein the transfer from step (e) to (f) occurs without opening the system; (g) cryopreserving the infusion bag containing the harvested TIL population from step (f) using a cryopreservation process; (h) administering a therapeutically effective dose of the third population of TILs from the infusion bag of step (g) to the subject; (i) administering at least one KRAS inhibitor to the subject.

[0008] In some embodiments, the present disclosure provides a method of treating cancer in a patient or subject in need thereof, comprising administering a population of tumor-infiltrating lymphocytes (TILs) and at least one KRAS inhibitor, the method comprising: (a) obtaining and / or receiving a first population of TILs from a surgical resection, needle biopsy, core biopsy, mini-biopsy, or other means for obtaining a sample containing a mixture of tumor cells and TIL cells from a patient or subject; (b) adding the first population of TILs to the closed system; (c) performing a first expansion by culturing the first TIL population in a cell culture medium containing IL-2 to generate a second TIL population, wherein the first expansion is performed in a closed container that provides a first gas permeable surface area, and the first expansion is performed for about 3 to 11 days to obtain a second TIL population, and the transition from step (b) to step (c) occurs without opening the system to generate a second TIL population; (d) performing a second expansion by supplementing the cell culture medium of the second TIL population with additional IL-2, OKT-3, and antigen-presenting cells (APCs) to generate a third TIL population, wherein the second expansion is performed for about 7 to 11 days to obtain a third TIL population, and wherein the second expansion is performed in a closed container that provides a second gas-permeable surface area, and the transition from step (c) to step (d) occurs without opening the system to generate the third TIL population; (e) harvesting the third population of TILs obtained from step (d), wherein the transition from step (d) to step (e) occurs without opening the system; (f) transferring the harvested third population of TILs from step (e) to an infusion bag, wherein the transfer from step (e) to (f) occurs without opening the system; (g) cryopreserving the infusion bag containing the harvested TIL population from step (f) using a cryopreservation process; (h) administering a therapeutically effective dose of the third population of TILs from the infusion bag of step (g) to the subject; (i) administering at least one KRAS inhibitor to the subject.

[0009] In some embodiments, the present disclosure provides a method of treating cancer in a patient or subject in need thereof, comprising administering a population of tumor-infiltrating lymphocytes (TILs) and at least one KRAS inhibitor, the method comprising: (a) optionally, by surgical resection, needle biopsy, core biopsy, mini-biopsy, or other means for obtaining a sample containing a mixture of tumor cells and TIL cells from the tumor, removing a tumor from a subject or patient, wherein the tumor comprises a first TIL population; (b) adding tumor fragments to the closed system; (c) performing a first expansion by culturing the first TIL population in a cell culture medium containing IL-2 to generate a second TIL population, wherein the first expansion is performed in a closed container that provides a first gas permeable surface area, and the first expansion is performed for about 3 to 11 days to obtain a second TIL population, and the transition from step (b) to step (c) occurs without opening the system to generate the second TIL population; (d) performing a second expansion by supplementing the cell culture medium of the second TIL population with additional IL-2, OKT-3, and antigen-presenting cells (APCs) to generate a third TIL population, wherein the second expansion is performed for about 7 to 11 days to obtain a third TIL population, and wherein the second expansion is performed in a closed container that provides a second gas-permeable surface area, and the transition from step (c) to step (d) occurs without opening the system to generate the third TIL population; (e) harvesting the third population of TILs obtained from step (d), wherein the transition from step (d) to step (e) occurs without opening the system; (f) transferring the harvested third population of TILs from step (e) to an infusion bag, wherein the transfer from step (e) to (f) occurs without opening the system; (g) cryopreserving the infusion bag containing the harvested TIL population from step (f) using a cryopreservation process; (h) administering a therapeutically effective dose of the third population of TILs from the infusion bag of step (g) to a subject or patient with cancer; (i) administering at least one KRAS inhibitor to the subject.

[0010] In some embodiments, the present disclosure provides a method of treating cancer in a patient or subject in need thereof, comprising administering a population of tumor-infiltrating lymphocytes (TILs) and at least one KRAS inhibitor, the method comprising: (a) obtaining and / or receiving a first population of TILs from a surgical resection, needle biopsy, core biopsy, mini-biopsy, or other means for obtaining a sample containing a mixture of tumor cells and TIL cells from a subject or patient; (c) contacting the first TIL population with a first cell culture medium; (d) performing an initial expansion (or first expansion by priming) of the first TIL population in a first cell culture medium to obtain a second TIL population, wherein the first cell culture medium comprises IL-2, and optionally the first expansion by priming occurs over a period of 1 to 8 days; (e) performing rapid expansion of the second TIL population in a second cell culture medium comprising IL-2, OKT-3 (anti-CD3 antibody), and optionally irradiated allogeneic peripheral blood mononuclear cells (PBMCs), and the rapid expansion is performed over a period of no more than 14 days, and optionally the second TIL expansion can proceed for 1 day, 2 days, 3 days, 4 days, 5 days, 6 days, 7 days, 8 days, 9 days, or 10 days after initiation of the rapid second expansion to obtain a third TIL population; (f) harvesting a third population of TILs; (g) administering a therapeutically effective portion of the third population of TILs to a subject or patient with cancer; (i) administering at least one KRAS inhibitor to the subject.

[0011] In some embodiments, the present disclosure provides a method of treating cancer in a patient or subject in need thereof, comprising administering a population of tumor-infiltrating lymphocytes (TILs) and at least one KRAS inhibitor, the method comprising: (a) optionally, removing a tumor from a subject or patient by surgical resection, needle biopsy, core biopsy, mini-biopsy, or other means for obtaining a sample containing a mixture of tumor cells and TIL cells from the tumor, wherein the subject or patient has previously been treated with at least one KRAS inhibitor; (b) fragmenting the tumor into tumor fragments; (c) contacting the tumor fragment with a first cell culture medium; (d) performing an initial expansion (or first expansion by priming) of the first TIL population in a first cell culture medium to obtain a second TIL population, wherein the second TIL population is at least 5 times more numerous than the first TIL population, the first cell culture medium comprising IL-2, and optionally the first expansion by priming occurs over a period of 1 to 8 days; (e) performing rapid expansion of the second TIL population in a second cell culture medium to obtain a third TIL population, wherein the third TIL population is at least 50-fold more numerous than the second TIL population 7-8 days after initiation of rapid expansion, the second cell culture medium comprising IL-2, OKT-3 (an anti-CD3 antibody), and optionally irradiated allogeneic peripheral blood mononuclear cells (PBMCs), the rapid expansion being performed over a period of 14 days or less, and optionally the second TIL expansion being allowed to proceed for 1 day, 2 days, 3 days, 4 days, 5 days, 6 days, 7 days, 8 days, 9 days, or 10 days after initiation of rapid second expansion; (f) harvesting a third population of TILs; (g) administering a therapeutically effective portion of the third population of TILs to a subject or patient with cancer; (i) administering at least one KRAS inhibitor to the subject.

[0012] In some embodiments, the second TIL population is at least 50-fold more numerous than the first TIL population.

[0013] In some embodiments, the patient or subject has a cancer that is NSCLC, wherein the NSCLC is unresectable, metastatic, resistant and / or refractory to a KRAS inhibitor.

[0014] In some embodiments, the patient or subject has a KRAS gene mutation.

[0015] In some embodiments, the patient or subject has a cancer that exhibits the p.G12C mutation.

[0016] In some embodiments, the cancer has been previously treated with a KRAS inhibitor.

[0017] In some embodiments, the cancer has not been previously treated with a KRAS inhibitor.

[0018] In some embodiments, the KRAS inhibitor is selected from the group consisting of AMG-510 (sotorasib), AMG-510 racemate (sotorasib racemate), MRTX849 (adagrasib), JNJ-74699157 / ARS-3248, JDQ443, LY3499446, LY3537982, RLY-1971 / GDC-6036, BBP-454, BI 1701963, BI 1823911, mRNA-5671 / V941, D-1553, BI-2852, BI-3406, ARS-1620, BAY-293, MRTX-1257, PROTAC K-Ras Degrader-1, LC-2, ARS-853, ARS-1323, ARS-1323-alkyne, ARS-1630, K-Ras G12C-IN-2, KRAS inhibitor-6, KRAS inhibitor 8, KRAS inhibitor 7, KRAS G12C inhibitor 15, KRAS G12C inhibitor 5, KRAS G12C inhibitor 13, KRAS G12C inhibitor 17, KRAS G12C inhibitor 16, KRAS G12C inhibitor 14, KRas G12C inhibitor 4, KRas G12C inhibitor 1, KRas G12C inhibitor 3, KRas G12C inhibitor 2, 6H05, SAH-SOS1ATFA, KRAS inhibitor 10, SAH-SOS1A, atorvastatin-PEG3-FITC, C6ME, CS-0115617, HY-130260, HY-135864, HY-135866, Cmpd2, CS-0115618, CS-0115620, EX-A4387, CS-01 06134, HY-135865, 2241719-75-3, HY-125873, CS-0046138, CS-0046137, CS-01 01474, HY-125875, CS-0102608, CS-0102610, CS-0102606, HY-112493, CS-0046 139, 1-{4-[6-chloro-8-fluoro-7-(5-methyl-1h-indazol-4-yl)quinazolin-4-yl]piperazin-1-yl}propan-1-one, HY-126292, HY-112491, CS-0046136, HY-114168, HY-125874, HY-112494, CS-0102607, BCP29475|2206735-61-5, HY-112492, CS-0078097, 2158296-45-6, HY-125872, and 2158297-63-1, and pharmaceutically acceptable salts thereof.

[0019] In some embodiments, the cancer has been previously treated with a PD-1 inhibitor and / or a PD-L1 inhibitor or a biosimilar thereof.

[0020] In some embodiments, the PD-1 inhibitor is selected from the group consisting of nivolumab, pembrolizumab, and biosimilars thereof.

[0021] In some embodiments, the PD-L1 inhibitor is selected from the group consisting of avelumab, atezolizumab, durvalumab, and biosimilars thereof.

[0022] In some embodiments, the cancer has not been previously treated with a PD-1 inhibitor and / or a PD-L1 inhibitor or a biosimilar thereof.

[0023] In some embodiments, the cancer has been previously treated with a CTLA-4 inhibitor or a biosimilar thereof.

[0024] In some embodiments, the CTLA-4 inhibitor is selected from the group consisting of ipilumumab, tremelimumab, and biosimilars thereof.

[0025] In some embodiments, the cancer has been previously treated with a chemotherapy regimen.

[0026] In some embodiments, the chemotherapy regimen includes dacarbazine or temozolimide.

[0027] In some embodiments, the first or initial expansion is carried out over a period of about 11 days.

[0028] In some embodiments, IL-2 is present in the cell culture medium at an initial concentration of 1000 IU / mL to 6000 IU / mL in the first expansion or initial expansion.

[0029] In some embodiments, in the second or rapid expansion step, IL-2 is present at an initial concentration of 1000 IU / mL to 6000 IU / mL and the OKT-3 antibody is present at an initial concentration of about 30 ng / mL.

[0030] In some embodiments, the first or initial expansion is performed using a gas-permeable container.

[0031] In some embodiments, the second or rapid expansion is performed using a gas-permeable container.

[0032] In some embodiments, the first cell culture medium further comprises a cytokine selected from the group consisting of IL-4, IL-7, IL-15, IL-21, and combinations thereof.

[0033] In some embodiments, the second cell culture medium further comprises a cytokine selected from the group consisting of IL-4, IL-7, IL-15, IL-21, and combinations thereof.

[0034] In some embodiments, the methods provided herein further comprise treating the patient with a non-myeloablative lymphodepletion regimen prior to administering the TILs to the patient.

[0035] In some embodiments, the non-myeloablative lymphodepletion regimen includes cyclophosphamide at 60 mg / m 2 / day for 2 days, followed by fludarabine at 25 mg / m 2 / day for 5 days.

[0036] In some embodiments, the non-myeloablative lymphodepletion regimen includes cyclophosphamide at 60 mg / m 2 Fludarabine at a dose of 25 mg / m 2 / day for 2 days, followed by fludarabine at 25 mg / m 2 / day for 3 days.

[0037] In some embodiments, cyclophosphamide is administered with mesna.

[0038] In some embodiments, the method further comprises treating the patient with an IL-2 regimen starting the day after administering the third population of TILs to the patient.

[0039] In some embodiments, the methods provided herein further comprise treating the patient with an IL-2 regimen starting on the same day that the third population of TILs is administered to the patient.

[0040] In some embodiments, the IL-2 regimen is a high-dose IL-2 regimen comprising 600,000 or 720,000 IU / kg aldesleukin, or a biosimilar or variant thereof, administered as a 15-minute bolus intravenous infusion every 8 hours until tolerated.

[0041] In some embodiments, a therapeutically effective population of TILs is administered and comprises about 2.3×10 10 to about 13.7×10 10 TILs.

[0042] In some embodiments, the initial expansion occurs over a period of 21 days or less.

[0043] In some embodiments, the initial expansion is carried out over a period of 7 days or less.

[0044] In some embodiments, rapid expansion occurs over a period of 7 days or less.

[0045] In some embodiments, the first expansion in step (c) and the second expansion in step (d) are each carried out separately within a period of 11 days.

[0046] In some embodiments, steps (a) through (f) are carried out in about 10 days to about 22 days.

[0047] In some embodiments, the subject has received prior treatment that included administration of a KRAS inhibitor prior to tumor resection.

[0048] In some embodiments, the subject has undergone prior treatment including administration of a KRAS inhibitor prior to surgical resection.

[0049] In some embodiments, the subject has undergone previous treatment including administration of a KRAS inhibitor prior to resection of the cancer.

[0050] In some embodiments, the prior treatment comprises administration of sotorasib or adagrasib, or a pharmaceutically acceptable salt thereof, at a dose of about 500 to 1500 mg.

[0051] In some embodiments, sotorasib was administered at a dose of about 960 mg.

[0052] In some embodiments, adagrasib was administered at a dose of about 600 mg.

[0053] In some embodiments, the prior treatment comprises sotorasib or adagrasib, or a pharmaceutically acceptable salt thereof, administered twice daily.

[0054] In some embodiments, the at least one KRAS inhibitor is administered simultaneously with a therapeutically effective dose of the third population of TILs.

[0055] In some embodiments, administration of the at least one KRAS inhibitor is maintained after administration of the therapeutically effective dose of the third population of TILs.

[0056] In some embodiments, the at least one KRAS inhibitor is administered after administering the therapeutically effective dose of the third population of TILs.

[0057] In some embodiments, the subject is administered at least one KRAS inhibitor at least one week after administering the therapeutically effective dose of the third population of TILs.

[0058] In some embodiments, the patient also received at least one KRAS inhibitor prior to administering the therapeutically effective dose of the third population of TILs.

[0059] In some embodiments, the at least one KRAS inhibitor is not administered simultaneously with the therapeutically effective dose of the third population of TILs.

[0060] In some embodiments, the at least one KRAS inhibitor comprises sotrasib or adagrasib, or a pharmaceutically acceptable salt thereof, administered at a dose of about 500 to 1500 mg.

[0061] In some embodiments, sotorasib is administered at a dose of about 960 mg.

[0062] In some embodiments, adagrasib is administered at a dose of about 600 mg.

[0063] In some embodiments, sotorasib or adagrasib is administered twice daily.

[0064] In some embodiments, the cancer is selected from the group consisting of glioblastoma (GBM), gastrointestinal cancer, melanoma, ovarian cancer, endometrial cancer, thyroid cancer, colorectal cancer, cervical cancer, non-small cell lung cancer (NSCLC), lung cancer, bladder cancer, breast cancer, endometrial cancer, cholangiocarcinoma, cancer caused by human papillomavirus, head and neck cancer (including head and neck squamous cell carcinoma (HNSCC)), renal cancer, renal cell carcinoma, multiple myeloma, chronic lymphocytic leukemia, acute lymphocytic leukemia, diffuse large B-cell lymphoma, non-Hodgin's lymphoma, Hodgin's lymphoma, follicular lymphoma, and mantle cell lymphoma.

[0065] In some embodiments, the cancer is selected from the group consisting of cutaneous melanoma, ocular melanoma, uveal melanoma, and conjunctival melanoma.

[0066] In some embodiments, the cancer is selected from the group consisting of pleomorphic xanthoastrocytoma, dysembryoplastic neuroepithelial tumor, ganglioglioma, and pilocytic astrocytoma.

[0067] In some embodiments, the cancer is endometrial adenocarcinoma with non-small cell lung cancer (NSCLC).

[0068] In some embodiments, the cancer is endometrial adenocarcinoma with prominent mucinous differentiation (ECMD).

[0069] In some embodiments, the cancer is papillary thyroid cancer.

[0070] In some embodiments, the cancer is serous low-grade or borderline ovarian cancer.

[0071] In some embodiments, the cancer is hairy cell leukemia.

[0072] In some embodiments, the cancer is Langerhans cell histiocytosis.

[0073] In some embodiments, the cancer is a cancer with a p.G12C mutation in the KRAS protein.

[0074] In some embodiments, the cancer is non-small cell lung cancer (NSCLC) with the p.G12C mutation.

[0075] In some embodiments, the methods provided herein further comprise treating the patient with an IL-2 regimen after administering the third population of TILs to the patient.

[0076] In some embodiments, the methods provided herein further comprise treating the patient with an IL-2 regimen on the same day that the third population of TILs is administered to the patient.

[0077] In some embodiments, the IL-2 regimen includes nembareukin.

[0078] In some embodiments, the patient has previously received checkpoint inhibitor therapy.

[0079] In some embodiments, the patient has previously received KRAS inhibitor therapy.

[0080] In some embodiments, the patient has NSCLC.

[0081] In some embodiments, provided herein is a method of treating NSCLC in a patient in need thereof, comprising administering a population of tumor-infiltrating lymphocytes (TILs) and at least one KRAS inhibitor, wherein the patient or subject has received at least one prior therapy, wherein the at least one prior therapy comprises checkpoint inhibitor therapy.

[0082] In some embodiments, provided herein are methods of treating NSCLC in a patient or subject in need thereof, comprising administering a population of tumor-infiltrating lymphocytes (TILs) and one or more KRAS inhibitors, the method comprising: (a) obtaining and / or receiving a first population of TILs from a tumor excised from a patient by processing a tumor sample obtained from the patient into a plurality of tumor fragments; (b) adding the first population of TILs to the closed system; (c) performing a first expansion by culturing the first TIL population in a cell culture medium containing IL-2 to generate a second TIL population, wherein the first expansion is performed in a closed container that provides a first gas permeable surface area, and the first expansion is performed for about 3 to 14 days to obtain a second TIL population, and the transition from step (b) to step (c) occurs without opening the system to generate the second TIL population; (d) performing a second expansion by supplementing the cell culture medium of the second TIL population with additional IL-2, OKT-3, and antigen-presenting cells (APCs) to generate a third TIL population, wherein the second expansion is performed for about 7 to 14 days to obtain a third TIL population, the third TIL population being a therapeutic TIL population, and wherein the second expansion is performed in a closed container that provides a second gas-permeable surface area, and wherein the transition from step (c) to step (d) occurs without opening the system. (e) harvesting the therapeutic TIL population obtained from step (d), wherein the transition from step (d) to step (e) occurs without opening the system; (f) transferring the harvested TIL population from step (e) to an infusion bag, wherein the transfer from step (e) to (f) occurs without opening the system; (g) cryopreserving the infusion bag containing the harvested TIL population from step (f) using a cryopreservation process; (h) administering one or more KRAS inhibitors and a therapeutically effective dose of the third population of TILs to the subject from the infusion bag in step (g); The patient has received at least one prior therapy, wherein the at least one prior therapy includes checkpoint inhibitor therapy.

[0083] In some embodiments, provided herein are methods of treating NSCLC in a patient in need thereof, comprising administering one or more KRAS inhibitors and a population of tumor-infiltrating lymphocytes (TILs), the method comprising: (a) obtaining a first population of TILs from a tumor excised from a subject by processing a tumor sample obtained from the subject into a plurality of tumor fragments; (b) adding tumor fragments to the closed system; (c) performing a first expansion by culturing the first TIL population in a cell culture medium containing IL-2 to generate a second TIL population, wherein the first expansion is performed in a closed container that provides a first gas permeable surface area, and the first expansion is performed for about 3 to 11 days to obtain a second TIL population, and the transition from step (b) to step (c) occurs without opening the system to generate the second TIL population; (d) performing a second expansion by supplementing the cell culture medium of the second TIL population with additional IL-2, OKT-3, and antigen-presenting cells (APCs) to generate a third TIL population, wherein the second expansion is performed for about 7 to 11 days to obtain a third TIL population, and wherein the second expansion is performed in a closed container that provides a second gas-permeable surface area, and the transition from step (c) to step (d) occurs without opening the system to generate the third TIL population; (e) harvesting the third population of TILs obtained from step (d), wherein the transition from step (d) to step (e) occurs without opening the system; (f) transferring the harvested third population of TILs from step (e) to an infusion bag, wherein the transfer from step (e) to (f) occurs without opening the system; (g) cryopreserving the infusion bag containing the harvested TIL population from step (f) using a cryopreservation process; (h) administering to the patient from the infusion bag of step (g) one or more KRAS inhibitors and a therapeutically effective dose of the third population of TILs; The patient has received at least one prior therapy, wherein the at least one prior therapy includes checkpoint inhibitor therapy.

[0084] In some embodiments, provided herein are methods of treating NSCLC in a patient in need thereof, comprising administering one or more KRAS inhibitors and a population of tumor-infiltrating lymphocytes (TILs), the method comprising: (a) obtaining and / or receiving a first population of TILs from a surgical resection, needle biopsy, core biopsy, mini-biopsy, or other means for obtaining a sample containing a mixture of tumor cells and TIL cells from a patient or subject; (b) adding the first population of TILs to the closed system; (c) performing a first expansion by culturing the first TIL population in a cell culture medium containing IL-2 to generate a second TIL population, wherein the first expansion is performed in a closed container that provides a first gas permeable surface area, and the first expansion is performed for about 3 to 11 days to obtain a second TIL population, and the transition from step (b) to step (c) occurs without opening the system to generate the second TIL population; (d) performing a second expansion by supplementing the cell culture medium of the second TIL population with additional IL-2, OKT-3, and antigen-presenting cells (APCs) to generate a third TIL population, wherein the second expansion is performed for about 7 to 11 days to obtain a third TIL population, and wherein the second expansion is performed in a closed container that provides a second gas-permeable surface area, and the transition from step (c) to step (d) occurs without opening the system to generate the third TIL population; (e) harvesting the third population of TILs obtained from step (d), wherein the transition from step (d) to step (e) occurs without opening the system; (f) transferring the harvested third population of TILs from step (e) to an infusion bag, wherein the transfer from step (e) to (f) occurs without opening the system; (g) cryopreserving the infusion bag containing the harvested TIL population from step (f) using a cryopreservation process; (h) administering to the patient from the infusion bag of step (g) one or more KRAS inhibitors and a therapeutically effective dose of the third population of TILs; The patient has received at least one prior therapy, wherein the at least one prior therapy includes checkpoint inhibitor therapy.

[0085] In some embodiments, provided herein are methods of treating NSCLC in a patient in need thereof, comprising administering one or more KRAS inhibitors and a population of tumor-infiltrating lymphocytes (TILs), the method comprising: (a) optionally, removing a tumor from a patient by surgical resection, needle biopsy, core biopsy, mini-biopsy, or other means for obtaining a sample containing a mixture of tumor cells and TIL cells from the tumor, wherein the tumor comprises a first TIL population; (b) adding tumor fragments to the closed system; (c) performing a first expansion by culturing the first TIL population in a cell culture medium containing IL-2 to generate a second TIL population, wherein the first expansion is performed in a closed container that provides a first gas permeable surface area, and the first expansion is performed for about 3 to 11 days to obtain a second TIL population, and the transition from step (b) to step (c) occurs without opening the system to generate the second TIL population; (d) performing a second expansion by supplementing the cell culture medium of the second TIL population with additional IL-2, OKT-3, and antigen-presenting cells (APCs) to generate a third TIL population, wherein the second expansion is performed for about 7 to 11 days to obtain a third TIL population, and wherein the second expansion is performed in a closed container that provides a second gas-permeable surface area, and the transition from step (c) to step (d) occurs without opening the system to generate the third TIL population; (e) harvesting the third population of TILs obtained from step (d), wherein the transition from step (d) to step (e) occurs without opening the system; (f) transferring the harvested third population of TILs from step (e) to an infusion bag, wherein the transfer from step (e) to (f) occurs without opening the system; (g) cryopreserving the infusion bag containing the harvested TIL population from step (f) using a cryopreservation process; (h) administering one or more KRAS inhibitors and a therapeutically effective dose of the third population of TILs from the infusion bag of step (g) to a patient with NSCLC; The patient has received at least one prior therapy, wherein the at least one prior therapy includes checkpoint inhibitor therapy.

[0086] In some embodiments, provided herein are methods of treating NSCLC in a patient in need thereof, comprising administering one or more KRAS inhibitors and a population of tumor-infiltrating lymphocytes (TILs), the method comprising: (a) obtaining and / or receiving a first population of TILs from a surgical resection, needle biopsy, core biopsy, mini-biopsy, or other means for obtaining a sample containing a mixture of tumor cells and TIL cells from a subject or patient; (b) contacting the first TIL population with a first cell culture medium; (c) performing an initial expansion (or first expansion by priming) of the first TIL population in a first cell culture medium to obtain a second TIL population, wherein the second TIL population is at least 5 times more numerous than the first TIL population, the first cell culture medium comprising IL-2, and optionally the first expansion by priming occurs over a period of 1 to 8 days; (d) performing rapid expansion of the second TIL population in a second cell culture medium to obtain a third TIL population, wherein the third TIL population is at least 50-fold more numerous than the second TIL population 7-8 days after initiation of rapid expansion, the second cell culture medium comprising IL-2, OKT-3 (an anti-CD3 antibody), and optionally irradiated allogeneic peripheral blood mononuclear cells (PBMCs), the rapid expansion being performed over a period of 14 days or less, and optionally the second TIL expansion can proceed for 1 day, 2 days, 3 days, 4 days, 5 days, 6 days, 7 days, 8 days, 9 days, or 10 days after initiation of rapid second expansion; (e) harvesting a third population of TILs; (f) administering one or more KRAS inhibitors and a therapeutically effective portion of the third TIL population to a patient with NSCLC; The patient has received at least one prior therapy, wherein the at least one prior therapy includes checkpoint inhibitor therapy.

[0087] In some embodiments, provided herein are methods of treating NSCLC in a patient in need thereof, comprising administering one or more KRAS inhibitors and a population of tumor-infiltrating lymphocytes (TILs), the method comprising: (a) optionally, removing a tumor from a subject or patient by surgical resection, needle biopsy, core biopsy, mini-biopsy, or other means for obtaining a sample containing a mixture of tumor cells and TIL cells from the tumor, wherein the patient has previously been treated for a tumor comprising a first TIL population; (b) fragmenting the tumor into tumor fragments; (c) contacting the tumor fragment with a first cell culture medium; (d) performing an initial expansion (or first expansion by priming) of the first TIL population in a first cell culture medium to obtain a second TIL population, wherein the second TIL population is at least 5 times more numerous than the first TIL population, the first cell culture medium comprising IL-2, and optionally the first expansion by priming occurs over a period of 1 to 8 days; (e) performing rapid expansion of the second TIL population in a second cell culture medium to obtain a third TIL population, wherein the third TIL population is at least 50-fold more numerous than the second TIL population 7-8 days after initiation of rapid expansion, the second cell culture medium comprising IL-2, OKT-3 (an anti-CD3 antibody), and optionally irradiated allogeneic peripheral blood mononuclear cells (PBMCs), the rapid expansion being performed over a period of 14 days or less, and optionally the second TIL expansion being allowed to proceed for 1 day, 2 days, 3 days, 4 days, 5 days, 6 days, 7 days, 8 days, 9 days, or 10 days after initiation of rapid second expansion; (f) harvesting a third population of TILs; (g) administering one or more KRAS inhibitors and a therapeutically effective portion of the third TIL population to a patient with NSCLC; The patient or subject has received at least one prior therapy, wherein the at least one prior therapy includes checkpoint inhibitor therapy.

[0088] In some embodiments, the NSCLC is unresectable, metastatic, resistant and / or refractory to a KRAS inhibitor.

[0089] In some embodiments, the patient has a KRAS gene mutation,

[0090] In some embodiments, the patient has NSCLC that exhibits the p.G12C mutation.

[0091] In some embodiments, the at least one prior therapy further comprises a KRAS inhibitor therapy.

[0092] In some embodiments, the method further comprises treating the patient with an IL-2 regimen after administering the third population of TILs to the patient.

[0093] In some embodiments, the method further comprises treating the patient with an IL-2 regimen on the same day that the third population of TILs is administered to the patient.

[0094] In some embodiments, the IL-2 regimen includes nembareukin.

[0095] In some embodiments, the method further comprises treating the patient with a non-myeloablative lymphodepletion regimen prior to administering the TILs to the patient.

[0096] In some embodiments, the non-myeloablative lymphodepletion regimen comprises administering cyclophosphamide at a dose of 60 mg / m2 / day for two days, followed by fludarabine at a dose of 25 mg / m2 / day for five days.

[0097] In some embodiments, the non-myeloablative lymphodepletion regimen comprises administering cyclophosphamide at a dose of 60 mg / m2 / day and fludarabine at a dose of 25 mg / m2 / day for two days, followed by fludarabine at a dose of 25 mg / m2 / day for three days.

[0098] In some embodiments, cyclophosphamide is administered with mesna.

[0099] In some embodiments, the tumor is resected from a patient who was pre-treated with one or more KRAS inhibitors prior to tumor resection.

[0100] In some embodiments, the NSCLC is unresectable, metastatic, resistant and / or refractory to a KRAS inhibitor.

[0101] In some embodiments, the patient or subject has a KRAS gene mutation.

[0102] In some embodiments, the patient or subject has a cancer that exhibits the p.G12C mutation.

[0103] In some embodiments, the cancer has been previously treated with a KRAS inhibitor.

[0104] In some embodiments, the cancer has not been previously treated with a KRAS inhibitor.

[0105] In some embodiments, the KRAS inhibitor is selected from the group consisting of AMG-510 (sotorasib), AMG-510 racemate (sotorasib racemate), MRTX849 (adagrasib), JNJ-74699157 / ARS-3248, JDQ443, LY3499446, LY3537982, RLY-1971 / GDC-6036, BBP-454, BI 1701963, BI 1823911, mRNA-5671 / V941, D-1553, BI-2852, BI-3406, ARS-1620, BAY-293, MRTX-1257, PROTAC K-Ras Degrader-1, LC-2, ARS-853, ARS-1323, ARS-1323-alkyne, ARS-1630, K-Ras G12C-IN-2, KRAS inhibitor-6, KRAS inhibitor 8, KRAS inhibitor 7, KRAS G12C inhibitor 15, KRAS G12C inhibitor 5, KRAS G12C inhibitor 13, KRAS G12C inhibitor 17, KRAS G12C inhibitor 16, KRAS G12C inhibitor 14, KRas G12C inhibitor 4, KRas G12C inhibitor 1, KRas G12C inhibitor 3, KRas G12C inhibitor 2, 6H05, SAH-SOS1ATFA, KRAS inhibitor 10, SAH-SOS1A, atorvastatin-PEG3-FITC, C6ME, CS-0115617, HY-130260, HY-135864, HY-135866, Cmpd2, CS-0115618, CS-0115620, EX-A4387, CS-01 06134, HY-135865, 2241719-75-3, HY-125873, CS-0046138, CS-0046137, CS-01 01474, HY-125875, CS-0102608, CS-0102610, CS-0102606, HY-112493, CS-0046 139, 1-{4-[6-chloro-8-fluoro-7-(5-methyl-1h-indazol-4-yl)quinazolin-4-yl]piperazin-1-yl}propan-1-one, HY-126292, HY-112491, CS-0046136, HY-114168, HY-125874, HY-112494, CS-0102607, BCP29475|2206735-61-5, HY-112492, CS-0078097, 2158296-45-6, HY-125872, and 2158297-63-1, and pharmaceutically acceptable salts thereof.

[0106] In some embodiments, the KRAS inhibitor is selected from the group consisting of AMG-510 (sotorasib), AMG-510 racemate (sotorasibraracemic), and MRTX849 (adagrasib).

[0107] In some embodiments, provided herein is a method of treating cancer in a patient or subject in need thereof, comprising: (a) treating the patient with a nonmyeloablative lymphodepleting regimen that includes melphalan; and (b) administering a population of tumor-infiltrating lymphocytes (TILs) and one or more KRAS inhibitors; (c) treating the patient with an IL-2 regimen after administration of the population of TILs, wherein the patient or subject has cancer.

[0108] In some embodiments, provided herein are methods of treating cancer in a patient in need thereof, comprising administering a population of tumor-infiltrating lymphocytes (TILs) and one or more KRAS inhibitors, the method comprising: (a) obtaining and / or receiving a first population of TILs from a tumor resected from a subject by processing a tumor sample obtained from the subject into a plurality of tumor fragments; (b) adding the first population of TILs to the closed system; (c) performing a first expansion by culturing the first TIL population in a cell culture medium containing IL-2 to generate a second TIL population, wherein the first expansion is performed in a closed container that provides a first gas permeable surface area, and the first expansion is performed for about 3 to 14 days to obtain a second TIL population, and the transition from step (b) to step (c) occurs without opening the system to generate the second TIL population; (d) performing a second expansion by supplementing the cell culture medium of the second TIL population with additional IL-2, OKT-3, and antigen-presenting cells (APCs) to generate a third TIL population, wherein the second expansion is performed for about 7 to 14 days to obtain a third TIL population, the third TIL population being a therapeutic TIL population, and wherein the second expansion is performed in a closed container that provides a second gas-permeable surface area, and wherein the transition from step (c) to step (d) occurs without opening the system. (e) harvesting the therapeutic TIL population obtained from step (d), wherein the transition from step (d) to step (e) occurs without opening the system; (f) transferring the harvested TIL population from step (e) to an infusion bag, wherein the transfer from step (e) to (f) occurs without opening the system; (g) cryopreserving the infusion bag containing the harvested TIL population from step (f) using a cryopreservation process; (h) administering to the patient from the infusion bag of step (g) one or more KRAS inhibitors and a therapeutically effective dose of the third population of TILs; (i) treating the patient with an IL-2 regimen after administration of the TIL population; prior to administering to the patient a therapeutically effective dose of the third population of TILs, the patient is treated with a non-myeloablative lymphodepleting regimen comprising melphalan; The patient has cancer.

[0109] In some embodiments, provided herein are methods of treating cancer in a patient in need thereof, comprising administering a population of tumor-infiltrating lymphocytes (TILs) and one or more KRAS inhibitors, the method comprising: (a) obtaining a first population of TILs from a tumor resected from a patient by processing a tumor sample obtained from the subject into a plurality of tumor fragments; (b) adding tumor fragments to the closed system; (c) performing a first expansion by culturing the first TIL population in a cell culture medium containing IL-2 to generate a second TIL population, wherein the first expansion is performed in a closed container that provides a first gas permeable surface area, and the first expansion is performed for about 3 to 11 days to obtain a second TIL population, and the transition from step (b) to step (c) occurs without opening the system to generate the second TIL population; (d) performing a second expansion by supplementing the cell culture medium of the second TIL population with additional IL-2, OKT-3, and antigen-presenting cells (APCs) to generate a third TIL population, wherein the second expansion is performed for about 7 to 11 days to obtain a third TIL population, and wherein the second expansion is performed in a closed container that provides a second gas-permeable surface area, and the transition from step (c) to step (d) occurs without opening the system to generate the third TIL population; (e) harvesting the third population of TILs obtained from step (d), wherein the transition from step (d) to step (e) occurs without opening the system; (f) transferring the harvested third population of TILs from step (e) to an infusion bag, wherein the transfer from step (e) to (f) occurs without opening the system; (g) cryopreserving the infusion bag containing the harvested TIL population from step (f) using a cryopreservation process; (h) administering to the patient from the infusion bag of step (g) one or more KRAS inhibitors and a therapeutically effective dose of the third population of TILs; (i) treating the patient with an IL-2 regimen after administration of the TIL population; prior to administering to the patient a therapeutically effective dose of the third population of TILs, the patient is treated with a non-myeloablative lymphodepleting regimen comprising melphalan; The patient has cancer.

[0110] In some embodiments, provided herein are methods of treating cancer in a patient in need thereof, comprising administering a population of tumor-infiltrating lymphocytes (TILs) and one or more KRAS inhibitors, the method comprising: (a) obtaining and / or receiving a first population of TILs from a surgical resection, needle biopsy, core biopsy, mini-biopsy, or other means for obtaining a sample containing a mixture of tumor cells and TIL cells from a patient; (b) adding the first population of TILs to the closed system; (c) performing a first expansion by culturing the first TIL population in a cell culture medium containing IL-2 to generate a second TIL population, wherein the first expansion is performed in a closed container that provides a first gas permeable surface area, and the first expansion is performed for about 3 to 11 days to obtain a second TIL population, and the transition from step (b) to step (c) occurs without opening the system to generate the second TIL population; (d) performing a second expansion by supplementing the cell culture medium of the second TIL population with additional IL-2, OKT-3, and antigen-presenting cells (APCs) to generate a third TIL population, wherein the second expansion is performed for about 7 to 11 days to obtain a third TIL population, and wherein the second expansion is performed in a closed container that provides a second gas-permeable surface area, and the transition from step (c) to step (d) occurs without opening the system to generate the third TIL population; (e) harvesting the third population of TILs obtained from step (d), wherein the transition from step (d) to step (e) occurs without opening the system; (f) transferring the harvested third population of TILs from step (e) to an infusion bag, wherein the transfer from step (e) to (f) occurs without opening the system; (g) cryopreserving the infusion bag containing the harvested TIL population from step (f) using a cryopreservation process; (h) administering to the patient from the infusion bag of step (g) one or more KRAS inhibitors and a therapeutically effective dose of the third population of TILs; (i) treating the patient with an IL-2 regimen after administration of the TIL population; prior to administering to the patient a therapeutically effective dose of the third population of TILs, the patient is treated with a non-myeloablative lymphodepleting regimen comprising melphalan; The patient has cancer.

[0111] In some embodiments, provided herein are methods of treating cancer in a patient in need thereof, comprising administering a population of tumor-infiltrating lymphocytes (TILs) and one or more KRAS inhibitors, the method comprising: (a) optionally, by surgical resection, needle biopsy, core biopsy, mini-biopsy, or other means for obtaining a sample containing a mixture of tumor cells and TIL cells from the tumor, removing a tumor from a subject or patient, wherein the tumor comprises a first TIL population; (b) adding tumor fragments to the closed system; (c) performing a first expansion by culturing the first TIL population in a cell culture medium containing IL-2 to generate a second TIL population, wherein the first expansion is performed in a closed container that provides a first gas permeable surface area, and the first expansion is performed for about 3 to 11 days to obtain a second TIL population, and the transition from step (b) to step (c) occurs without opening the system to generate the second TIL population; (d) performing a second expansion by supplementing the cell culture medium of the second TIL population with additional IL-2, OKT-3, and antigen-presenting cells (APCs) to generate a third TIL population, wherein the second expansion is performed for about 7 to 11 days to obtain a third TIL population, and wherein the second expansion is performed in a closed container that provides a second gas-permeable surface area, and the transition from step (c) to step (d) occurs without opening the system to generate the third TIL population; (e) harvesting the third population of TILs obtained from step (d), wherein the transition from step (d) to step (e) occurs without opening the system; (f) transferring the harvested third population of TILs from step (e) to an infusion bag, wherein the transfer from step (e) to (f) occurs without opening the system; (g) cryopreserving the infusion bag containing the harvested TIL population from step (f) using a cryopreservation process; (h) administering one or more KRAS inhibitors and a therapeutically effective dose of the third population of TILs from the infusion bag in step (g) to a subject or patient with cancer; (i) treating the patient with an IL-2 regimen after administration of the TIL population; prior to administering to the patient a therapeutically effective dose of the third population of TILs, the patient is treated with a non-myeloablative lymphodepleting regimen comprising melphalan; The patient has cancer.

[0112] In some embodiments, provided herein are methods of treating cancer in a patient in need thereof, comprising administering a population of tumor-infiltrating lymphocytes (TILs) and one or more KRAS inhibitors, the method comprising: (a) obtaining and / or receiving a first population of TILs from a surgical resection, needle biopsy, core biopsy, mini-biopsy, or other means for obtaining a sample containing a mixture of tumor cells and TIL cells from a patient; (b) contacting the first TIL population with a first cell culture medium; (c) performing an initial expansion (or first expansion by priming) of the first TIL population in a first cell culture medium to obtain a second TIL population, wherein the first cell culture medium comprises IL-2, and optionally the first expansion by priming occurs over a period of 1 to 8 days; (d) performing rapid expansion of the second TIL population in a second cell culture medium comprising IL-2, OKT-3 (an anti-CD3 antibody), and optionally irradiated allogeneic peripheral blood mononuclear cells (PBMCs), wherein the rapid expansion is performed over a period of no more than 14 days, and optionally, the second TIL expansion can proceed for 1 day, 2 days, 3 days, 4 days, 5 days, 6 days, 7 days, 8 days, 9 days, or 10 days after initiation of the rapid second expansion to obtain a third TIL population; (e) harvesting a third population of TILs; (f) administering a therapeutically effective portion of the third population of TILs to a subject or patient with cancer; (g) treating the patient with an IL-2 regimen after administering a therapeutically effective portion of the third population of TILs; prior to administering to the patient a therapeutically effective portion of the third TIL population, the patient is treated with a non-myeloablative lymphodepleting regimen comprising melphalan; The patient has cancer.

[0113] In some embodiments, provided herein are methods of treating cancer in a patient in need thereof, comprising administering a population of tumor-infiltrating lymphocytes (TILs) and one or more KRAS inhibitors, the method comprising: (a) optionally, removing a tumor from a patient by surgical resection, needle biopsy, core biopsy, mini-biopsy, or other means for obtaining a sample containing a mixture of tumor cells and TIL cells from the tumor, wherein the patient has previously been treated for a tumor comprising a first TIL population; (b) fragmenting the tumor into tumor fragments; (c) contacting the tumor fragment with a first cell culture medium; (d) performing an initial expansion (or first expansion by priming) of the first TIL population in a first cell culture medium to obtain a second TIL population, wherein the first cell culture medium comprises IL-2, and optionally the first expansion by priming occurs over a period of 1 to 8 days; (e) performing rapid expansion of the second TIL population in a second cell culture medium comprising IL-2, OKT-3 (anti-CD3 antibody), and optionally irradiated allogeneic peripheral blood mononuclear cells (PBMCs), wherein the rapid expansion is performed over a period of no more than 14 days, and optionally, the second TIL expansion can proceed for 1 day, 2 days, 3 days, 4 days, 5 days, 6 days, 7 days, 8 days, 9 days, or 10 days after initiation of the rapid second expansion to obtain a third TIL population; (f) harvesting a third population of TILs; (g) administering a therapeutically effective portion of the third population of TILs to a subject or patient with cancer; (h) treating the patient with an IL-2 regimen after administering a therapeutically effective portion of the third population of TILs; prior to administering to the patient a therapeutically effective portion of the third TIL population, the patient is treated with a non-myeloablative lymphodepleting regimen comprising melphalan; The patient has cancer.

[0114] In some embodiments, melphalan is administered intravenously at a dose of about 100 mg / m2 for two consecutive days.

[0115] In some embodiments, the IL-2 regimen comprises administering a low dose of IL-2 daily for up to 14 days following administration of the TIL population.

[0116] In some embodiments, the second TIL population is at least 50-fold more numerous than the first TIL population.

[0117] In some embodiments, the cancer is selected from the group consisting of glioblastoma (GBM), gastrointestinal cancer, melanoma, ovarian cancer, endometrial cancer, thyroid cancer, colorectal cancer, cervical cancer, non-small cell lung cancer (NSCLC), lung cancer, bladder cancer, breast cancer, endometrial cancer, cholangiocarcinoma, cancer caused by human papillomavirus, head and neck cancer (including head and neck squamous cell carcinoma (HNSCC)), renal cancer, renal cell carcinoma, multiple myeloma, chronic lymphocytic leukemia, acute lymphocytic leukemia, diffuse large B-cell lymphoma, non-Hodgin's lymphoma, Hodgin's lymphoma, follicular lymphoma, and mantle cell lymphoma.

[0118] In some embodiments, the cancer is selected from the group consisting of cutaneous melanoma, ocular melanoma, uveal melanoma, and conjunctival melanoma.

[0119] In some embodiments, the cancer is selected from the group consisting of pleomorphic xanthoastrocytoma, dysembryoplastic neuroepithelial tumor, ganglioglioma, and pilocytic astrocytoma.

[0120] In some embodiments, the cancer is endometrial adenocarcinoma with non-small cell lung cancer (NSCLC).

[0121] In some embodiments, the cancer is endometrial adenocarcinoma with prominent mucinous differentiation (ECMD).

[0122] In some embodiments, the cancer is papillary thyroid cancer.

[0123] In some embodiments, the cancer is serous low-grade or borderline ovarian cancer.

[0124] In some embodiments, the cancer is hairy cell leukemia.

[0125] In some embodiments, the cancer is Langerhans cell histiocytosis.

[0126] In some embodiments, the cancer is a cancer with a p.G12C mutation in the KRAS protein.

[0127] In some embodiments, the cancer is non-small cell lung cancer (NSCLC) with the p.G12C mutation.

[0128] In some embodiments, the tumor is resected from a patient who was pre-treated with one or more KRAS inhibitors prior to tumor resection.

[0129] In some embodiments, the NSCLC is unresectable, metastatic, resistant and / or refractory to a KRAS inhibitor.

[0130] In some embodiments, the patient or subject has a KRAS gene mutation.

[0131] In some embodiments, the patient or subject has a cancer that exhibits the p.G12C mutation.

[0132] In some embodiments, the cancer has been previously treated with a KRAS inhibitor.

[0133] In some embodiments, the cancer has not been previously treated with a KRAS inhibitor.

[0134] In some embodiments, the KRAS inhibitor is selected from the group consisting of AMG-510 (sotorasib), AMG-510 racemate (sotorasib racemate), MRTX849 (adagrasib), JNJ-74699157 / ARS-3248, JDQ443, LY3499446, LY3537982, RLY-1971 / GDC-6036, BBP-454, BI 1701963, BI 1823911, mRNA-5671 / V941, D-1553, BI-2852, BI-3406, ARS-1620, BAY-293, MRTX-1257, PROTAC K-Ras Degrader-1, LC-2, ARS-853, ARS-1323, ARS-1323-alkyne, ARS-1630, K-Ras G12C-IN-2, KRAS inhibitor-6, KRAS inhibitor 8, KRAS inhibitor 7, KRAS G12C inhibitor 15, KRAS G12C inhibitor 5, KRAS G12C inhibitor 13, KRAS G12C inhibitor 17, KRAS G12C inhibitor 16, KRAS G12C inhibitor 14, KRas G12C inhibitor 4, KRas G12C inhibitor 1, KRas G12C inhibitor 3, KRas G12C inhibitor 2, 6H05, SAH-SOS1ATFA, KRAS inhibitor 10, SAH-SOS1A, atorvastatin-PEG3-FITC, C6ME, CS-0115617, HY-130260, HY-135864, HY-135866, Cmpd2, CS-0115618, CS-0115620, EX-A4387, CS-01 06134, HY-135865, 2241719-75-3, HY-125873, CS-0046138, CS-0046137, CS-01 01474, HY-125875, CS-0102608, CS-0102610, CS-0102606, HY-112493, CS-0046 139, 1-{4-[6-chloro-8-fluoro-7-(5-methyl-1h-indazol-4-yl)quinazolin-4-yl]piperazin-1-yl}propan-1-one, HY-126292, HY-112491, CS-0046136, HY-114168, HY-125874, HY-112494, CS-0102607, BCP29475|2206735-61-5, HY-112492, CS-0078097, 2158296-45-6, HY-125872, and 2158297-63-1, and pharmaceutically acceptable salts thereof.

[0135] In some embodiments, the KRAS inhibitor is selected from the group consisting of AMG-510 (sotorasib), AMG-510 racemate (sotorasibraracemic), and MRTX849 (adagrasib).

[0136] In some embodiments, provided herein are methods of expanding tumor infiltrating lymphocytes (TILs) into a therapeutic TIL population, the methods comprising: (a) obtaining and / or receiving a first population of TILs from a tumor resected from a subject or patient by processing a tumor sample obtained from the subject or patient into a plurality of tumor fragments, wherein the subject or patient has previously been treated with at least one KRAS inhibitor; (b) adding the first TIL population to the closed system; (c) performing a first expansion by culturing the first TIL population in a cell culture medium containing IL-2 to generate a second TIL population, wherein the first expansion is performed in a closed container that provides a first gas permeable surface area, and the first expansion is performed for about 3 to 14 days to obtain a second TIL population, and the transition from step (b) to step (c) occurs without opening the system to generate the second TIL population; (d) performing a second expansion by supplementing the cell culture medium of the second TIL population with additional IL-2, OKT-3, and antigen-presenting cells (APCs) to generate a third TIL population, wherein the second expansion is performed for about 7 to 14 days to obtain a third TIL population, the third TIL population being a therapeutic TIL population, and wherein the second expansion is performed in a closed container that provides a second gas-permeable surface area, and the transition from step (c) to step (d) occurs without opening the system. (e) harvesting the therapeutic TIL population obtained from step (d), wherein the transition from step (d) to step (e) occurs without opening the system; (f) transferring the harvested TIL population from step (e) to an infusion bag, wherein the transfer from step (e) to (f) occurs without opening the system; (g) cryopreserving the infusion bag containing the harvested TIL population from step (f) using a cryopreservation process.

[0137] In some embodiments, provided herein are methods of expanding tumor infiltrating lymphocytes (TILs) into a therapeutic TIL population, the methods comprising: (a) obtaining a first population of TILs from a tumor resected from a subject by processing a tumor sample obtained from the subject into a plurality of tumor fragments, wherein the subject has previously been treated with at least one KRAS inhibitor; (b) adding tumor fragments to the closed system; (c) performing a first expansion by culturing the first TIL population in a cell culture medium comprising IL-2 to generate a second TIL population, wherein the first expansion is performed in a closed container providing a first gas permeable surface area, and wherein the first expansion is performed for about 3 to 11 days to obtain a second TIL population, wherein the second TIL population is at least 50-fold more numerous than the first TIL population, and wherein the transition from step (b) to step (c) occurs without opening the system; (d) performing a second expansion by supplementing the cell culture medium of the second TIL population with additional IL-2, OKT-3, and antigen-presenting cells (APCs) to generate a third TIL population, wherein the second expansion is performed for about 7 to 11 days to obtain a third TIL population, and wherein the second expansion is performed in a closed container that provides a second gas-permeable surface area, and the transition from step (c) to step (d) occurs without opening the system to generate the third TIL population; (e) harvesting the third population of TILs obtained from step (d), wherein the transition from step (d) to step (e) occurs without opening the system; and (f) transferring the harvested third population of TILs from step (e) to an infusion bag, wherein the transfer from step (e) to (f) occurs without opening the system; (g) cryopreserving the infusion bag containing the harvested TIL population from step (f) using a cryopreservation process.

[0138] In some embodiments, provided herein are methods of expanding tumor infiltrating lymphocytes (TILs) into a therapeutic TIL population, the methods comprising: (a) obtaining and / or receiving a first population of TILs from a surgical resection, needle biopsy, core biopsy, mini-biopsy, or other means for obtaining a sample containing a mixture of tumor cells and TIL cells from a patient or subject, wherein the subject or patient has previously been treated with at least one KRAS inhibitor; (b) adding the first TIL population to the closed system; (c) performing a first expansion by culturing the first TIL population in a cell culture medium containing IL-2 to generate a second TIL population, wherein the first expansion is performed in a closed container that provides a first gas permeable surface area, and the first expansion is performed for about 3 to 11 days to obtain a second TIL population, and the transition from step (b) to step (c) occurs without opening the system to generate the second TIL population; (d) performing a second expansion by supplementing the cell culture medium of the second TIL population with additional IL-2, OKT-3, and antigen-presenting cells (APCs) to generate a third TIL population, wherein the second expansion is performed for about 7 to 11 days to obtain a third TIL population, and wherein the second expansion is performed in a closed container that provides a second gas-permeable surface area, and the transition from step (c) to step (d) occurs without opening the system to generate the third TIL population; (e) harvesting the third population of TILs obtained from step (d), wherein the transition from step (d) to step (e) occurs without opening the system; and (f) transferring the harvested third population of TILs from step (e) to an infusion bag, wherein the transfer from step (e) to (f) occurs without opening the system; (g) cryopreserving the infusion bag containing the harvested TIL population from step (f) using a cryopreservation process.

[0139] In some embodiments, provided herein are methods of expanding tumor infiltrating lymphocytes (TILs) into a therapeutic TIL population, the methods comprising: (a) optionally, removing a tumor from a subject or patient by surgical resection, needle biopsy, core biopsy, mini-biopsy, or other means to obtain a sample containing a mixture of tumor cells and TIL cells from said tumor, wherein said tumor comprises a first TIL population, and said subject or patient has previously been treated with at least one KRAS inhibitor; (b) adding tumor fragments to the closed system; (c) performing a first expansion by culturing the first TIL population in a cell culture medium containing IL-2 to generate a second TIL population, wherein the first expansion is performed in a closed container that provides a first gas permeable surface area, and the first expansion is performed for about 3 to 11 days to obtain a second TIL population, and the transition from step (b) to step (c) occurs without opening the system to generate the second TIL population; (d) performing a second expansion by supplementing the cell culture medium of the second TIL population with additional IL-2, OKT-3, and antigen-presenting cells (APCs) to generate a third TIL population, wherein the second expansion is performed for about 7 to 11 days to obtain a third TIL population, and wherein the second expansion is performed in a closed container that provides a second gas-permeable surface area, and the transition from step (c) to step (d) occurs without opening the system to generate the third TIL population; (e) harvesting the third population of TILs obtained from step (d), wherein the transition from step (d) to step (e) occurs without opening the system; and (f) transferring the harvested third population of TILs from step (e) to an infusion bag, wherein the transfer from step (e) to (f) occurs without opening the system; (g) cryopreserving the infusion bag containing the harvested TIL population from step (f) using a cryopreservation process.

[0140] In some embodiments, provided herein are methods of expanding tumor infiltrating lymphocytes (TILs) into a therapeutic TIL population, the methods comprising: (a) obtaining and / or receiving a first population of TILs from a surgical resection, needle biopsy, core biopsy, mini-biopsy, or other means for obtaining a sample containing a mixture of tumor cells and TIL cells from a subject or patient, wherein the subject or patient has previously been treated with at least one KRAS inhibitor; (c) contacting the first TIL population with a first cell culture medium; (d) performing an initial expansion (or first expansion by priming) of the first TIL population in a first cell culture medium to obtain a second TIL population, wherein the first cell culture medium comprises IL-2, and optionally the first expansion by priming occurs over a period of 1 to 8 days; (e) performing rapid expansion of the second TIL population in a second cell culture medium to obtain a third TIL population, wherein the second cell culture medium comprises IL-2, OKT-3 (an anti-CD3 antibody), and optionally irradiated allogeneic peripheral blood mononuclear cells (PBMCs), and wherein the rapid expansion is performed over a period of 14 days or less, and optionally, the second TIL expansion is allowed to proceed for 1 day, 2 days, 3 days, 4 days, 5 days, 6 days, 7 days, 8 days, 9 days, or 10 days after initiation of the rapid second expansion; (f) harvesting a third population of TILs.

[0141] In some embodiments, provided herein are methods of expanding tumor infiltrating lymphocytes (TILs) into a therapeutic TIL population, the methods comprising: (a) optionally, removing a tumor from a subject or patient by surgical resection, needle biopsy, core biopsy, mini-biopsy, or other means for obtaining a sample containing a mixture of tumor cells and TIL cells from the tumor, wherein the subject or patient has previously been treated with at least one KRAS inhibitor; (b) fragmenting the tumor into tumor fragments; (c) contacting the tumor fragment with a first cell culture medium; (d) performing an initial expansion (or first expansion by priming) of the first TIL population in a first cell culture medium to obtain a second TIL population, wherein the second TIL population is at least 5 times more numerous than the first TIL population, and the first cell culture medium comprises IL-2, and optionally the first expansion by priming occurs over a period of 1 to 8 days; (e) performing rapid expansion of the second TIL population in a second cell culture medium to obtain a third TIL population, wherein the third TIL population is at least 50-fold more numerous than the second TIL population 7-8 days after initiation of rapid expansion, the second cell culture medium comprising IL-2, OKT-3 (an anti-CD3 antibody), and optionally irradiated allogeneic peripheral blood mononuclear cells (PBMCs), the rapid expansion being performed over a period of 14 days or less, and optionally the second TIL expansion being allowed to proceed for 1 day, 2 days, 3 days, 4 days, 5 days, 6 days, 7 days, 8 days, 9 days, or 10 days after initiation of rapid second expansion; (f) harvesting a third population of TILs.

[0142] In some embodiments, provided herein is an expanded population of tumor-infiltrating lymphocytes (TILs) obtainable by expanding a population of TILs from a tumor resected from a subject or patient, wherein prior to tumor resection, the subject or patient has been treated with at least one KRAS inhibitor.

[0143] In some embodiments, provided herein are expanded populations of tumor infiltrating lymphocytes (TILs) obtainable by the expansion methods provided herein.

[0144] In some embodiments, the present disclosure provides for the use of a population of tumor-infiltrating lymphocytes (TILs) in the manufacture of a medicament for use in combination with at least one KRAS inhibitor to treat cancer.

[0145] In some embodiments, the present disclosure provides for the use of a population of tumor-infiltrating lymphocytes (TILs) in the manufacture of a medicament for use in combination with at least one KRAS inhibitor to treat cancer.

[0146] In some embodiments, the present disclosure provides for the use of a population of tumor-infiltrating lymphocytes (TILs), wherein the TILs are expanded according to any of the methods described herein, in the manufacture of a medicament for use in combination with at least one KRAS inhibitor to treat cancer. [Brief explanation of the drawings]

[0147] [Figure 1] An exemplary Gen2 (Process 2A) chart providing an overview of steps A-F. [Figure 2A] 1 is a process flow diagram of an embodiment of Gen2 (Process 2A) for TIL fabrication. [Figure 2B] 1 is a process flow diagram of an embodiment of Gen2 (Process 2A) for TIL fabrication. [Figure 2C] 1 is a process flow diagram of an embodiment of Gen2 (Process 2A) for TIL fabrication. [Figure 3] 1 shows a diagram of an embodiment of an exemplary manufacturing process (approximately 22 days) for cryopreserved TILs. [Figure 4] 1 shows a diagram of an embodiment of Gen2 (Process 2A), a 22-day process for TIL fabrication. [Figure 5] 1 is a comparison table of steps A-F from exemplary embodiments of Process 1C and Gen2 (Process 2A) for TIL fabrication. [Figure 6] Detailed comparison of Process 1C and Gen2 (Process 2A) embodiments for TIL fabrication. [Figure 7] 1. Exemplary Gen3 TIL fabrication process. [Figure 8A]A comparison of embodiments of the 2A process (an approximately 22 day process) and the Gen3 process (an approximately 14-16 day process) for TIL fabrication is shown. [Figure 8B] An exemplary Process Gen3 chart providing an overview of steps A-F (approximately a 14-16 day process). [Figure 8C] A chart providing three exemplary Gen3 processes along with an overview of steps A-F (approximately 14- to 16-day processes) for each of the three process variations. [Figure 8D] An exemplary modified Gen2-like process (approximately a 22-day process) providing an overview of steps A-F. [Figure 9] 1 provides an experimental flow chart for the comparison between Gen2 (Process 2A) and Gen3 processes. [Figure 10] 1 shows a comparison of various Gen2 (Process 2A) and Gen3.1 process embodiments. [Figure 11] 1 is a table illustrating various features of embodiments of the Gen2, Gen2.1, and Gen3.0 processes. [Figure 12] Summary of media conditions for an embodiment of the Gen3 process, designated Gen3.1. [Figure 13] 1 is a table illustrating various features of embodiments of the Gen2, Gen2.1, and Gen3.0 processes. [Figure 14] 1 is a table comparing various features of embodiments of the Gen2 and Gen3.0 processes. [Figure 15] 1 is a table providing media use in various embodiments of the described expansion process. [Figure 16] Schematic of an exemplary embodiment of the Gen3 process (16-day process). [Figure 17] Schematic of an exemplary embodiment of a method for expanding T cells from hematopoietic malignancies using the Gen3 expansion platform. [Figure 18]Structures IA and IB are provided. The cylinders refer to individual polypeptide binding domains. Structures IA and IB comprise three linearly linked TNFRSF-binding domains, e.g., derived from an antibody that binds to 4-1BBL or 4-1BB, that fold together to form a trivalent protein, which is then linked to a second trivalent protein by IgG1-Fc (comprising the CH3 and CH2 domains), which is then used to link two of the trivalent proteins together by disulfide bonds (small, elongated ovals), stabilizing the structure and providing an agonist that can bring together the intracellular signaling domains of six receptors and signaling proteins to form a signaling complex. The TNFRSF-binding domains shown as cylinders can be, for example, scFv domains comprising VH and VL chains connected by a linker that may contain hydrophilic residues and Gly and Ser sequences for flexibility, and Glu and Lys for solubility. [Figure 19] Schematic of an exemplary embodiment of the Gen3 process (16-day process). [Figure 20] 1 provides a process overview of an exemplary embodiment of the Gen3.1 process (16-day process). [Figure 21] Schematic of an exemplary embodiment of the Gen3.1 testing process (16-17 day process). [Figure 22] Schematic of an exemplary embodiment of the Gen3 process (16-day process). [Figure 23] 1 is a comparison table of an exemplary Gen2 process and an exemplary Gen3 process. [Figure 24] Schematic of an exemplary embodiment of the preparation timeline for the Gen3 process (16-17 day process). [Figure 25] Schematic of an exemplary embodiment of the Gen3 process (14-16 day process). [Figure 26A] Schematic of an exemplary embodiment of the Gen3 process (16-day process). [Figure 26B] Schematic of an exemplary embodiment of the Gen3 process (16-day process). [Figure 27] Schematic of an exemplary embodiment of the Gen3 process (16-day process). [Figure 28] Comparison of Gen2, Gen2.1, and Gen3 process (16-day process) embodiments. [Figure 29] Comparison of Gen2, Gen2.1, and Gen3 process (16-day process) embodiments. [Figure 30] Components of a Gen3 embodiment. [Figure 31] Flowchart comparison of Gen3 embodiments (Gen3.0, Gen3.1 control, Gen3.1 test). [Figure 32] Components of an exemplary embodiment of the Gen3 process (16-17 day process) are shown. [Figure 33] Approval criteria table. [Figure 34] Study design diagram for Cohort 1. Cohort 1: KRAS p.G12C inhibitor naive patients with STK11 or CDKN2A / B+ low expression and co-mutation with thyroid transcription factor-1 (TTF-1): Patients are pretreated with a KRAS p.G12C inhibitor (sotorasib 960 mg orally once daily or adagrasib 600 mg orally twice daily) for 3-4 weeks before TIL resection. Patients resume KRAS p.G12C inhibitor treatment after TIL resection and until the onset of NMALD. Then, 3-4 weeks after TIL infusion, NMALD is resumed until progression or toxicity. The diagram shows NMALD as a 5-day regimen. The 7-day regimen is as follows: NMA LD regimen consists of 2 days of intravenous (IV) cyclophosphamide (60 mg / kg) with mesna (per local standard of care or USPI / SmPC) on days -7 and 6, and 5 days of fludarabine IV (25 mg / m2, days -5 to 1). [Figure 35]Study design diagram for Cohort 2. Cohort 2: KRAS p.G12C inhibitor pretreated patients with STK11 or CDKN2A / B+ low expression of thyroid transcription factor-1 (TTF-1) co-mutations: Patients will resume the KRAS p.G12C inhibitor (sotorasib 960 mg orally once daily or adagrasib 600 mg orally twice daily) 3–4 weeks after TIL infusion until progression or toxicity. The diagram shows NMA-LD as a 5-day regimen. The 7-day regimen is as follows: NMA-LD regimen consists of 2 days of intravenous (IV) cyclophosphamide (60 mg / kg) with mesna (local standard of care or per USPI / SmPC) on days -7 and -6, and 5 days of fludarabine IV (25 mg / m², days -5 to -1). [Figure 36] Study design diagram for Cohort 3. Cohort 3: KRAS p.G12C inhibitor naive with TP53 co-mutation: Patients will begin a KRAS p.G12C inhibitor (sotorasib 960 mg orally once daily or adagrasib 600 mg orally twice daily) after TIL resection and until initiation of NMALD. Treatment will then resume 3–4 weeks after TIL infusion until progression or toxicity. The diagram below shows NMA-LD as a 5-day regimen. The 7-day regimen is as follows: NMA-LD regimen consists of 2 days of intravenous (IV) cyclophosphamide (60 mg / kg) with mesna (local standard of care or per USPI / SmPC) on days -7 and -6, and 5 days of fludarabine IV (25 mg / m2, days -5 to -1). [Figure 37]Study design diagram for Cohort 4. Cohort 4: KRAS p.G12C inhibitor pretreated, harboring TP53 co-mutation: Patients will resume the KRAS p.G12C inhibitor (sotorasib 960 mg orally once daily or adagrasib 600 mg orally twice daily) 3–4 weeks after TIL infusion until progression or toxicity. The diagram below shows NMA-LD as a 5-day regimen. The 7-day regimen is as follows: NMA-LD regimen consists of 2 days of intravenous (IV) cyclophosphamide (60 mg / kg) with mesna (local standard of care or per USPI / SmPC) on days -7 and 6, and 5 days of fludarabine IV (25 mg / m2, days -5 to 1).

[0148] Brief Description of Sequence Listing SEQ ID NO: 1 is the amino acid sequence of the heavy chain of muromonab.

[0149] SEQ ID NO: 2 is the amino acid sequence of the light chain of muromonab.

[0150] SEQ ID NO: 3 is the amino acid sequence of recombinant human IL-2 protein.

[0151] SEQ ID NO: 4 is the amino acid sequence of aldesleukin.

[0152] SEQ ID NO: 5 is an IL-2 form.

[0153] SEQ ID NO: 6 is the amino acid sequence of nemvaleukin alpha.

[0154] SEQ ID NO: 7 is an IL-2 form.

[0155] SEQ ID NO: 8 is a mucin domain polypeptide.

[0156] SEQ ID NO: 9 is the amino acid sequence of recombinant human IL-4 protein.

[0157] SEQ ID NO: 10 is the amino acid sequence of recombinant human IL-7 protein.

[0158] SEQ ID NO: 11 is the amino acid sequence of recombinant human IL-15 protein.

[0159] SEQ ID NO: 12 is the amino acid sequence of recombinant human IL-21 protein.

[0160] SEQ ID NO: 13 is the IL-2 sequence.

[0161] SEQ ID NO: 14 is the IL-2 mutein sequence.

[0162] SEQ ID NO: 15 is the sequence of an IL-2 mutein.

[0163] SEQ ID NO: 16 is HCDR1_IL-2 of IgG.IL2R67A.H1.

[0164] SEQ ID NO: 17 is HCDR2 of IgG.IL2R67A.H1.

[0165] SEQ ID NO: 18 is the HCDR3 of IgG.IL2R67A.H1.

[0166] SEQ ID NO: 19 is HCDR1_IL-2 Kabat of IgG.IL2R67A.H1.

[0167] SEQ ID NO: 20 is the HCDR2 Kabat of IgG.IL2R67A.H1.

[0168] SEQ ID NO: 21 is the HCDR3 Kabat of IgG.IL2R67A.H1.

[0169] SEQ ID NO: 22 is the HCDR1_IL-2 clotia of IgG.IL2R67A.H1.

[0170] SEQ ID NO: 23 is the HCDR2 clone of IgG.IL2R67A.H1.

[0171] SEQ ID NO: 24 is the HCDR3 clone of IgG.IL2R67A.H1.

[0172] SEQ ID NO: 25 is HCDR1_IL-2 IMGT of IgG.IL2R67A.H1.

[0173] SEQ ID NO: 26 is HCDR2 IMGT of IgG.IL2R67A.H1.

[0174] SEQ ID NO: 27 is the HCDR3 IMGT of IgG.IL2R67A.H1.

[0175] SEQ ID NO: 28 is the V H It's a chain.

[0176] SEQ ID NO: 29 is the heavy chain of IgG.IL2R67A.H1.

[0177] SEQ ID NO: 30 is the LCDR1 Kabat of IgG.IL2R67A.H1.

[0178] SEQ ID NO: 31 is the LCDR2 Kabat of IgG.IL2R67A.H1.

[0179] SEQ ID NO: 32 is the LCDR3 Kabat of IgG.IL2R67A.H1.

[0180] SEQ ID NO: 33 is the LCDR1 Clothiase of IgG.IL2R67A.H1.

[0181] SEQ ID NO: 34 is the LCDR2 clone of IgG.IL2R67A.H1.

[0182] SEQ ID NO: 35 is the LCDR3 clone of IgG.IL2R67A.H1.

[0183] SEQ ID NO: 36 is V L It's a chain.

[0184] SEQ ID NO: 37 is the light chain.

[0185] SEQ ID NO: 38 is the light chain.

[0186] SEQ ID NO: 39 is the light chain.

[0187] SEQ ID NO: 40 is the amino acid sequence of human 4-1BB.

[0188] SEQ ID NO: 41 is the amino acid sequence of mouse 4-1BB.

[0189] SEQ ID NO: 42 is the heavy chain of the 4-1BB agonist monoclonal antibody utomilumab (PF-05082566).

[0190] SEQ ID NO: 43 is the light chain of the 4-1BB agonist monoclonal antibody utomilumab (PF-05082566).

[0191] SEQ ID NO: 44 represents the heavy chain variable region (V) of the 4-1BB agonist monoclonal antibody utomilumab (PF-05082566). H )

[0192] SEQ ID NO: 45 is the light chain variable region (V) of the 4-1BB agonist monoclonal antibody utomilumab (PF-05082566). L )

[0193] SEQ ID NO: 46 is the heavy chain CDR1 of the 4-1BB agonist monoclonal antibody utomilumab (PF-05082566).

[0194] SEQ ID NO: 47 is the heavy chain CDR2 of the 4-1BB agonist monoclonal antibody utomilumab (PF-05082566).

[0195] SEQ ID NO: 48 is the heavy chain CDR3 of the 4-1BB agonist monoclonal antibody utomilumab (PF-05082566).

[0196] SEQ ID NO: 49 is the light chain CDR1 of the 4-1BB agonist monoclonal antibody utomilumab (PF-05082566).

[0197] SEQ ID NO: 50 is the light chain CDR2 of the 4-1BB agonist monoclonal antibody utomilumab (PF-05082566).

[0198] SEQ ID NO: 51 is the light chain CDR3 of the 4-1BB agonist monoclonal antibody utomilumab (PF-05082566).

[0199] SEQ ID NO: 52 is the heavy chain of the 4-1BB agonist monoclonal antibody urelumab (BMS-663513).

[0200] SEQ ID NO: 53 is the light chain of the 4-1BB agonist monoclonal antibody urelumab (BMS-663513).

[0201] SEQ ID NO: 54 is the heavy chain variable region (VH) of the 4-1BB agonist monoclonal antibody urelumab (BMS-663513).

[0202] SEQ ID NO: 55 is the light chain variable region (VL) of the 4-1BB agonist monoclonal antibody urelumab (BMS-663513).

[0203] SEQ ID NO: 56 is the heavy chain CDR1 of the 4-1BB agonist monoclonal antibody urelumab (BMS-663513).

[0204] SEQ ID NO: 57 is the heavy chain CDR2 of the 4-1BB agonist monoclonal antibody urelumab (BMS-663513).

[0205] SEQ ID NO: 58 is the heavy chain CDR3 of the 4-1BB agonist monoclonal antibody urelumab (BMS-663513).

[0206] SEQ ID NO: 59 is the light chain CDR1 of the 4-1BB agonist monoclonal antibody urelumab (BMS-663513).

[0207] SEQ ID NO: 60 is the light chain CDR2 of the 4-1BB agonist monoclonal antibody urelumab (BMS-663513).

[0208] SEQ ID NO: 61 is the light chain CDR3 of the 4-1BB agonist monoclonal antibody urelumab (BMS-663513).

[0209] SEQ ID NO: 62 is the Fc domain of the TNFRSF agonist fusion protein.

[0210] SEQ ID NO: 63 is the linker of the TNFRSF agonist fusion protein.

[0211] SEQ ID NO: 64 is the linker of the TNFRSF agonist fusion protein.

[0212] SEQ ID NO: 65 is the linker of the TNFRSF agonist fusion protein.

[0213] SEQ ID NO: 66 is the linker of the TNFRSF agonist fusion protein.

[0214] SEQ ID NO: 67 is the linker for the TNFRSF agonist fusion protein.

[0215] SEQ ID NO: 68 is the linker for the TNFRSF agonist fusion protein.

[0216] SEQ ID NO: 69 is the linker for the TNFRSF agonist fusion protein.

[0217] SEQ ID NO: 70 is the linker of the TNFRSF agonist fusion protein.

[0218] SEQ ID NO: 71 is the linker for the TNFRSF agonist fusion protein.

[0219] SEQ ID NO: 72 is the linker for the TNFRSF agonist fusion protein.

[0220] SEQ ID NO: 73 is the Fc domain of the TNFRSF agonist fusion protein.

[0221] SEQ ID NO: 74 is the linker for the TNFRSF agonist fusion protein.

[0222] SEQ ID NO: 75 is the linker for the TNFRSF agonist fusion protein.

[0223] SEQ ID NO: 76 is the linker for the TNFRSF agonist fusion protein.

[0224] SEQ ID NO: 77 is the 4-1BB ligand (4-1BBL) amino acid sequence.

[0225] SEQ ID NO: 78 is the soluble portion of the 4-1BBL polypeptide.

[0226] SEQ ID NO: 79 is the heavy chain variable region (V) of 4-1BB agonist antibody 4B4-1-1 version 1 H )

[0227] SEQ ID NO: 80 represents the light chain variable region (V) of 4-1BB agonist antibody 4B4-1-1 version 1 L )

[0228] SEQ ID NO: 81 is the heavy chain variable region (V H )

[0229] SEQ ID NO: 82 is the light chain variable region (V) of 4-1BB agonist antibody 4B4-1-1 version 2 L )

[0230] SEQ ID NO: 83 identifies the heavy chain variable region (V H )

[0231] SEQ ID NO: 84 identifies the light chain variable region (V L )

[0232] SEQ ID NO: 85 is the amino acid sequence of human OX40.

[0233] SEQ ID NO: 86 is the amino acid sequence of mouse OX40.

[0234] SEQ ID NO: 87 is the heavy chain of the OX40 agonist monoclonal antibody tabolixizumab (MEDI-0562).

[0235] SEQ ID NO: 88 is the light chain of the OX40 agonist monoclonal antibody tabolixizumab (MEDI-0562).

[0236] SEQ ID NO: 89 is the heavy chain variable region (V) of the OX40 agonist monoclonal antibody tabolixizumab (MEDI-0562). H )

[0237] SEQ ID NO: 90 is the light chain variable region (V) of the OX40 agonist monoclonal antibody tabolixizumab (MEDI-0562). L )

[0238] SEQ ID NO: 91 is the heavy chain CDR1 of the OX40 agonist monoclonal antibody tabolixizumab (MEDI-0562).

[0239] SEQ ID NO: 92 is the heavy chain CDR2 of the OX40 agonist monoclonal antibody tabolixizumab (MEDI-0562).

[0240] SEQ ID NO: 93 is the heavy chain CDR3 of the OX40 agonist monoclonal antibody tabolixizumab (MEDI-0562).

[0241] SEQ ID NO: 94 is the light chain CDR1 of the OX40 agonist monoclonal antibody tabolixizumab (MEDI-0562).

[0242] SEQ ID NO: 95 is the light chain CDR2 of the OX40 agonist monoclonal antibody tabolixizumab (MEDI-0562).

[0243] SEQ ID NO: 96 is the light chain CDR3 of the OX40 agonist monoclonal antibody tabolixizumab (MEDI-0562).

[0244] SEQ ID NO: 97 is the heavy chain of the OX40 agonist monoclonal antibody 11D4.

[0245] SEQ ID NO: 98 is the light chain of the OX40 agonist monoclonal antibody 11D4.

[0246] SEQ ID NO: 99 is the heavy chain variable region (V) of the OX40 agonist monoclonal antibody 11D4. H )

[0247] SEQ ID NO: 100 is the light chain variable region (V) of the OX40 agonist monoclonal antibody 11D4. L )

[0248] SEQ ID NO: 101 is the heavy chain CDR1 of the OX40 agonist monoclonal antibody 11D4.

[0249] SEQ ID NO: 102 is the heavy chain CDR2 of the OX40 agonist monoclonal antibody 11D4.

[0250] SEQ ID NO: 103 is the heavy chain CDR3 of the OX40 agonist monoclonal antibody 11D4.

[0251] SEQ ID NO: 104 is the light chain CDR1 of the OX40 agonist monoclonal antibody 11D4.

[0252] SEQ ID NO: 105 is the light chain CDR2 of the OX40 agonist monoclonal antibody 11D4.

[0253] SEQ ID NO: 106 is the light chain CDR3 of the OX40 agonist monoclonal antibody 11D4.

[0254] SEQ ID NO: 107 is the heavy chain of the OX40 agonist monoclonal antibody 18D8.

[0255] SEQ ID NO: 108 is the light chain of the OX40 agonist monoclonal antibody 18D8.

[0256] SEQ ID NO: 109 is the heavy chain variable region (V) of the OX40 agonist monoclonal antibody 18D8. H )

[0257] SEQ ID NO: 110 is the light chain variable region (V) of the OX40 agonist monoclonal antibody 18D8. L )

[0258] SEQ ID NO: 111 is the heavy chain CDR1 of the OX40 agonist monoclonal antibody 18D8.

[0259] SEQ ID NO: 112 is the heavy chain CDR2 of the OX40 agonist monoclonal antibody 18D8.

[0260] SEQ ID NO: 113 is the heavy chain CDR3 of the OX40 agonist monoclonal antibody 18D8.

[0261] SEQ ID NO: 114 is the light chain CDR1 of the OX40 agonist monoclonal antibody 18D8.

[0262] SEQ ID NO: 115 is the light chain CDR2 of the OX40 agonist monoclonal antibody 18D8.

[0263] SEQ ID NO: 116 is the light chain CDR3 of the OX40 agonist monoclonal antibody 18D8.

[0264] SEQ ID NO: 117 is the heavy chain variable region (V) of the OX40 agonist monoclonal antibody Hu119-122. H )

[0265] SEQ ID NO: 118 is the light chain variable region (V) of the OX40 agonist monoclonal antibody Hu119-122. L )

[0266] SEQ ID NO: 119 is the heavy chain CDR1 of the OX40 agonist monoclonal antibody Hu119-122.

[0267] SEQ ID NO: 120 is the heavy chain CDR2 of the OX40 agonist monoclonal antibody Hu119-122.

[0268] SEQ ID NO: 121 is the heavy chain CDR3 of the OX40 agonist monoclonal antibody Hu119-122.

[0269] SEQ ID NO: 122 is the light chain CDR1 of the OX40 agonist monoclonal antibody Hu119-122.

[0270] SEQ ID NO: 123 is the light chain CDR2 of the OX40 agonist monoclonal antibody Hu119-122.

[0271] SEQ ID NO: 124 is the light chain CDR3 of the OX40 agonist monoclonal antibody Hu119-122.

[0272] SEQ ID NO: 125 is the heavy chain variable region (V) of the OX40 agonist monoclonal antibody Hu106-222. H )

[0273] SEQ ID NO: 126 is the light chain variable region (V) of the OX40 agonist monoclonal antibody Hu106-222. L )

[0274] SEQ ID NO: 127 is the heavy chain CDR1 of the OX40 agonist monoclonal antibody Hu106-222.

[0275] SEQ ID NO: 128 is the heavy chain CDR2 of the OX40 agonist monoclonal antibody Hu106-222.

[0276] SEQ ID NO: 129 is the heavy chain CDR3 of the OX40 agonist monoclonal antibody Hu106-222.

[0277] SEQ ID NO: 130 is the light chain CDR1 of the OX40 agonist monoclonal antibody Hu106-222.

[0278] SEQ ID NO: 131 is the light chain CDR2 of the OX40 agonist monoclonal antibody Hu106-222.

[0279] SEQ ID NO: 132 is the light chain CDR3 of the OX40 agonist monoclonal antibody Hu106-222.

[0280] SEQ ID NO: 133 is the OX40 ligand (OX40L) amino acid sequence.

[0281] SEQ ID NO: 134 is the soluble portion of the OX40L polypeptide.

[0282] SEQ ID NO: 135 is an alternative soluble portion of the OX40L polypeptide.

[0283] SEQ ID NO: 136 is the heavy chain variable region (V H )

[0284] SEQ ID NO: 137 is the light chain variable region (V) of OX40 agonist monoclonal antibody 008. L )

[0285] SEQ ID NO: 138 is the heavy chain variable region (V) of OX40 agonist monoclonal antibody 011. H )

[0286] SEQ ID NO: 139 is the light chain variable region (V) of the OX40 agonist monoclonal antibody 011. L )

[0287] SEQ ID NO: 140 is the heavy chain variable region (V) of the OX40 agonist monoclonal antibody 021. H )

[0288] SEQ ID NO: 141 is the light chain variable region (V) of the OX40 agonist monoclonal antibody 021. L )

[0289] SEQ ID NO: 142 is the heavy chain variable region (V) of the OX40 agonist monoclonal antibody 023. H )

[0290] SEQ ID NO: 143 is the light chain variable region (V) of the OX40 agonist monoclonal antibody 023. L )

[0291] SEQ ID NO: 144 is the heavy chain variable region (V H )

[0292] SEQ ID NO: 145 is the light chain variable region (V) of the OX40 agonist monoclonal antibody L )

[0293] SEQ ID NO: 146 is the heavy chain variable region (V H )

[0294] SEQ ID NO: 147 is the light chain variable region (V) of the OX40 agonist monoclonal antibody L )

[0295] SEQ ID NO: 148 is the heavy chain variable region (V H )

[0296] SEQ ID NO: 149 is the heavy chain variable region (V) of a humanized OX40 agonist monoclonal antibody. H )

[0297] SEQ ID NO: 150 is the light chain variable region (V) of a humanized OX40 agonist monoclonal antibody. L )

[0298] SEQ ID NO: 151 is the light chain variable region (V) of a humanized OX40 agonist monoclonal antibody. L )

[0299] SEQ ID NO: 152 is the heavy chain variable region (V H )

[0300] SEQ ID NO: 153 is the heavy chain variable region (V) of a humanized OX40 agonist monoclonal antibody. H )

[0301] SEQ ID NO: 154 is the light chain variable region (V) of a humanized OX40 agonist monoclonal antibody. L )

[0302] SEQ ID NO: 155 is the light chain variable region (V) of a humanized OX40 agonist monoclonal antibody. L )

[0303] SEQ ID NO: 156 is the heavy chain variable region (V H )

[0304] SEQ ID NO: 157 is the light chain variable region (V) of the OX40 agonist monoclonal antibody L )

[0305] SEQ ID NO: 158 is the heavy chain amino acid sequence of the PD-1 inhibitor nivolumab.

[0306] SEQ ID NO: 159 is the light chain amino acid sequence of the PD-1 inhibitor nivolumab.

[0307] SEQ ID NO: 160 represents the heavy chain variable region (V) of the PD-1 inhibitor nivolumab H ) amino acid sequence.

[0308] SEQ ID NO: 161 represents the light chain variable region (V) of the PD-1 inhibitor nivolumab L ) amino acid sequence.

[0309] SEQ ID NO: 162 is the heavy chain CDR1 amino acid sequence of the PD-1 inhibitor nivolumab.

[0310] SEQ ID NO: 163 is the heavy chain CDR2 amino acid sequence of the PD-1 inhibitor nivolumab.

[0311] SEQ ID NO: 164 is the heavy chain CDR3 amino acid sequence of the PD-1 inhibitor nivolumab.

[0312] SEQ ID NO: 165 is the light chain CDR1 amino acid sequence of the PD-1 inhibitor nivolumab.

[0313] SEQ ID NO: 166 is the light chain CDR2 amino acid sequence of the PD-1 inhibitor nivolumab.

[0314] SEQ ID NO: 167 is the light chain CDR3 amino acid sequence of the PD-1 inhibitor nivolumab.

[0315] SEQ ID NO: 168 is the heavy chain amino acid sequence of the PD-1 inhibitor pembrolizumab.

[0316] SEQ ID NO: 169 is the light chain amino acid sequence of the PD-1 inhibitor pembrolizumab.

[0317] SEQ ID NO: 170 represents the heavy chain variable region (V) of the PD-1 inhibitor pembrolizumab H ) amino acid sequence.

[0318] SEQ ID NO: 171 is the light chain variable region (V) of the PD-1 inhibitor pembrolizumab L ) amino acid sequence.

[0319] SEQ ID NO: 172 is the heavy chain CDR1 amino acid sequence of the PD-1 inhibitor pembrolizumab.

[0320] SEQ ID NO: 173 is the heavy chain CDR2 amino acid sequence of the PD-1 inhibitor pembrolizumab.

[0321] SEQ ID NO: 174 is the heavy chain CDR3 amino acid sequence of the PD-1 inhibitor pembrolizumab.

[0322] SEQ ID NO: 175 is the light chain CDR1 amino acid sequence of the PD-1 inhibitor pembrolizumab.

[0323] SEQ ID NO: 176 is the light chain CDR2 amino acid sequence of the PD-1 inhibitor pembrolizumab.

[0324] SEQ ID NO: 177 is the light chain CDR3 amino acid sequence of the PD-1 inhibitor pembrolizumab.

[0325] SEQ ID NO: 178 is the heavy chain amino acid sequence of the PD-L1 inhibitor durvalumab.

[0326] SEQ ID NO: 179 is the light chain amino acid sequence of the PD-L1 inhibitor durvalumab.

[0327] SEQ ID NO: 180 represents the heavy chain variable region (V) of the PD-L1 inhibitor durvalumab H ) amino acid sequence.

[0328] SEQ ID NO: 181 represents the light chain variable region (V) of the PD-L1 inhibitor durvalumab L ) amino acid sequence.

[0329] SEQ ID NO: 182 is the heavy chain CDR1 amino acid sequence of the PD-L1 inhibitor durvalumab.

[0330] SEQ ID NO: 183 is the heavy chain CDR2 amino acid sequence of the PD-L1 inhibitor durvalumab.

[0331] SEQ ID NO: 184 is the heavy chain CDR3 amino acid sequence of the PD-L1 inhibitor durvalumab.

[0332] SEQ ID NO: 185 is the light chain CDR1 amino acid sequence of the PD-L1 inhibitor durvalumab.

[0333] SEQ ID NO: 186 is the light chain CDR2 amino acid sequence of the PD-L1 inhibitor durvalumab.

[0334] SEQ ID NO: 187 is the light chain CDR3 amino acid sequence of the PD-L1 inhibitor durvalumab.

[0335] SEQ ID NO: 188 is the heavy chain amino acid sequence of the PD-L1 inhibitor avelumab.

[0336] SEQ ID NO: 189 is the light chain amino acid sequence of the PD-L1 inhibitor avelumab.

[0337] SEQ ID NO: 190 represents the heavy chain variable region (V) of the PD-L1 inhibitor avelumab H ) amino acid sequence.

[0338] SEQ ID NO: 191 is the light chain variable region (V) of the PD-L1 inhibitor avelumab L ) amino acid sequence.

[0339] SEQ ID NO: 192 is the heavy chain CDR1 amino acid sequence of the PD-L1 inhibitor avelumab.

[0340] SEQ ID NO: 193 is the heavy chain CDR2 amino acid sequence of the PD-L1 inhibitor avelumab.

[0341] SEQ ID NO: 194 is the heavy chain CDR3 amino acid sequence of the PD-L1 inhibitor avelumab.

[0342] SEQ ID NO: 195 is the light chain CDR1 amino acid sequence of the PD-L1 inhibitor avelumab.

[0343] SEQ ID NO: 196 is the light chain CDR2 amino acid sequence of the PD-L1 inhibitor avelumab.

[0344] SEQ ID NO: 197 is the light chain CDR3 amino acid sequence of the PD-L1 inhibitor avelumab.

[0345] SEQ ID NO: 198 is the heavy chain amino acid sequence of the PD-L1 inhibitor atezolizumab.

[0346] SEQ ID NO: 199 is the light chain amino acid sequence of the PD-L1 inhibitor atezolizumab.

[0347] SEQ ID NO: 200 represents the heavy chain variable region (V) of the PD-L1 inhibitor atezolizumab H ) amino acid sequence.

[0348] SEQ ID NO: 201 is the light chain variable region (V) of the PD-L1 inhibitor atezolizumab L ) amino acid sequence.

[0349] SEQ ID NO: 202 is the heavy chain CDR1 amino acid sequence of the PD-L1 inhibitor atezolizumab.

[0350] SEQ ID NO: 203 is the heavy chain CDR2 amino acid sequence of the PD-L1 inhibitor atezolizumab.

[0351] SEQ ID NO: 204 is the heavy chain CDR3 amino acid sequence of the PD-L1 inhibitor atezolizumab.

[0352] SEQ ID NO: 205 is the light chain CDR1 amino acid sequence of the PD-L1 inhibitor atezolizumab.

[0353] SEQ ID NO: 206 is the light chain CDR2 amino acid sequence of the PD-L1 inhibitor atezolizumab.

[0354] SEQ ID NO: 207 is the light chain CDR3 amino acid sequence of the PD-L1 inhibitor atezolizumab.

[0355] SEQ ID NO: 208 is the heavy chain amino acid sequence of the CTLA-4 inhibitor ipilimumab.

[0356] SEQ ID NO: 209 is the light chain amino acid sequence of the CTLA-4 inhibitor ipilimumab.

[0357] SEQ ID NO: 210 represents the heavy chain variable region (V) of the CTLA-4 inhibitor ipilimumab H ) amino acid sequence.

[0358] SEQ ID NO: 211 represents the light chain variable region (V) of the CTLA-4 inhibitor ipilimumab L ) amino acid sequence.

[0359] SEQ ID NO: 212 is the heavy chain CDR1 amino acid sequence of the CTLA-4 inhibitor ipilimumab.

[0360] SEQ ID NO: 213 is the heavy chain CDR2 amino acid sequence of the CTLA-4 inhibitor ipilimumab.

[0361] SEQ ID NO: 214 is the heavy chain CDR3 amino acid sequence of the CTLA-4 inhibitor ipilimumab.

[0362] SEQ ID NO: 215 is the light chain CDR1 amino acid sequence of the CTLA-4 inhibitor ipilimumab.

[0363] SEQ ID NO: 216 is the light chain CDR2 amino acid sequence of the CTLA-4 inhibitor ipilimumab.

[0364] SEQ ID NO: 217 is the light chain CDR3 amino acid sequence of the CTLA-4 inhibitor ipilimumab.

[0365] SEQ ID NO: 218 is the heavy chain amino acid sequence of the CTLA-4 inhibitor tremelimumab.

[0366] SEQ ID NO: 219 is the light chain amino acid sequence of the CTLA-4 inhibitor tremelimumab.

[0367] SEQ ID NO: 220 represents the heavy chain variable region (V) of the CTLA-4 inhibitor tremelimumab H ) amino acid sequence.

[0368] SEQ ID NO: 221 represents the light chain variable region (V) of the CTLA-4 inhibitor tremelimumab L ) amino acid sequence.

[0369] SEQ ID NO: 222 is the heavy chain CDR1 amino acid sequence of the CTLA-4 inhibitor tremelimumab.

[0370] SEQ ID NO: 223 is the heavy chain CDR2 amino acid sequence of the CTLA-4 inhibitor tremelimumab.

[0371] SEQ ID NO: 224 is the heavy chain CDR3 amino acid sequence of the CTLA-4 inhibitor tremelimumab.

[0372] SEQ ID NO: 225 is the light chain CDR1 amino acid sequence of the CTLA-4 inhibitor tremelimumab.

[0373] SEQ ID NO: 226 is the light chain CDR2 amino acid sequence of the CTLA-4 inhibitor tremelimumab.

[0374] SEQ ID NO: 227 is the light chain CDR3 amino acid sequence of the CTLA-4 inhibitor tremelimumab.

[0375] SEQ ID NO: 228 is the heavy chain amino acid sequence of the CTLA-4 inhibitor zalifrelimab.

[0376] SEQ ID NO: 229 is the light chain amino acid sequence of the CTLA-4 inhibitor zalifrelimab.

[0377] SEQ ID NO: 230 is the heavy chain variable region (V) of the CTLA-4 inhibitor zalifrelimab H ) amino acid sequence.

[0378] SEQ ID NO: 231 is the light chain variable region (V) of the CTLA-4 inhibitor zalifrelimab L ) amino acid sequence.

[0379] SEQ ID NO: 232 is the heavy chain CDR1 amino acid sequence of the CTLA-4 inhibitor zalifrelimab.

[0380] SEQ ID NO: 233 is the heavy chain CDR2 amino acid sequence of the CTLA-4 inhibitor zalifrelimab.

[0381] SEQ ID NO: 234 is the heavy chain CDR3 amino acid sequence of the CTLA-4 inhibitor zalifrelimab.

[0382] SEQ ID NO: 235 is the light chain CDR1 amino acid sequence of the CTLA-4 inhibitor zalifrelimab.

[0383] SEQ ID NO: 236 is the light chain CDR2 amino acid sequence of the CTLA-4 inhibitor zalifrelimab.

[0384] SEQ ID NO: 237 is the light chain CDR3 amino acid sequence of the CTLA-4 inhibitor zalifrelimab.

[0385] I. Definition Unless otherwise defined, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this invention belongs. All patents and publications referenced herein are incorporated by reference in their entirety.

[0386] As used herein, the terms "co-administration," "co-administering," "administered in combination," "administering in combination," "simultaneous," and "concurrent" encompass administration of two or more active pharmaceutical ingredients (e.g., multiple TILs in preferred embodiments of the present invention) to a subject such that both active pharmaceutical ingredients and / or their metabolites are present in the subject at the same time. Concurrent administration includes simultaneous administration in separate compositions, administration at different times in separate compositions, or administration in a composition in which two or more active pharmaceutical ingredients are present. Concurrent administration in separate compositions and administration in a composition in which both agents are present are preferred.

[0387] The term "in vivo" refers to events that take place inside a subject's body.

[0388] The term "in vitro" refers to events that occur outside a subject's body. In vitro assays include cell-based assays, in which living or dead cells are used, and can also include cell-free assays, in which no intact cells are used.

[0389] The term "ex vivo" refers to events involving the administration of a therapy or treatment to cells, tissues, and / or organs that have been removed from a subject's body. Suitably, the cells, tissues, and / or organs may be returned to the subject's body by surgical or therapeutic methods.

[0390] The term "rapid expansion" refers to an increase in the number of antigen-specific TILs of at least about 3-fold (or 4, 5, 6, 7, 8, or 9-fold) over a one-week period, more preferably at least about 10-fold (or 20, 30, 40, 50, 60, 70, 80, or 90-fold) over a one-week period, or most preferably at least about 100-fold over a one-week period. Several rapid expansion protocols are described herein. Several rapid expansion protocols are outlined herein.

[0391] As used herein, "tumor infiltrating lymphocytes" or "TILs" refers to a population of cells that are initially acquired as leukocytes that have left the bloodstream of a subject and migrated to a tumor. TILs include CD8 + Cytotoxic T cells (lymphocytes), Th1 and Th17 CD4 + These include, but are not limited to, T cells, natural killer cells, dendritic cells, and M1 macrophages. TILs include both primary and secondary TILs. "Primary TILs" are those obtained from a patient tissue sample as outlined herein (sometimes referred to as "freshly harvested"), and "secondary TILs" are any expanded or propagated TIL cell populations discussed herein, including, but not limited to, bulk TILs and expanded TILs ("REP TILs" or "post-REP TILs"). TIL cell populations may include genetically modified TILs.

[0392] As used herein, a "population of cells" (including TILs) refers to several cells that share a common trait. Generally, a population is generally on the order of 1 x 10 6 ~1×10 10 The number of TILs ranges from 1 x 10 to 1 x 10, with different TIL populations containing different numbers. For example, the initial growth of primary TILs in the presence of IL-2 is approximately 1 x 10 8 REP expansion typically yields a bulk TIL population of 1.5 x 10 cells for injection. 9 ~1.5×10 10 This is done to provide a population of cells.

[0393] As used herein, "cryopreserved TILs" refers to TILs, either primary, bulk, or expanded (REP TILs), that are processed and stored at temperatures ranging from approximately -150°C to -60°C. General methods for cryopreservation are described elsewhere herein, including in the Examples. For clarity, "cryopreserved TILs" can be distinguished from frozen tissue samples that may be used as a source of primary TILs.

[0394] As used herein, "thawed cryopreserved TILs" refers to a population of TILs that have been previously cryopreserved and then processed to return to room temperature or above, including but not limited to, cell culture temperature or a temperature at which the TILs can be administered to a patient.

[0395] TILs can generally be defined either biochemically using cell surface markers or functionally by their ability to infiltrate tumors and achieve therapy. TILs can generally be classified by expression of one or more of the following biomarkers: CD4, CD8, TCRαβ, CD27, CD28, CD56, CCR7, CD45Ra, CD95, PD-1, and CD25. Additionally and alternatively, TILs can be functionally defined by their ability to infiltrate solid tumors upon reintroduction into patients.

[0396] The term "cryopreservation media" or "cryopreservation medium" refers to any medium that can be used for cryopreserving cells. Such media can include media containing 7% 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 that contains DMSO. In some embodiments, CS10 medium contains 10% DMSO.

[0397] The term "central memory T cells" refers to cells that are CD45R0+ and CCR7 (CCR7 高 ) and CD62L (CD62 高 ) is a subset of T cells that constitutively express the CD4 receptor. The surface phenotype of central memory T cells also includes TCR, CD3, CD127 (IL-7R), and IL-15R. Transcription factors of central memory T cells include BCL-6, BCL-6B, MBD2, and BMI1. After TCR triggering, central memory T cells primarily secrete IL-2 and CD40L as effector molecules. Central memory T cells predominate in the CD4 compartment in the blood and are proportionally enriched in lymph nodes and tonsils in humans.

[0398] The term "effector memory T cells" refers to T cells that are CD45R0+ like central memory T cells, but have lost constitutive expression of CCR7 (CCR7 低 ), heterogeneous or low CD62L expression (CD62L 低), refers 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. Transcription factors of central memory T cells include BLIMP1. Effector memory T cells rapidly secrete high levels of inflammatory cytokines after antigen stimulation, including interferon gamma, IL-4, and IL-5. Effector memory T cells predominate in the CD8 compartment in the blood and are proportionally enriched in the lung, liver, and intestine in humans. CD8+ effector memory T cells carry large amounts of perforin.

[0399] The term "closed system" refers to a system that is closed to the external environment. Any closed system suitable for cell culture methods can be used in the methods of the present invention. Closed systems include, but are not limited to, closed G-vessels. Once tumor segments are added to the closed system, the system is not opened to the external environment until the TILs are ready to be administered to a patient.

[0400] The terms "fragmenting," "fragments," and "fragmented" as used herein to describe processes for destroying tumors include mechanical fragmentation methods such as crushing, slicing, dividing, and mincing tumor tissue, as well as any other method for disrupting the physical structure of tumor tissue.

[0401] The terms "peripheral blood mononuclear cells" and "PBMCs" refer to peripheral blood cells with round nuclei, including lymphocytes (T cells, B cells, NK cells) and monocytes. When used as antigen-presenting cells (PBMCs are a type of antigen-presenting cell), the peripheral blood mononuclear cells are preferably irradiated allogeneic peripheral blood mononuclear cells.

[0402] The terms "peripheral blood lymphocytes" and "PBLs" refer to T cells expanded from peripheral blood. In some embodiments, PBLs are isolated from whole blood or apheresis products from a donor. In some embodiments, PBLs are isolated from whole blood or apheresis products from a donor by positive or negative selection of a T cell phenotype, such as a CD3+CD45+ T cell phenotype.

[0403] The term "anti-CD3 antibody" refers to an antibody or variant thereof, e.g., a monoclonal antibody, including a human, humanized, chimeric, or murine antibody, directed against the CD3 receptor in the T cell antigen receptor of mature T cells. Anti-CD3 antibodies include OKT-3, also known as muromonab. Anti-CD3 antibodies also include the UHCT1 clone, also known as T3 and CD3ε. Other anti-CD3 antibodies include, for example, otelixizumab, teplizumab, and visilizumab.

[0404] The term "OKT-3" (also referred to herein as "OKT3") refers to a monoclonal antibody, including a human, humanized, chimeric, or murine antibody against the CD3 receptor in the T cell antigen receptor of mature T cells, or a biosimilar or variant thereof, including commercially available forms such as OKT-3 (30 ng / mL, MACS GMP CD3 pure, Miltenyi Biotech, Inc., San Diego, CA, USA) and muromonab, or variants, conservative amino acid substitutions, glycoforms, or biosimilars thereof. The amino acid sequences of the heavy and light chains of muromonab are shown in Table 1 (SEQ ID NO: 1 and SEQ ID NO: 2). A hybridoma capable of producing OKT-3 has been deposited with the American Type Culture Collection and assigned ATCC accession number CRL8001. A hybridoma capable of producing OKT-3 has also been deposited with the European Collection of Authenticated Cell Cultures (ECACC) and assigned catalog number 86022706. [Table 1]

[0405] 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 human and mammalian forms, conservative amino acid substitutions, glycoforms, biosimilars, and variants. IL-2 is described, for example, in Nelson, J. Immunol. 2004, 172, 3983-88 and Malek, Annu. Rev. Immunol. 2008, 26, 453-79, the disclosures of which are incorporated herein by reference. The amino acid sequence of recombinant human IL-2 suitable for use in the present invention is shown in Table 2 (SEQ ID NO: 3). For example, the term IL-2 encompasses human recombinant IL-2 forms such as aldesleukin (PROLEUKIN, commercially available from multiple suppliers at 22 million IU per single-use vial), as well as the recombinant IL-2 form (catalog number CYT-209-b) marketed by CellGenix, Inc., Portsmouth, NH, USA (CELLGRO GMP) or ProSpec-Tany TechnoGene Ltd., East Brunswick, NJ, USA, and other commercial equivalents from other vendors. Aldesleukin (des-alanyl-1, serine-125 human IL-2) is a non-glycosylated human recombinant IL-2 form with a molecular weight of approximately 15 kDa. The amino acid sequence of aldesleukin suitable for use in the present invention is shown in Table 2 (SEQ ID NO: 4). The term IL-2 also refers to the pegylated IL-2 prodrug bempegaldesleukin (NKTR-214, an IL-2 prodrug in which an average of six lysine residues are replaced with [(2,7-bis{[methylpoly(oxyethylene)]carbamoyl}-9H-fluoren-9-yl)methoxy]carbonyl). 6The present invention also encompasses pegylated forms of IL-2 described herein, including pegylated human recombinant IL-2 such as SEQ ID NO: 4, which is available from Nektar Therapeutics (South San Francisco, CA, USA) or can be prepared by methods known in the art, such as the method described in Example 19 of International Patent Application Publication No. WO 2018 / 132496 A1 or Example 1 of U.S. Patent Application Publication No. US 2019 / 0275133 A1, the disclosures of which are incorporated herein by reference. Benpegaldesleukin (NKTR-214) and other pegylated IL-2 molecules suitable for use in the present invention are described in U.S. Patent Application Publication No. US 2014 / 0328791 A1 and International Patent Application Publication No. WO 2012 / 065086 A1, the disclosures of which are incorporated herein by reference. Alternative forms of conjugated IL-2 suitable for use in the present invention are described in U.S. Patent Nos. 4,766,106, 5,206,344, 5,089,261, and 4,902,502, the disclosures of which are incorporated herein by reference. Formulations of IL-2 suitable for use in the present invention are described in U.S. Patent No. 6,706,289, the disclosure of which is incorporated herein by reference.

[0406] In some embodiments, a suitable IL-2 form for use in the present invention is THOR-707, available from Synthorx, Inc. The preparation and properties of THOR-707 and additional alternative forms of IL-2 suitable for use in the present invention are described in U.S. Patent Application Publication Nos. US2020 / 0181220A1 and US2020 / 0330601A1, the disclosures of which are incorporated herein by reference. In some embodiments, a suitable IL-2 form for use in the present invention is an interleukin-2 (IL-2) complex comprising an isolated and purified IL-2 polypeptide and a conjugation moiety that binds to the isolated and purified IL-2 polypeptide at an amino acid position selected from K35, T37, R38, T41, F42, K43, F44, Y45, E61, E62, E68, K64, P65, V69, L72, and Y107, wherein the numbering of the amino acid residues corresponds to SEQ ID NO:5. In some embodiments, the amino acid position is selected from T37, R38, T41, F42, F44, Y45, E61, E62, E68, K64, P65, V69, L72, and Y107. In some embodiments, the amino acid position is selected from T37, R38, T41, F42, F44, Y45, E61, E62, E68, P65, V69, L72, and Y107. In some embodiments, the amino acid position is selected from T37, T41, F42, F44, Y45, P65, V69, L72, and Y107. In some embodiments, the amino acid position is selected from R38 and K64. In some embodiments, the amino acid position is selected from E61, E62, and E68. In some embodiments, the amino acid position is E62. In some embodiments, an amino acid residue selected from K35, T37, R38, T41, F42, K43, F44, Y45, E61, E62, E68, K64, P65, V69, L72, and Y107 is further mutated to lysine, cysteine, or histidine. In some embodiments, the amino acid residue is mutated to cysteine. In some embodiments, the amino acid residue is mutated to lysine. In some embodiments, K35, T37, R38, T41, F42, K43, F44, Y45, E61, E62, E68, K64, P65, V69, L72, and Y107 are ...and Y107 are further mutated to an unnatural amino acid. In some embodiments, the unnatural amino acid is selected from N6-azidoethoxy-L-lysine (AzK), N6-propargylethoxy-L-lysine (PraK), BCN-L-lysine, norbornene lysine, TCO-lysine, methyltetrazine lysine, allyloxycarbonyl lysine, 2-amino-8-oxononanoic acid, 2-amino-8-oxooctanoic acid, p-acetyl-L-phenylalanine, p-azidomethyl-L-phenylalanine (pAMF), p-iodo-L-phenylalanine, m-acetylphenylalanine, 2-amino-8-oxononanoic acid, p-propargyloxyphenylalanine, p-propargyl-phenylalanine, 3-methyl-phenylalanine, L-dopa, fluorinated phenylalanine, isopropyl and selenocysteine, or selenocysteine. In some embodiments, the IL-2 complex has a reduced affinity for the IL-2 receptor alpha (IL-2Rα) subunit compared to a wild-type IL-2 polypeptide. In some embodiments, the reduced affinity is about a 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 95%, 99%, or greater than a 99% reduction in binding affinity for IL-2Rα compared to a wild-type IL-2 polypeptide. In some embodiments, the reduced affinity is about a 1-fold, 2-fold, 3-fold, 4-fold, 5-fold, 6-fold, 7-fold, 8-fold, 9-fold, 10-fold, 30-fold, 50-fold, 100-fold, 200-fold, 300-fold, 500-fold, or greater reduction in binding affinity for IL-2Rα compared to a wild-type IL-2 polypeptide.1000-fold or more. In some embodiments, the conjugated moiety impairs or blocks the binding of IL-2 to IL-2Rα. In some embodiments, the conjugated moiety comprises a water-soluble polymer. In some embodiments, the additional conjugated moiety comprises a water-soluble polymer. In some embodiments, each of the water-soluble polymers independently comprises polyethylene glycol (PEG), poly(propylene glycol) (PPG), copolymers of ethylene glycol and propylene glycol, poly(oxyethylated polyol), poly(olefinic alcohol), poly(vinylpyrrolidone), poly(hydroxyalkyl methacrylamide), poly(hydroxyalkyl methacrylate), poly(saccharide), poly(α-hydroxy acid), poly(vinyl alcohol), polyphosphazene, polyoxazoline (POZ), poly(N-acryloylmorpholine), or a combination thereof. In some embodiments, each of the water-soluble polymers independently comprises PEG. In some embodiments, the PEG is linear PEG or branched PEG. In some embodiments, each of the water-soluble polymers independently comprises a polysaccharide. In some embodiments, the polysaccharide comprises dextran, polysialic acid (PSA), hyaluronic acid (HA), amylose, heparin, heparan sulfate (HS), dextrin, or hydroxyethyl starch (HES). In some embodiments, each of the water soluble polymers independently comprises a glycan. In some embodiments, each of the water soluble polymers independently comprises a polyamine. In some embodiments, the conjugation moiety comprises a protein. In some embodiments, the additional conjugation moieties comprise a protein. In some embodiments, each of the proteins independently comprises albumin, transferrin, or transthyretin. In some embodiments, each of the proteins independently comprises an Fc portion. In some embodiments, each of the proteins independently comprises an Fc portion of an IgG. In some embodiments, the conjugation moiety comprises a polypeptide. In some embodiments, the additional conjugation moieties comprise a polypeptide. In some embodiments, each of the proteins independently comprises an XTEN peptide, a glycine-rich homoamino acid polymer (HAP),The conjugated moiety may comprise a PAS polypeptide, an elastin-like polypeptide (ELP), a CTP peptide, or a gelatin-like protein (GLK) polymer. In some embodiments, the isolated and purified IL-2 polypeptide is modified by glutamylation. In some embodiments, the conjugated moiety is directly attached to the isolated and purified IL-2 polypeptide. In some embodiments, the conjugated moiety is indirectly attached to the isolated and purified IL-2 polypeptide via a linker. In some embodiments, the linker comprises a homobifunctional linker. In some embodiments, the homobifunctional linker is selected from the group consisting of the Romant reagents dithiobis(succinimidyl propionate) DSP, 3'3'-dithiobis(sulfosuccinimidyl propionate) (DTSSP), disuccinimidyl suberate (DSS), bis(sulfosuccinimidyl) suberate (BS), disuccinimidyl tartrate (DST), disulfosuccinimidyl tartrate (sulfoDS T), ethylene glycobis(succinimidyl succinate) (EGS), disuccinimidyl glutarate (DSG), N,N'-disuccinimidyl carbonate (DSC), dimethyl adipimidate (DMA), dimethyl pimelimidate (DMP), dimethyl suberimidate (DMS), dimethyl-3,3'-dithiobispropionimidate (DTBP), 1,4-di-(3'-(2'-pyridyldithio) (e) propionamido) butane (DPDPB), bismaleimidohexane (BMH), halogenated aryl-containing compounds (DFDNB), such as 1,5-difluoro-2,4-dinitrobenzene or 1,3-difluoro-4,6-dinitrobenzene, 4,4'-difluoro-3,3'-dinitrophenyl sulfone (DFDNPS), bis-[β-(4-azidosalicylamido)ethyl] disulfide (BASED), formaldehyde, glutaraldehyde, 1,4-butanediol diglycidyl ether, adipic acid dihydrazide, carbohydrazide, o-toluidine, 3,3'-dimethylbenzidine, benzidine, α,α'-p-diaminodiphenyl, diiodo-p-xylene sulfonic acid, N,N'-ethylene-bis(iodoacetamide), or N,N'-hexamethylenebis(iodoacetamide). In some embodiments, the linker isIn some embodiments, the heterobifunctional linker includes N-succinimidyl 3-(2-pyridyldithio)propionate (sPDP), long-chain N-succinimidyl 3-(2-pyridyldithio)propionate (LC-sPDP), water-soluble long-chain N-succinimidyl 3-(2-pyridyldithio)propionate (sulfo-LC-sPDP), succinimidyloxycarbonyl-α-methyl-α-(2-pyridyldithio)toluene (sMPT), sulfosuccinimidyl-6-[α-methyl-α-(2-pyridyldithio)toluamide]hexamethyl. Succinimidyl-4-(N-maleimidomethyl)cyclohexane-1-carboxylate (sulfo-LC-sMPT), succinimidyl-4-(N-maleimidomethyl)cyclohexane-1-carboxylate (sMCC), sulfosuccinimidyl-4-(N-maleimidomethyl)cyclohexane-1-carboxylate (sulfo-sMCC), m-maleimidobenzoyl-N-hydroxysuccinimide ester (MB), m-maleimidobenzoyl-N-hydroxysulfosuccinimide ester (sulfo-MB), N-succinimidyl (4-iodoacetyl)aminobenzoate (sIAB), sulfosuccinimide Succinimidyl (4-iodoacetyl)aminobenzoate (sulfo-sIAB), succinimidyl-4-(p-maleimidophenyl)butyrate (sMPB), sulfosuccinimidyl-4-(p-maleimidophenyl)butyrate (sulfo-sMPB), N-(γ-maleimidobutyryloxy)succinimide ester (GMB), N-(γ-maleimidobutyryloxy)sulfosuccinimide ester (sulfo-GMB), succinimidyl 6-((iodoacetyl)amino)hexanoate (sIAX), succinimidyl 6-[6-(( (iodoacetyl)amino)hexanoyl)amino]hexanoate (slAXX), succinimidyl 4-(((iodoacetyl)amino)methyl)cyclohexane-1-carboxylate (sIAC), succinimidyl 6-(((((4-iodoacetyl)amino)methyl)cyclohexane-1-carbonyl)amino)hexanoate (sIACX), p-nitrophenyl iodoacetate (NPIA), carbonyl-reactive and sulfhydryl-reactive crosslinkers, such as 4-(4-N-maleimidophenyl)butyric acid hydrazide (MPBH),4-(N-maleimidomethyl)cyclohexane-1-carboxyl-hydrazide-8 (M2C2H), 3-(2-pyridyldithio)propionylhydrazide (PDPH), N-hydroxysuccinimidyl-4-azidosalicylate (NHs-AsA), N-hydroxysulfosuccinimidyl-4-azidosalicylate (sulfo-NHs-AsA), sulfosuccinimidyl-(4-, N-hydroxysuccinimidyl-4-azidobenzoate (sulfo-NHs-LC-AsA), sulfosuccinimidyl-2-(p-azidosalicylamido)ethyl-1,3'-dithiopropionate (sAsD), N-hydroxysuccinimidyl-4-azidobenzoate (HsAB), N-hydroxysulfosuccinimidyl-4-azidobenzoate (sulfo-HsAB), N-succinimidyl-6-(4'-azido-2'-nitrophenylamino)hexanoate (sANPAH), sulfosuccinimidyl-6-(4'-azido-2'-nitrophenylamino)hexanoate (sulfo-NHs-LC-AsA), sulfosuccinimidyl-2-(p-azidosalicylamido)ethyl-1,3'-dithiopropionate (sAsD), sulfosuccinimidyl-4-azidobenzoate (HsAB), sulfosuccinimidyl-6-(4'-azido-2'-nitrophenylamino)hexanoate (sANPAH), sulfosuccinimidyl-6-(4'-azido-2'-nitrophenylamino)hexanoate (sulfo-NHs-LC-AsA), sulfosuccinimidyl-2-(p-azidosalicylamido)ethyl-1,3'-dithiopropionate (sAsD), sulfosuccinimidyl-4-azidobenzoate (sulfo-HsAB), sulfosuccinimidyl-6-(4'-azido-2'-nitrophenylamino)hexanoate (sANPAH), sulfosuccinimidyl-6-(4'-azido-2'-nitrophenylamino)hexanoate (sulfo-NHs-LC-AsA ... Succinimidyl-6-(4'-azido-2'-nitrophenylamino)hexanoate (sulfo-sANPAH), N-5-azido-2-nitrobenzoyloxysuccinimide (ANB-NO), sulfosuccinimidyl-2-(m-azido-o-nitrobenzamido)-ethyl-1,3'-dithiopropionate (sAND), N-succinimidyl-4(4-azidophenyl)1,3'-dithiopropionate (sADP), N-sulfosuccinimidyl(4-azidophenyl)- 1,3'-dithiopropionate (sulfo-sADP), sulfosuccinimidyl 4-(ρ-azidophenyl)butyrate (sulfo-sAPB), sulfosuccinimidyl 2-(7-azido-4-methylcoumarin-3-acetamido)ethyl-1,3'-dithiopropionate (sAED), sulfosuccinimidyl 7-azido-4-methylcoumarin-3-acetate (sulfo-sAMCA), p-nitrophenyl diazopyruvate (pNPDP), p-nitrophenyl diazo-3 ,3,3-trifluoropropionate (PNP-DTP), 1-(ρ-azidosalicylamido)-4-(iodoacetamido)butane (AsIB), N-[4-(ρ-azidosalicylamido)butyl]-3'-(2'-pyridyldithio)propionamide (APDP), benzophenone-4-iodoacetamide, p-azidobenzoylhydrazide (ABH), 4-(ρ-azidosalicylamido)butylamine (AsBA), or p-azidophenylglyoxal (APG). In some embodiments, the linker optionally comprises a non-cleavable linker, including a dipeptide linker. In some embodiments, the dipeptide linker comprises Val-Cit, Phe-Lys, Val-Ala, or Val-Lys.In some embodiments, the linker comprises a non-cleavable linker. In some embodiments, the linker optionally comprises a maleimide group comprising maleimidocaproyl (mc), succinimidyl-4-(N-maleimidomethyl)cyclohexane-1-carboxylate (sMCC), or sulfosuccinimidyl-4-(N-maleimidomethyl)cyclohexane-1-carboxylate (sulfo-sMCC). In some embodiments, the linker further comprises a spacer. In some embodiments, the spacer comprises p-aminobenzyl alcohol (PAB), p-aminobenzyloxycarbonyl (PABC), a derivative, or an analog thereof. In some embodiments, the conjugated moiety is capable of extending the serum half-life of the IL-2 conjugate. In some embodiments, the additional conjugated moiety is capable of extending the serum half-life of the IL-2 conjugate. In some embodiments, the IL-2 form suitable for use in the present invention is a fragment of any of the IL-2 forms described herein. In some embodiments, IL-2 forms suitable for use in the present invention are pegylated as disclosed in U.S. Patent Application Publication Nos. US2020 / 0181220A1 and US2020 / 0330601A1. In some embodiments, IL-2 forms suitable for use in the present invention are IL-2 conjugates comprising an IL-2 polypeptide comprising N6-azidoethoxy-L-lysine (AzK) covalently attached to a conjugation moiety comprising polyethylene glycol (PEG), wherein the IL-2 polypeptide comprises an amino acid sequence having at least 80% sequence identity to SEQ ID NO:5, and AzK substitutes an amino acid at position K35, F42, F44, K43, E62, P65, R38, T41, E68, Y45, V69, or L72 relative to amino acid position within SEQ ID NO:5. In some embodiments, the IL-2 polypeptide comprises a one-residue N-terminal deletion relative to SEQ ID NO:5. In some embodiments, forms of IL-2 suitable for use in the present invention lack IL-2R alpha chain association but retain normal binding to the intermediate affinity IL-2R beta-gamma signaling complex.In some embodiments, a suitable IL-2 form for use in the present invention is an IL-2 conjugate comprising an IL-2 polypeptide comprising N6-azidoethoxy-L-lysine (AzK) covalently attached to a conjugation moiety comprising polyethylene glycol (PEG), wherein the IL-2 polypeptide comprises an amino acid sequence having at least 90% sequence identity to SEQ ID NO:5, and wherein AzK substitutes an amino acid at position K35, F42, F44, K43, E62, P65, R38, T41, E68, Y45, V69, or L72 relative to amino acid position within SEQ ID NO:5. In some embodiments, a suitable IL-2 form for use in the present invention is an IL-2 conjugate comprising an IL-2 polypeptide comprising N6-azidoethoxy-L-lysine (AzK) covalently attached to a conjugation moiety comprising polyethylene glycol (PEG), wherein the IL-2 polypeptide comprises an amino acid sequence having at least 95% sequence identity to SEQ ID NO:5, and wherein AzK substitutes an amino acid at position K35, F42, F44, K43, E62, P65, R38, T41, E68, Y45, V69, or L72 relative to amino acid position within SEQ ID NO:5. In some embodiments, a suitable IL-2 form for use in the present invention is an IL-2 conjugate comprising an IL-2 polypeptide comprising N6-azidoethoxy-L-lysine (AzK) covalently attached to a conjugation moiety comprising polyethylene glycol (PEG), wherein the IL-2 polypeptide comprises an amino acid sequence having at least 98% sequence identity to SEQ ID NO:5, and wherein AzK substitutes an amino acid at position K35, F42, F44, K43, E62, P65, R38, T41, E68, Y45, V69, or L72 relative to amino acid position in SEQ ID NO:5.

[0407] In some embodiments, a form of IL-2 suitable for use in the present invention is nembareukin alfa, also known as ALKS-4230 (SEQ ID NO: 6), available from Alkermes, Inc. Nembareukin alfa is a nucleotide analogue of IL-2 that is linked via a peptidyl linker ( 60 GG 61 ) and fused to human interleukin-2 fragment (62-132) via a peptidyl linker ( 133 GSGGGS 138Human interleukin-2 receptor α-chain fragment (139-303) fused via a glycosylated variant (Cys) produced in Chinese hamster ovary (CHO) cells. 125 >Ser 51 ; fused to human interleukin 2 (IL-2) (4-74)-peptide (62-132) via a G2 peptide linker (60-61) and to human interleukin 2 receptor alpha chain (IL2R subunit alpha, IL2Rα, IL2RA) (1-165)-peptide (139-303) via a GSG3S peptide linker (133-138), and produced in Chinese hamster ovary (CHO) cells, alpha-glycosylated human interleukin 2 (IL-2) (75-133)-peptide [Cys 125(51)>Ser]-mutant (1-59). The amino acid sequence of nemvaleukin alpha is set forth in SEQ ID NO: 6. In some embodiments, nemvaleukin alpha exhibits the following post-translational modifications: disulfide bridges at the following positions: 31-116, 141-285, 184-242, 269-301, 166-197, or 166-199, 168-199, or 168-197 (using the numbering of SEQ ID NO: 6), and glycosylation sites at the following positions: N187, N206, T212, using the numbering of SEQ ID NO: 6. The preparation and properties of nemvaleukin alpha, as well as additional alternative forms of IL-2 suitable for use in the present invention, are described in U.S. Patent Application Publication No. US2021 / 0038684A1 and U.S. Patent No. 10,183,979, the disclosures of which are incorporated herein by reference. In some embodiments, an IL-2 form suitable for use in the present invention is a protein having at least 80%, at least 90%, at least 95%, or at least 90% sequence identity to SEQ ID NO:6. In some embodiments, an IL-2 form suitable for use in the present invention has the amino acid sequence set forth in SEQ ID NO:6 or conservative amino acid substitutions thereof. In some embodiments, an IL-2 form suitable for use in the present invention is a fusion protein comprising amino acids 24-452 of SEQ ID NO:7, or a variant, fragment, or derivative thereof. In some embodiments, an IL-2 form suitable for use in the present invention is a fusion protein comprising an amino acid sequence having at least 80%, at least 90%, at least 95%, or at least 90% sequence identity to amino acids 24-452 of SEQ ID NO:7, or a variant, fragment, or derivative thereof. Other IL-2 forms suitable for use in the present invention are described in U.S. Pat. No. 10,183,979, the disclosure of which is incorporated herein by reference.Optionally, in some embodiments, a form of IL-2 suitable for use in the invention is a fusion protein comprising a first fusion partner linked to a second fusion partner by a mucin domain polypeptide linker, wherein the first fusion partner is IL-1Rα or a protein having at least 98% amino acid sequence identity to IL-1Rα and having receptor antagonist activity for IL-Rα, the second fusion partner comprises all or a portion of an immunoglobulin comprising an Fc region, and the mucin domain polypeptide linker comprises SEQ ID NO:8 or an amino acid sequence having at least 90% sequence identity to SEQ ID NO:8, and wherein the half-life of the fusion protein is improved compared to fusion of the first fusion partner with the second fusion partner in the absence of the mucin domain polypeptide linker. [Table 2]

[0408] In some embodiments, IL-2 forms suitable for use in the present invention comprise a heavy chain variable region (V) comprising complementarity determining regions HCDR1, HCDR2, and HCDR3. H ) and a light chain variable region (V L ) and V H or V L and an IL-2 molecule or a fragment thereof grafted onto the CDRs of a heavy chain variable region (V) comprising complementarity determining regions HCDR1, HCDR2, and HCDR3. H ) and a light chain variable region (V L ) and V H or V Land an IL-2 molecule or fragment thereof grafted onto the CDRs of, wherein the IL-2 molecule is a mutein, and the antibody cytokine grafted protein preferentially expands T effector cells over regulatory T cells. In some embodiments, the IL-2 regimen comprises administration of an antibody described in U.S. Patent Application Publication No. US2020 / 0270334A1, the disclosure of which is incorporated herein by reference. In some embodiments, the antibody cytokine grafted protein comprises a heavy chain variable region (V) comprising complementarity determining regions HCDR1, HCDR2, and HCDR3. H ) and a light chain variable region (V L ) and an IL-2 molecule or a fragment thereof grafted into the CDR of VH or VL, wherein the IL-2 molecule is a mutein, and the antibody cytokine grafted protein preferentially expands T effector cells over regulatory T cells, and the antibody further comprises an IgG class heavy chain and an IgG class light chain selected from the group consisting of an IgG class light chain comprising SEQ ID NO: 39 and an IgG class heavy chain comprising SEQ ID NO: 38, an IgG class light chain comprising SEQ ID NO: 37 and an IgG class heavy chain comprising SEQ ID NO: 29, an IgG class light chain comprising SEQ ID NO: 39 and an IgG class heavy chain comprising SEQ ID NO: 29, an IgG class light chain comprising SEQ ID NO: 37 and an IgG class heavy chain comprising SEQ ID NO: 38.

[0409] In some embodiments, the IL-2 molecule or fragment thereof is V H In some embodiments, the IL-2 molecule or fragment thereof is a mutein. H In some embodiments, the IL-2 molecule or fragment thereof is a mutein. H In some embodiments, the IL-2 molecule or fragment thereof is a mutein. L In some embodiments, the IL-2 molecule or a fragment thereof is grafted onto LCDR1 of V LIn some embodiments, the IL-2 molecule or fragment thereof is grafted onto LCDR2 of V L The LCDR3 of the IL-2 molecule is grafted onto the LCDR3 of the IL-2 molecule, and the IL-2 molecule is a mutein.

[0410] The insertion of the IL-2 molecule can be at or near the N-terminal region of the CDR, the middle region of the CDR, or at or near the C-terminal region of the CDR. In some embodiments, the antibody cytokine graft protein comprises an IL-2 molecule incorporated into the CDR, where the IL2 sequence does not frameshift the CDR sequence. In some embodiments, the antibody cytokine graft protein comprises an IL-2 molecule incorporated into the CDR, where the IL-2 sequence replaces all or part of the CDR sequence. The replacement with the IL-2 molecule can be at or near the N-terminal region of the CDR, the middle region of the CDR, or the C-terminal region of the CDR. The replacement with the IL-2 molecule can be as little as one or two amino acids of the CDR sequence, or the entire CDR sequence.

[0411] In some embodiments, the IL-2 molecule is directly grafted onto the CDR without a peptide linker and without additional amino acids between the CDR sequence and the IL-2 sequence. In some embodiments, the IL-2 molecule is indirectly grafted onto the CDR using a peptide linker with one or more additional amino acids between the CDR sequence and the IL-2 sequence.

[0412] In some embodiments, the IL-2 molecule described herein is an IL-2 mutein. In some cases, the IL-2 mutein comprises an R67A substitution. In some embodiments, the IL-2 mutein comprises the amino acid sequence of SEQ ID NO: 14 or SEQ ID NO: 15. In some embodiments, the IL-2 mutein comprises the amino acid sequence of Table 1 of U.S. Patent Application Publication No. US2020 / 0270334A1, the disclosure of which is incorporated herein by reference.

[0413] In some embodiments, the antibody cytokine transplant protein comprises an HCDR1 selected from the group consisting of SEQ ID NO: 16, SEQ ID NO: 19, SEQ ID NO: 22, and SEQ ID NO: 25. In some embodiments, the antibody cytokine transplant protein comprises an HCDR1 selected from the group consisting of SEQ ID NO: 7, SEQ ID NO: 10, SEQ ID NO: 13, and SEQ ID NO: 16. In some embodiments, the antibody cytokine transplant protein comprises an HCDR2 selected from the group consisting of SEQ ID NO: 17, SEQ ID NO: 20, SEQ ID NO: 23, and SEQ ID NO: 26. In some embodiments, the antibody cytokine transplant protein comprises an HCDR3 selected from the group consisting of SEQ ID NO: 18, SEQ ID NO: 21, SEQ ID NO: 24, and SEQ ID NO: 27. In some embodiments, the antibody cytokine transplant protein comprises a V comprising the amino acid sequence of SEQ ID NO: 28. H In some embodiments, the antibody cytokine transplant protein comprises a heavy chain comprising the amino acid sequence of SEQ ID NO: 29. In some embodiments, the antibody cytokine transplant protein comprises a V region comprising the amino acid sequence of SEQ ID NO: 36. L In some embodiments, the antibody cytokine transplant protein comprises a light chain comprising the amino acid sequence of SEQ ID NO: 37. In some embodiments, the antibody cytokine transplant protein comprises a V region comprising the amino acid sequence of SEQ ID NO: 28. H V comprising the region and the amino acid sequence of SEQ ID NO: 36 LIn some embodiments, the antibody cytokine transplant protein comprises a heavy chain region comprising the amino acid sequence of SEQ ID NO:29 and a light chain region comprising the amino acid sequence of SEQ ID NO:37. In some embodiments, the antibody cytokine transplant protein comprises a heavy chain region comprising the amino acid sequence of SEQ ID NO:29 and a light chain region comprising the amino acid sequence of SEQ ID NO:39. In some embodiments, the antibody cytokine transplant protein comprises a heavy chain region comprising the amino acid sequence of SEQ ID NO:38 and a light chain region comprising the amino acid sequence of SEQ ID NO:37. In some embodiments, the antibody cytokine transplant protein comprises a heavy chain region comprising the amino acid sequence of SEQ ID NO:38 and a light chain region comprising the amino acid sequence of SEQ ID NO:39. In some embodiments, the antibody cytokine transplant protein comprises IgG.IL2F71A.H1 or IgG.IL2R67A.H1 of U.S. Patent Application Publication No. 2020 / 0270334A1, or a variant, derivative, or fragment thereof, or conservative amino acid substitutions thereof, or a protein having at least 80%, at least 90%, at least 95%, or at least 98% sequence identity thereto. In some embodiments, the antibody component of the antibody cytokine transplant proteins described herein comprises immunoglobulin sequences, framework sequences, or CDR sequences of palivizumab. In some embodiments, the antibody cytokine transplant proteins described herein have a longer serum half-life than a wild-type IL-2 molecule, such as, but not limited to, aldesleukin or an equivalent molecule. In some embodiments, the antibody cytokine transplant proteins described herein have a sequence set forth in Table 3. [Table 3-1] [Table 3-2]

[0414] 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, as well as 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 additional IL-4 in a positive feedback loop. IL-4 also stimulates B cell proliferation and class II MHC expression, and induces class switching from B cells to IgE and IgG1 expression. Recombinant human IL-4 suitable for use in the present invention is 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 sequence of recombinant human IL-4 suitable for use in the present invention is shown in Table 2 (SEQ ID NO: 9).

[0415] 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 IL-7 receptor, a heterodimer consisting of the IL-7 receptor alpha and the common gamma chain receptor, which 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 the present invention is commercially available from several suppliers, including ProSpec-Tany TechnoGene Ltd., East Brunswick, NJ, USA (catalog number CYT-254) and ThermoFisher Scientific, Inc., Waltham, MA, USA (human IL-15 recombinant protein, catalog number Gibco PHC0071). The amino acid sequence of a recombinant human IL-7 suitable for use in the present invention is shown in Table 2 (SEQ ID NO: 10).

[0416] 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 human and mammalian forms, conservative amino acid substitutions, glycoforms, biosimilars, and variants. IL-15 is described, for example, in Fehniger and Caligiuri, Blood 2001, 97, 14-32, the disclosure of which is incorporated herein by reference. IL-15 shares β and γ signaling receptor subunits with IL-2. Recombinant human IL-15 is a single, non-glycosylated polypeptide chain containing 114 amino acids (and an N-terminal methionine) with a molecular weight of 12.8 kDa. Recombinant human IL-15 is commercially available from several suppliers, including ProSpec-Tany TechnoGene Ltd., East Brunswick, NJ, USA (catalog number CYT-230-b) and ThermoFisher Scientific, Inc., Waltham, MA, USA (human IL-15 recombinant protein, catalog number 34-8159-82). The amino acid sequence of recombinant human IL-15 suitable for use in the present invention is shown in Table 2 (SEQ ID NO: 11).

[0417] 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 human and mammalian forms, conservative amino acid substitutions, glycoforms, biosimilars, and variants. IL-21 is described, for example, in Spolski and Leonard, Nat. Rev. Drug. Disc. 2014, 13, 379-95, the disclosure of which is incorporated herein by reference. IL-21 is produced primarily by natural killer T cells and activated human CD4+ 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 sources, including ProSpec-Tany TechnoGene Ltd., East Brunswick, NJ, USA (catalog number CYT-408-b) and ThermoFisher Scientific, Inc., Waltham, MA, USA (human IL-21 recombinant protein, catalog number 14-8219-80). The amino acid sequence of recombinant human IL-21 suitable for use in the present invention is shown in Table 2 (SEQ ID NO: 21).

[0418] When an "antitumor effective amount," "tumor inhibiting effective amount," or "therapeutic amount" is indicated, the exact amount of the composition of the present invention to be administered can be determined by a physician, taking into account individual differences in the age, weight, tumor size, extent of infection or metastasis, and health status of the patient (subject). Generally, tumor-infiltrating lymphocytes (e.g., secondary TILs or genetically modified cytotoxic lymphocytes) described herein are administered at a dose of 10 per kg of body weight. 4 ~10 11 cells (e.g., 10 per kg body weight) 5 ~10 6 , 10 5 ~10 10 , 10 5 ~10 11 , 10 6 ~10 10 , 10 6 ~10 11 , 10 7 ~1011 , 10 7 ~10 10 , 10 8 ~10 11 , 10 8 ~10 10 , 10 9 ~10 11 , or 10 9 ~10 10 It may be stated that the TIL (optionally including genetically modified cytotoxic lymphocytes) compositions may be administered at doses of 1000-150 ...

[0419] The terms "hematological malignancies," "blood system malignancies," or terms of related meaning, refer to cancers and tumors of mammalian hematopoietic and lymphatic tissues, including, but not limited to, blood, bone marrow, lymph nodes, and lymphatic tissues. Hematological malignancies are also referred to as "liquid tumors." Hematological malignancies may include, but are not limited to, acute lymphoblastic leukemia (ALL), chronic lymphocytic lymphoma (CLL), small lymphocytic lymphoma (SLL), acute myeloid leukemia (AML), chronic myelogenous leukemia (CML), multiple myeloma, acute monocytic leukemia (AMoL), Hodgkin's lymphoma, and non-Hodgkin's lymphoma. The term "B-cell hematological malignancies" refers to hematological malignancies affecting B cells.

[0420] The term "liquid tumor" refers to an abnormal mass of cells that is fluid in nature. Liquid tumor cancers include, but are not limited to, leukemia, myeloma, and lymphoma, as well as other hematological malignancies. TILs obtained from liquid tumors may also be referred to herein as bone marrow-infiltrating lymphocytes (MILs). TILs obtained from liquid tumors, including those circulating in peripheral blood, may also be referred to herein as PBLs. The terms MILs, TILs, and PBLs are used interchangeably herein and differ only based on the tissue type from which the cells are derived.

[0421] The term "microenvironment" as used herein may refer to the solid or hematological tumor microenvironment as a whole, or to individual subsets of cells within the microenvironment. As used herein, the tumor microenvironment refers to a complex mixture of "cells, soluble factors, signaling molecules, extracellular matrix, and mechanical cues that promote neoplastic transformation, support tumor growth and invasion, protect tumors from host immunity, foster therapeutic resistance, and provide a niche for successful and dominant metastasis," as described in Swartz, et al., Cancer Res., 2012, 72, 2473. Tumors express antigens that are recognized by T cells, but tumor clearance by the immune system is rare due to immunosuppression by the microenvironment.

[0422] In some embodiments, the invention includes methods of treating cancer with a TIL population, wherein the patient is pretreated with non-myeloablative chemotherapy prior to infusion of TILs according to the invention. In some embodiments, a TIL population may be provided, wherein the patient is pretreated with non-myeloablative chemotherapy prior to infusion of TILs according to the invention. In some embodiments, the non-myeloablative chemotherapy is cyclophosphamide 60 mg / kg / day for 2 days (27 and 26 days before TIL infusion) and fludarabine 25 mg / m2 / day for 5 days (27-23 days before TIL infusion). In some embodiments, after non-myeloablative chemotherapy and TIL infusion according to the invention (day 0), the patient receives an intravenous infusion of IL-2 at 720,000 IU / kg every 8 hours to physiological tolerance.

[0423] Experimental findings indicate that lymphodepletion prior to adoptive transfer of tumor-specific T lymphocytes plays an important role in enhancing therapeutic efficacy by eliminating regulatory T cells and competing elements of the immune system ("cytokine sinks"). Accordingly, some embodiments of the present invention utilize a lymphodepletion step (sometimes referred to as "immunosuppressive conditioning") on patients prior to introducing the TILs of the present invention.

[0424] The term "effective amount" or "therapeutically effective amount" refers to an amount of a compound or combination of compounds described herein sufficient to achieve the intended use, including, but not limited to, disease treatment. A therapeutically effective amount may vary depending on the intended use (in vitro or in vivo), the subject and condition being treated (e.g., the subject's weight, age, and sex), the severity of the condition, or the method of administration. The term also applies to a dose that induces a specific response in target cells (e.g., reduced platelet adhesion and / or cell migration). The specific dose will vary depending on the particular compound selected, the dosing regimen to be followed, whether the compound is administered in combination with other compounds, the timing of administration, the tissue to which it is administered, and the physical delivery system by which the compound is delivered.

[0425] The terms "treatment," "treating," "treating," and the like refer to obtaining a desired pharmacological and / or physiological effect. The effect may be prophylactic, in terms of completely or partially preventing the disease or condition, and / or therapeutic, in terms of partially or completely curing the disease and / or side effects caused by the disease. As used herein, "treatment" encompasses any treatment of disease in a mammal, particularly a human, and includes (a) preventing the disease from occurring in a subject who may be susceptible to the disease but has not yet been diagnosed with it; (b) suppressing the disease, i.e., arresting its onset or progression; and (c) palliating the disease, i.e., causing regression of the disease and / or alleviating one or more disease symptoms. "Treatment" is also intended to encompass the delivery of an agent to provide a pharmacological effect even in the absence of a disease or condition. For example, "treatment" encompasses the delivery of a composition capable of eliciting an immune response or conferring immunity in the absence of a pathology, e.g., in the case of a vaccine.

[0426] "Non-myeloablative chemotherapy," "non-myeloablative lymphodepletion," "NMALD," "NMA LD," "NMA-LD," and any variants of the foregoing, are used interchangeably to refer to chemotherapy regimens designed to deplete a patient's lymphoid immune cells while avoiding depletion of a patient's myeloid immune cells. Typically, a patient undergoes a course of non-myeloablative chemotherapy before administering tumor-infiltrating lymphocytes to the patient, as described herein.

[0427] The term "heterologous," when used with reference to portions of a nucleic acid or protein, indicates that the nucleic acid or protein comprises two or more subsequences that are not found in the same relationship to each other in nature. For example, nucleic acids are typically produced recombinantly, with two or more sequences from unrelated genes arranged to create a new functional nucleic acid, e.g., a promoter from one source and a coding region from another source, or coding regions from different sources. Similarly, a heterologous protein indicates that the protein comprises two or more subsequences that are not found in the same relationship to each other in nature (e.g., a fusion protein).

[0428] The terms "sequence identity," "percent identity," and "percent sequence identity" (or their synonyms, e.g., "99% identical") in the context of two or more nucleic acids or polypeptides refer to two or more sequences or subsequences that are the same or have a specified percentage of the same nucleotide or amino acid residues when compared and aligned for maximum correspondence (introducing gaps, if necessary), without considering any conservative amino acid substitutions as part of the sequence identity. Percent identity can be measured using sequence comparison software or algorithms or by visual inspection. Various algorithms and software that can be used to obtain alignment of amino acid or nucleotide sequences are known in the art. Suitable programs for determining percent sequence identity include, for example, the BLAST suite of programs 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 to compare nucleic acid sequences, and BLASTP is used to compare amino acid sequences. ALIGN, ALIGN-2 (Genentech, South San Francisco, California), or MegAlign, available from DNASTAR, are additional publicly available software programs that can be used to align sequences. Those skilled in the art can determine the appropriate parameters for maximum alignment depending on the particular alignment software. In certain embodiments, the default parameters of the alignment software are used.

[0429] As used herein, the term "variant" includes, but is not limited to, an antibody or fusion protein comprising an amino acid sequence that differs from the amino acid sequence of a reference antibody by one or more substitutions, deletions, and / or additions at specific positions within or adjacent to the amino acid sequence of the reference antibody. A variant may contain one or more conservative substitutions in its amino acid sequence compared to the amino acid sequence of the reference antibody. Conservative substitutions may include, for example, substitutions of similarly charged or uncharged amino acids. A variant retains the ability of the reference antibody to specifically bind to an antigen. The term variant also includes pegylated antibodies or proteins.

[0430] As used herein, "tumor infiltrating lymphocytes" or "TILs" refers to a population of cells that are initially acquired as leukocytes that have left the bloodstream of a subject and migrated to a tumor. TILs include CD8 + Cytotoxic T cells (lymphocytes), Th1 and Th17 CD4 + These include, but are not limited to, T cells, natural killer cells, dendritic cells, and M1 macrophages. TILs include both primary and secondary TILs. "Primary TILs" are those obtained from a patient tissue sample as outlined herein (sometimes referred to as "freshly harvested"). "Secondary TILs" are any TIL cell populations that have been expanded or propagated as discussed herein, including, but not limited to, bulk TILs, expanded TILs ("REP TILs"), and "reREP TILs" as discussed herein. reREP TILs can include, for example, second expanded TILs or second additional expanded TILs (e.g., those described in step D of FIG. 8, including TILs designated as reREP TILs).

[0431] TILs can generally be defined either biochemically using cell surface markers or functionally by their ability to infiltrate tumors and achieve therapy. TILs can generally be classified by expression of one or more of the following biomarkers: CD4, CD8, TCRαβ, CD27, CD28, CD56, CCR7, CD45Ra, CD95, PD-1, and CD25. Additionally and alternatively, TILs can be functionally defined by their ability to infiltrate solid tumors upon reintroduction into patients. TILs can be further characterized by potency; for example, TILs can be considered potent if their interferon (IFN) release is greater than about 50 pg / mL, greater than about 100 pg / mL, greater than about 150 pg / mL, or greater than about 200 pg / mL. For example, TILs can be considered potent if interferon (IFNγ) release is greater than about 50 pg / mL, greater than about 100 pg / mL, greater than about 150 pg / mL, or greater than about 200 pg / mL, greater than about 300 pg / mL, greater than about 400 pg / mL, greater than about 500 pg / mL, greater than about 600 pg / mL, greater than about 700 pg / mL, greater than about 800 pg / mL, greater than about 900 pg / mL, or greater than about 1000 pg / mL.

[0432] The term "deoxyribonucleotide" encompasses natural and synthetic, unmodified and modified deoxyribonucleotides. Modifications include changes to the sugar moiety, the base moiety, and / or the linkages between deoxyribonucleotides in an oligonucleotide.

[0433] The term "RNA" defines a molecule containing at least one ribonucleotide residue. The term "ribonucleotide" defines a nucleotide having a hydroxyl group at the 2' position of a bD-ribofuranose moiety. The term RNA includes double-stranded RNA, single-stranded RNA, isolated RNA such as partially purified RNA, essentially pure RNA, synthetic RNA, recombinantly produced RNA, and altered RNA that differs from naturally occurring RNA by the addition, deletion, substitution, and / or alteration of one or more nucleotides. The nucleotides of the RNA molecules described herein can also include non-standard nucleotides, such as non-naturally occurring nucleotides or chemically synthesized nucleotides or deoxynucleotides. These altered RNAs may be referred to as analogs or analogs of naturally occurring RNA.

[0434] The term "pharmaceutically acceptable carrier" or "pharmaceutically acceptable excipient" is intended to include any and all solvents, dispersion media, coatings, antibacterial and antifungal agents, isotonic and absorption delaying agents, and inactive ingredients. The use of such pharmaceutically acceptable carriers or pharmaceutically acceptable excipients for active pharmaceutical ingredients is well known in the art. Except insofar as any conventional pharmaceutically acceptable carrier or pharmaceutically acceptable excipient is incompatible with the active pharmaceutical ingredient, its use in the therapeutic compositions of the invention is contemplated. Additional active pharmaceutical ingredients, such as other drugs, can also be incorporated into the compositions and methods described.

[0435] The terms "about" and "approximately" mean within a statistically significant range of 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 acceptable deviation encompassed by the term "about" or "approximately" depends on the particular system under study and can be readily understood by one of ordinary skill in the art. Furthermore, as used herein, the terms "about" and "approximately" mean that dimensions, sizes, formulations, parameters, shapes, and other quantities and characteristics are not, and need not be, exact, but may be approximate and / or larger or smaller, as appropriate, reflecting tolerances, conversion factors, rounding, measurement errors, etc., and other factors known to those of ordinary skill in the art. In general, a dimension, size, formulation, parameter, shape, or other quantity or characteristic is "about" or "approximately" whether or not expressly stated as such. It should be noted that embodiments of widely different sizes, shapes, and dimensions may employ the described configurations.

[0436] When used in the appended claims, the transitional terms "comprising," "consisting essentially of," and "consisting of" define the claim in its original and amended form, in terms of whether additional unrecited claim elements or steps, if any, are excluded from the claim. The term "comprising" is intended to be inclusive or open-ended and does not exclude any additional unrecited elements, methods, steps, or materials. The term "consisting of" excludes any element, step, or material other than those specified in the claim, and in the latter case, also excludes impurities normally associated with the specified material(s). The term "consisting essentially of" limits the claim to the specified element, step, or material(s) and does not materially affect the basic and novel feature(s) of the claimed invention. All compositions, methods, and kits described herein embodying the present invention may, in alternative embodiments, be more specifically defined by any of the transitional terms "comprising," "consisting essentially of," and "consisting of."

[0437] The terms "antibody" and its plural "antibodies" refer to whole immunoglobulins and any antigen-binding fragment ("antigen-binding portion") or single chains thereof. "Antibody" also refers to a glycoprotein comprising at least two heavy (H) chains and two light (L) chains interconnected by disulfide bonds, or antigen-binding portions thereof. Each heavy chain contains a heavy chain variable region (referred to herein as V H Each light chain is composed of a light chain variable region (abbreviated herein as V) and a heavy chain constant region. The heavy chain constant region is composed of three domains: CH1, CH2, and CH3. L The light chain constant region consists of one domain, C L The V of the antibody H and V L The regions can be further subdivided into regions of hypervariability, called complementarity-determining regions (CDRs) or hypervariable regions (HVRs), which may be interspersed with more conserved regions called framework regions (FRs). H and V L is composed of three CDRs and four FRs arranged from the amino terminus to the carboxy terminus in the following order: FR1, CDR1, FR2, CDR2, FR3, CDR3, FR4. The variable regions of the heavy and light chains contain binding domains that interact with one or more antigen epitopes. The constant region of the antibody may mediate the binding of the immunoglobulin to host tissues or factors, including various cells of the immune system (e.g., effector cells) and the first component (Clq) of the classical complement system.

[0438] The term "antigen" refers to a substance that induces an immune response. In some embodiments, an antigen is a molecule that can be bound by an antibody or TCR when presented by a major histocompatibility complex (MHC) molecule. As used herein, the term "antigen" also encompasses T cell epitopes. An antigen can additionally be recognized by the immune system. In some embodiments, an antigen can induce a humoral or cellular immune response, leading to the activation of B and / or T lymphocytes. In some cases, this may require that the antigen contain or be bound by a Th cell epitope. An antigen may also have one or more epitopes (e.g., B- and T-epitopes). In some embodiments, an antigen preferably reacts with a corresponding antibody or TCR, typically in a highly specific and selective manner, and not with many other antibodies or TCRs that may be induced by other antigens.

[0439] The terms "monoclonal antibody," "mAb," "monoclonal antibody composition," or their plurals, refer to a preparation of antibody molecules of single molecular composition. A monoclonal antibody composition exhibits a single binding specificity and affinity for a particular epitope. Monoclonal antibodies specific for a particular receptor can be produced using knowledge and techniques in the art by injecting a test subject with an appropriate antigen and then isolating hybridomas expressing antibodies with the desired sequence or functional characteristics. DNA encoding the monoclonal antibody is readily isolated and sequenced using conventional procedures (e.g., by using oligonucleotide probes capable of specifically binding to genes encoding the heavy and light chains of the monoclonal antibody). Hybridoma cells serve as a preferred source of such DNA. Once isolated, the DNA can be placed into an expression vector and then transfected into host cells such as E. coli cells, monkey COS cells, Chinese hamster ovary (CHO) cells, or myeloma cells that do not produce immunoglobulin protein to obtain the synthesis of the monoclonal antibody in the recombinant host cells. Recombinant production of antibodies is described in more detail below.

[0440] As used herein, the term "antigen-binding portion" or "antigen-binding fragment" of an antibody (or simply "antibody portion" or "fragment") refers to one or more fragments of an antibody that retain the ability to specifically bind to an antigen. It has been shown that the antigen-binding function of an antibody can be performed by fragments of a full-length antibody. Examples of binding fragments encompassed by the term "antigen-binding portion" of an antibody include: (i) V L , V H , C L (ii) a Fab fragment, which is a monovalent fragment consisting of two Fab fragments linked by a disulfide bridge at the hinge region; (iii) a V H and an Fd fragment consisting of the CH1 domain, (iv) a V of a single arm of an antibody L and V H Fv fragment consisting of domains, (v) V H or VL and (v) isolated complementarity-determining regions (CDRs). L and V H are encoded by separate genes, they can be synthesized using recombinant methods L and V H The domains may be joined by a synthetic linker that allows them to pair as a single protein chain to form a monovalent molecule known as a single-chain Fv (scFv; see, e.g., Bird, et al., Science 1988, 242, 423-426, and Huston, et al., Proc. Natl. Acad. Sci. USA 1988, 85, 5879-5883). Such scFv antibodies are also intended to be encompassed by the term "antigen-binding portion" or "antigen-binding fragment" of an antibody. These antibody fragments are obtained using conventional techniques known to those skilled in the art, and the fragments are screened for utility in the same manner as intact antibodies. In some embodiments, the scFv protein domain comprises a V H Part and V L The scFv molecule contains a V L If the domain is the N-terminal portion of the scFv molecule, V L -LV H , or V H If the domain is the N-terminal portion of the scFv molecule, V H -LV L Methods for producing scFv molecules and designing suitable peptide linkers are described in U.S. Pat. Nos. 4,704,692, 4,946,778, R. Raag and M. Whitlow, "Single Chain Fvs," FASEB Vol. 9:73-80 (1995), and RE Bird and BW Walker, Single Chain Antibody Variable Regions, TIBTECH, Vol. 9:132-137 (1991), the disclosures of which are incorporated herein by reference.

[0441] As used herein, the term "human antibody" is intended to include antibodies having variable regions in which both the framework and CDR regions are derived from human germline immunoglobulin sequences. Furthermore, if the antibody contains a constant region, the constant region also is derived from human germline immunoglobulin sequences. The human antibodies of the invention may include amino acid residues not encoded by human germline immunoglobulin sequences (e.g., mutations introduced by random or site-specific mutagenesis in vitro or by somatic mutation in vivo). As used herein, the term "human antibody" is not intended to include antibodies in which CDR sequences derived from the germline of another mammalian species, such as a mouse, have been grafted onto human framework sequences.

[0442] The term "human monoclonal antibody" refers to antibodies displaying a single binding specificity which have variable regions in which both the framework and CDR regions are derived from human germline immunoglobulin sequences. In some embodiments, human monoclonal antibodies are produced by hybridomas comprising B cells obtained from a transgenic non-human animal, e.g., a transgenic mouse, whose genome comprises human heavy chain and light chain transgenes fused to an immortalized cell.

[0443] As used herein, the term "recombinant human antibody" includes all human antibodies prepared, expressed, generated, or isolated by recombinant means, such as (a) antibodies isolated from animals (such as mice) that are transgenic or transchromosomal for human immunoglobulin genes or hybridomas prepared therefrom (described further below); (b) antibodies isolated from host cells transformed to express human antibodies, e.g., from transfectomas; (c) antibodies isolated from recombinant combinatorial human antibody libraries; and (d) antibodies prepared, expressed, generated, or isolated by any other means, including splicing human immunoglobulin gene sequences to other DNA sequences. Such recombinant human antibodies have variable regions in which the framework and CDR regions are derived from human germline immunoglobulin sequences. However, in certain embodiments, such recombinant human antibodies may be subjected to in vitro mutagenesis (or, when animals transgenic for human Ig sequences are used, in vivo somatic mutagenesis) to thereby modify the V sequences of the recombinant antibodies. H and V L The amino acid sequence of the region is human germline V H and V L These are sequences that are derived from and related to sequences, but may not naturally occur within the human antibody germline repertoire in vivo.

[0444] As used herein, "isotype" refers to the antibody class (e.g., IgM or IgG1) that is encoded by heavy chain constant region genes.

[0445] The phrases "an antibody that recognizes an antigen" and "an antibody that is specific for an antigen" are used interchangeably herein with the term "an antibody that specifically binds to an antigen."

[0446] The term "human antibody derivative" refers to any modified form of a human antibody, including a conjugate of the antibody with another active pharmaceutical ingredient or antibody. The terms "conjugate," "antibody drug conjugate," "ADC," or "immunoconjugate" refer to an antibody or fragment thereof conjugated to another therapeutic moiety, which can be conjugated to the antibodies described herein using methods available in the art.

[0447] The terms "humanized antibody," "humanized antibodies," and "humanization" are intended to refer to antibodies in which CDR sequences derived from the germline of another mammalian species, such as mouse, have been grafted onto human framework sequences. Additional framework region modifications can be made within the human framework sequences. Humanized forms of non-human (e.g., murine) antibodies are chimeric antibodies that contain minimal sequence derived from non-human immunoglobulin. In most cases, humanized antibodies are human immunoglobulins (recipient antibodies) in which residues from a hypervariable region of the recipient are replaced by residues from a hypervariable region of a non-human species (donor antibody) such as mouse, rat, rabbit, or non-human primate having the desired specificity, affinity, and capacity. In some instances, Fv framework (FR) residues of the human immunoglobulin are replaced by corresponding non-human residues. Furthermore, humanized antibodies may contain residues that are not found in the recipient or donor antibody. These modifications are made to further refine antibody performance. Generally, a humanized antibody will comprise substantially all of at least one, and typically two, variable domains, in which all or substantially all of the hypervariable loops correspond to those of a non-human immunoglobulin and all or substantially all of the FR regions are those of a human immunoglobulin sequence. The humanized antibody will also optionally comprise at least a portion of an immunoglobulin constant region (Fc), typically that of a human immunoglobulin. For further details, see Jones, et al., Nature 1986, 321, 522-525; Riechmann, et al., Nature 1988, 332, 323-329; and Presta, Curr. Op. Struct. Biol. 1992, 2, 593-596. The antibodies described herein can also be modified to use any Fc variant known to confer improved (e.g., reduced) effector function and / or FcR binding.Fc variants are described in, for example, International Patent Application Publication Nos. WO1988 / 07089A1, WO1996 / 14339A1, WO1998 / 05787A1, WO1998 / 23289A1, WO1999 / 51642A1, WO99 / 58572A1, WO2000 / 09560A2, WO2000 / 32767A1, WO2000 / 42072A2, and WO2002 / 4 4215A2, WO2002 / 060919A2, WO2003 / 074569A2, WO2004 / 016750A2, WO2004 / 029207A2, WO2004 / 03 5752A2, WO2004 / 063351A2, WO2004 / 074455A2, WO2004 / 099249A2, WO2005 / 040217A2, WO2005 / 07 0963A1, WO2005 / 077981A2, WO2005 / 092925A2, WO2005 / 123780A2, WO2006 / 019447A1, WO2006 / 047350A2, and WO2006 / 085967A2, as well as U.S. Pat. Nos. 5,648,260, 5,739,277, 5,834,250, 5,869,046, 6,096 ,871, 6,121,022, 6,194,551, 6,242,195, 6,277,375, 6,528,624, 6,538,124, 6,737,056, 6,821,505, 6,998,253, and 7,083,784 (the disclosures of which are incorporated herein by reference).

[0448] The term "chimeric antibody" is intended to refer to an antibody in which the variable region sequences are derived from one species and the constant region sequences are derived from another species, e.g., an antibody in which the variable region sequences are derived from a murine antibody and the constant region sequences are derived from a human antibody.

[0449] A "diabody" is a small antibody fragment that has two antigen-binding sites. The fragments bind to the same polypeptide chain (VH -V L or V L -V H ) in the light chain variable domain (V L ) connected to the heavy chain variable domain (V H ). When a linker that is too short to pair the two domains on the same chain is used, the domains are forced to pair with complementary domains on another chain and create two antigen-binding sites. Bispecific antibodies are more fully described in, for example, European Patent No. EP 404,097, International Patent Publication No. WO 93 / 11161, and Bolliger, et al., Proc. Natl. Acad. Sci. USA 1993, 90, 6444-6448.

[0450] The term "glycosylation" refers to modified derivatives of antibodies. An aglycosylated antibody lacks glycosylation. Glycosylation can be altered to, for example, increase the affinity of the antibody for an antigen. Such carbohydrate modifications can be achieved, for example, by altering one or more glycosylation sites within the antibody sequence. For example, one or more amino acid substitutions can be made that result in the elimination of one or more variable region framework glycosylation sites, thereby eliminating glycosylation at those sites. As described in U.S. Pat. Nos. 5,714,350 and 6,350,861, aglycosylation may increase the affinity of the antibody for the antigen. Additionally or alternatively, antibodies can be generated with altered types of glycosylation, such as hypofucosylated antibodies with reduced amounts of fucosyl residues or antibodies with increased bisecting GlcNac structures. Such altered glycosylation patterns have been demonstrated to increase antibody potency. Such carbohydrate modifications can be achieved, for example, by expressing the antibody in a host cell with altered glycosylation machinery. Cells with altered glycosylation machinery have been described in the art and can be used as host cells to express the recombinant antibodies of the present invention, thereby producing antibodies with altered glycosylation. For example, the cell lines Ms704, Ms705, and Ms709 lack the fucosyltransferase gene FUT8 (alpha(1,6) fucosyltransferase), such that antibodies expressed in these cell lines lack fucose on their carbohydrates. The Ms704, Ms705, and Ms709 FUT8- / - cell lines were generated by targeted disruption of the FUT8 gene in CHO / DG44 cells using two replacement vectors (see, e.g., U.S. Patent Publication No. 2004 / 0110704 or Yamane-Ohnuki, et al., Biotechnol. Bioeng., 2004, 87, 614-622).As another example, European Patent No. EP 1,176,195 describes cell lines with a functionally disrupted FUT8 gene encoding a fucosyltransferase, thereby resulting in antibodies expressed in such cell lines exhibiting hypofucosylation by reducing or eliminating alpha-1,6 bond-related enzymes. It also describes cell lines with reduced or no enzymatic activity for adding fucose to N-acetylglucosamine attached to the Fc region of antibodies, such as the rat myeloma cell line YB2 / 0 (ATCC CRL 1662). International Patent Publication No. WO 03 / 035835 describes a variant CHO cell line, Lec 13 cells, that has a reduced ability to attach fucose to Asn(297)-linked carbohydrates, further resulting in hypofucosylation of antibodies expressed in the host cells (see also Shields, et al., J. Biol. Chem. 2002, 277, 26733-26740). International Patent Publication No. WO 99 / 54342 describes cell lines engineered to express glycoprotein-modifying glycosyltransferases (e.g., beta(1,4)-N-acetylglucosaminyltransferase III (GnTIII)), such that antibodies expressed in the engineered cell lines exhibit increased bisecting GlcNac structures, resulting in increased ADCC activity of the antibodies (see also Umana, et al., Nat. Biotech. 1999, 17, 176-180). Alternatively, fucose residues on antibodies can be cleaved using a fucosidase enzyme. For example, the fucosidase alpha-L-fucosidase removes fucosyl residues from antibodies, as described in Tarentino, et al., Biochem. 1975, 14, 5516-5523.

[0451] "PEGylation" refers to a modified antibody or fragment thereof that has been reacted with polyethylene glycol (PEG), such as a reactive ester or aldehyde derivative of PEG, under conditions that result in one or more PEG groups being attached to the antibody or antibody fragment. PEGylation can, for example, increase the biological (e.g., serum) half-life of the antibody. Preferably, PEGylation is carried out via an acylation reaction or an alkylation reaction with a reactive PEG molecule (or an analogous reactive water-soluble polymer). As used herein, the term "polyethylene glycol" refers to a mono(C1-C2)-poly(ethylene glycol ... 10 PEG is intended to encompass any of the forms of PEG that have been used to derivatize other proteins, such as alkoxy- or aryloxy-polyethylene glycol, or polyethylene glycol-maleimide. The antibody to be pegylated may be an aglycosylated antibody. Methods for pegylation are known in the art and can be applied to the antibodies of the invention, for example, as described in European Patent Nos. EP 0 154 316 and EP 0 401 384, and U.S. Pat. No. 5,824,778 (the disclosures of each of which are incorporated herein by reference).

[0452] The term "biosimilar" refers to a biological product that is highly similar to a reference biological product approved in the United States, despite minor differences in clinically inactive components, including monoclonal antibodies or proteins, and that has no clinically meaningful differences between the biological product and the reference product in terms of product safety, purity, and potency. In Europe, a similar biological or "biosimilar" drug is a biological product similar to another biological product already approved for use by the European Medicines Agency. The term "biosimilar" is also used synonymously by regulatory agencies in other countries and regions. Biological products or biopharmaceuticals are medicines made by or derived from living sources, such as bacteria or yeast. They can consist of relatively small molecules, such as human insulin or erythropoietin, or complex molecules, such as monoclonal antibodies. For example, if the reference IL-2 protein is aldesleukin (proleukin), a protein approved by drug regulatory authorities for aldesleukin is a "biosimilar" of aldesleukin or a "biosimilar of" aldesleukin. In Europe, a similar biological or "biosimilar" medicinal product is a biological product similar to another biological product already authorized for use by the European Medicines Agency (EMA). The legal basis for similar biological uses in Europe is Article 6 of Regulation (EC) No. 726 / 2004, as amended, and Article 10(4) of Directive 2001 / 83 / EC. Therefore, in Europe, biosimilars may be authorized or approved for authorisation or authorization under Article 6 of Regulation (EC) No. 726 / 2004 and Article 10(4) of Directive 2001 / 83 / EC. The original biological product already authorized may be referred to as the "reference medicinal product" in Europe. Some of the requirements for a product to be considered a biosimilar are outlined in the CHMP guideline on biosimilar medicinal products. Additionally, product-specific guidelines, including those related to monoclonal antibody biosimilars, are provided by the EMA on a product-by-product basis and are available on its website.Biosimilars described herein may be similar to the reference medicinal product in terms of quality characteristics, biological activity, mechanism of action, safety profile, and / or efficacy. Furthermore, biosimilars may be used or intended for use to treat the same condition as the reference medicinal product. Thus, biosimilars described herein may be considered to have similar or very similar quality characteristics to the reference medicinal product. Alternatively, or in addition, biosimilars described herein may be considered to have similar or very similar biological activity to the reference medicinal product. Alternatively, or in addition, biosimilars described herein may be considered to have a similar or very similar safety profile to the reference medicinal product. Alternatively, or in addition, biosimilars described herein may be considered to have similar or very similar efficacy to the reference medicinal product. As described herein, biosimilars in Europe are compared to reference medicinal products authorized by the EMA. However, in some cases, biosimilars may be compared in specific studies to biopharmaceuticals authorized outside the European Economic Area (non-EEA authorized "comparators"). Such studies include, for example, specific clinical studies and in vivo non-clinical studies. As used herein, the term "biosimilar" also refers to a biopharmaceutical that has been or can be compared to a non-EEA-approved comparator. Particular biosimilars are proteins, such as antibodies, antibody fragments (e.g., antigen-binding portions), and fusion proteins. Protein biosimilars may have amino acid sequences with minor modifications to the amino acid structure (e.g., including amino acid deletions, additions, and / or substitutions) that do not significantly affect the function of the polypeptide. A biosimilar may have an amino acid sequence that has 97% or more sequence identity, e.g., 97%, 98%, 99%, or 100%, to the amino acid sequence of its reference pharmaceutical. A biosimilar may include one or more post-translational modifications, such as, but not limited to, glycosylation, oxidation, deamidation, and / or cleavage, that differ from the post-translational modifications of the reference pharmaceutical, provided that the differences do not result in a change in the safety and / or efficacy of the pharmaceutical. A biosimilar may have the same or a different glycosylation pattern as the reference pharmaceutical, provided that the differences do not result in a change in the safety and / or efficacy of the pharmaceutical.In particular, but not exclusively, biosimilars may have different glycosylation patterns if the differences address or are intended to address safety concerns associated with the reference drug. Additionally, biosimilars may deviate from the reference drug, for example, in its strength, dosage form, formulation, excipients, and / or presentation, provided that the drug's safety and efficacy are not compromised. Biosimilars may contain differences, for example, in their pharmacokinetic (PK) and / or pharmacodynamic (PD) profiles, compared to the reference drug, but are still considered sufficiently similar to the reference drug to be approved or deemed suitable for approval. In certain circumstances, biosimilars exhibit different binding characteristics compared to the reference drug, and these different binding characteristics are not considered by regulatory authorities, such as the EMA, to be a barrier to approval as a similar biological product. The term "biosimilar" is also used interchangeably by regulatory agencies in other countries and regions.

[0453] II. Gen2 TIL Manufacturing Process An exemplary family of TIL processes known as Gen2 (also known as Process 2A) that includes some of these features is shown in Figures 1 and 2. An embodiment of Gen2 is shown in Figure 2.

[0454] As discussed herein, the present invention may include steps related to restimulating cryopreserved TILs prior to transplantation into a patient to increase their metabolic activity, and thus their relative health, as well as methods for testing their metabolic health. As generally outlined herein, TILs are generally harvested from a patient sample and manipulated to expand their numbers prior to transplantation into a patient. In some embodiments, TILs may optionally be genetically engineered, as discussed below.

[0455] In some embodiments, TILs can be cryopreserved, and upon thawing, they can be restimulated to enhance their metabolism before infusion into patients.

[0456] In some embodiments, as discussed in detail below and in the Examples and Figures, the first expansion (including the process referred to as pre-REP and the process shown as step A in Figure 1) is shortened to 3-14 days, and the second expansion (including the process referred to as REP and the process shown as step B in Figure 1) is shortened to 7-14 days. In some embodiments, the first expansion (e.g., the expansion described as step B in Figure 1) is shortened to 11 days, and the second expansion (e.g., the expansion described as step D in Figure 1) is shortened to 11 days. In some embodiments, as discussed in detail below and in the Examples and Figures, the combined first and second expansion (e.g., the expansion described as steps B and D in Figure 1) is shortened to 22 days.

[0457] The "step" designations A, B, C, etc. below refer to Figure 1 and to certain specific embodiments described herein. The order of steps below and in Figure 1 is exemplary, and any combination or order of steps, as well as additional steps, repeated steps, and / or omission of steps, are contemplated by the present application and methods disclosed herein.

[0458] A. Step A: Obtaining a patient tumor sample Generally, TILs are initially obtained from a patient tumor sample, then expanded into 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.

[0459] Patient tumor samples can be obtained using methods known in the art, generally via surgical resection, needle biopsy, core biopsy, mini-biopsy, or other means for obtaining a sample containing a mixture of tumor cells and TIL cells. In some embodiments, multi-lesion sampling is used. In some embodiments, surgical resection, needle biopsy, core biopsy, mini-biopsy, or other means for obtaining a sample containing a mixture of tumor cells and TIL cells includes multi-lesion sampling (i.e., obtaining samples from one or more tumor sites and / or locations in a patient, as well as one or more tumors at the same or adjacent locations). Generally, tumor samples can be derived from any solid tumor, including primary tumors, invasive tumors, or metastatic tumors. Tumor samples can be liquid tumors, such as tumors obtained from hematological malignancies. Solid tumors can be of lung tissue. In some embodiments, useful TILs are obtained from non-small cell lung cancer (NSCLC). Solid tumors can be of skin tissue. In some embodiments, useful TILs are obtained from melanoma.

[0460] Once obtained, tumor samples are generally cut into sections of 1 to approximately 8 mm using sharp dissection. 3 fragmented into small pieces of about 2-3 mm 3are particularly useful. In some embodiments, TILs are cultured from these fragments using an enzymatic tumor digest. Such tumor digests can be generated by incubation in an enzymatic medium (e.g., Roswell Park Memorial Institute (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 dissociator). Tumor digests can be generated by placing the tumor in the enzymatic medium, mechanically dissociating the tumor for approximately 1 minute, followed by incubation at 37°C in 5% CO2 for 30 minutes, and then repeating the cycle of mechanical dissociation and incubation under the aforementioned conditions until only small tissue fragments are present. If the cell suspension contains a large number of red blood cells or dead cells at the end of this process, density gradient separation using FICOLL branched hydrophilic polysaccharide can be performed to remove these cells. Alternative methods known in the art can be used, such as those described in U.S. Patent Application Publication No. 2012 / 0244133A1, the disclosure of which is incorporated herein by reference. Any of the foregoing methods can be used in any of the embodiments described herein for methods of expanding TILs or methods of treating cancer.

[0461] The tumor dissociation enzyme mixture can include one or more dissociation (digestion) enzymes, such as, but not limited to, collagenase (including any blend or type of collagenase), Accutase™, Accumax™, hyaluronidase, neutral protease (dispase), chymotrypsin, chymopapain, trypsin, caseinase, elastase, papain, type XIV protease (pronase), deoxyribonuclease I (DNase), trypsin inhibitor, any other dissociation enzyme or proteolytic enzyme, and any combination thereof.

[0462] In some embodiments, the dissociation enzyme is reconstituted from a lyophilized enzyme, hi some embodiments, the lyophilized enzyme is reconstituted with an amount of a sterile buffer, such as HBSS.

[0463] In some cases, collagenase (such as animal-free type 1 collagenase) is reconstituted in 10 mL of sterile HBSS or another buffer. Lyophilized stock enzyme may be at a concentration of 2892 PZ U / vial. In some embodiments, collagenase is reconstituted in 5 mL to 15 mL of buffer. In some embodiments, the collagenase stock after reconstitution is in the range of about 100 U / mL to about 400 U / mL, e.g., about 100 U / mL to about 400 U / mL, about 100 U / mL to about 350 U / mL, about 100 U / mL to about 300 U / mL, about 150 U / mL to about 400 U / mL, about 100 U / mL, about 150 U / mL, about 200 U / mL, about 210 U / mL, about 220 U / mL, about 230 U / mL, about 240 U / mL, about 250 U / mL, about 260 U / mL, about 270 U / mL, about 280 U / mL, about 289.2 U / mL, about 300 U / mL, U / mL, about 350 PZ U / mL, or about 400 PZ U / mL.

[0464] In some embodiments, the neutral protease is reconstituted in 1 mL of sterile HBSS or another buffer. The lyophilized stock enzyme may be at a concentration of 175 DMC U / vial. In some embodiments, the neutral protease stock after reconstitution may range from about 100 DMC / mL to about 400 DMC / mL, e.g., from about 100 DMC / mL to about 400 DMC / mL, from about 100 DMC / mL to about 350 DMC / mL, from about 100 DMC / mL to about 300 DMC / mL, from about 150 DMC / mL to about 400 DMC / mL, from about 100 DMC / mL, or about 110 DMC / mL. C / mL, about 120 DMC / mL, about 130 DMC / mL, about 140 DMC / mL, about 150 DMC / mL, about 160 DMC / mL, about 170 DMC / mL, about 175 DMC / mL, about 180 DMC / mL, about 190 DMC / mL, about 200 DMC / mL, about 250 DMC / mL, about 300 DMC / mL, about 350 DMC / mL, or about 400 DMC / mL.

[0465] In some embodiments, DNAse I is reconstituted in 1 mL of sterile HBSS or another buffer. The lyophilized stock enzyme was at a concentration of 4 KU / vial. In some embodiments, the reconstituted DNase I stock ranges from about 1 KU / mL to 10 KU / mL, e.g., about 1 KU / mL, about 2 KU / mL, about 3 KU / mL, about 4 KU / mL, about 5 KU / mL, about 6 KU / mL, about 7 KU / mL, about 8 KU / mL, about 9 KU / mL, or about 10 KU / mL.

[0466] In some embodiments, enzyme stocks are variable and concentrations may need to be determined. In some embodiments, the concentration of the lyophilized stock can be verified. In some embodiments, the final amount of enzyme added to the digestion cocktail is adjusted based on the determined stock concentration.

[0467] In some embodiments, the enzyme mixture comprises about 10.2 ul of neutral protease (0.36 DMC U / mL), 21.3 μl of collagenase (1.2 PZ / mL), and 250 ul of DNAse I (200 U / mL) in about 4.7 mL of sterile HBSS.

[0468] As noted above, in some embodiments, the TILs are derived from solid tumors. In some embodiments, the solid tumor is not fragmented. In some embodiments, the solid tumor is not fragmented and is subjected to enzymatic digestion as a whole tumor. In some embodiments, the tumor is digested in an enzyme mixture comprising collagenase, DNase, and hyaluronidase. In some embodiments, the tumor is digested in an enzyme mixture comprising collagenase, DNase, and hyaluronidase for 1-2 hours. In some embodiments, the tumor is digested in an enzyme mixture comprising collagenase, DNase, and hyaluronidase for 1-2 hours at 37°C and 5% CO2. In some embodiments, the tumor is digested in an enzyme mixture comprising collagenase, DNase, and hyaluronidase for 1-2 hours at 37°C and 5% CO2 with rotation. In some embodiments, the tumor is digested overnight with constant rotation. In some embodiments, the tumor is digested overnight at 37°C and 5% CO2 with constant rotation. In some embodiments, the whole tumor is combined with the enzymes to form a tumor digestion reaction mixture.

[0469] In some embodiments, the tumor is reconstituted with lyophilized enzyme in a sterile buffer. In some embodiments, the buffer is sterile HBSS.

[0470] In some embodiments, the enzyme mixture includes collagenase. In some embodiments, the collagenase is collagenase IV. In some embodiments, the working stock of collagenase is a 10x working stock of 100 mg / mL.

[0471] In some embodiments, the enzyme mixture comprises DNAse, hi some embodiments, the working stock of DNAse is a 10x working stock of 10,000 IU / mL.

[0472] In some embodiments, the enzyme mixture comprises hyaluronidase. In some embodiments, the working stock of hyaluronidase is a 10x working stock of 10 mg / mL.

[0473] In some embodiments, the enzyme mixture comprises 10 mg / mL collagenase, 1000 IU / mL DNAse, and 1 mg / mL hyaluronidase.

[0474] In some embodiments, the enzyme mixture comprises 10 mg / mL collagenase, 500 IU / mL DNAse, and 1 mg / mL hyaluronidase.

[0475] Generally, the harvested cell suspension is referred to as a "primary cell population" or "freshly harvested" cell population.

[0476] In some embodiments, fragmentation comprises physical fragmentation, including, for example, dissection and digestion. In some embodiments, fragmentation is physical fragmentation. In some embodiments, fragmentation is dissection. In some embodiments, fragmentation is by digestion. In some embodiments, TILs may be initially cultured from enzymatic tumor digests and tumor fragments obtained from digesting or fragmenting a tumor sample obtained from a patient.

[0477] In some embodiments where the tumor is a solid tumor, after a tumor sample is obtained, for example, in step A (provided in FIG. 1), the tumor is subjected to physical fragmentation. In some embodiments, fragmentation occurs before cryopreservation. In some embodiments, fragmentation occurs after cryopreservation. In some embodiments, fragmentation occurs after obtaining the tumor in the absence of 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. In some embodiments, the tumor is fragmented and 30 or 40 fragments or pieces are placed in each container for the first expansion. In some embodiments, the tumor is fragmented and 40 fragments or pieces are placed in each container for the first expansion. In some embodiments, the plurality of fragments comprises about 4 to about 50 fragments, each fragment being about 27 mm. 3 In some embodiments, the plurality of pieces has a volume of about 1300 mm 3 ~approx. 1500mm 3 In some embodiments, the plurality of pieces comprises about 30 to about 60 pieces having a total volume of about 1350 mm 3 In some embodiments, the plurality of fragments comprises about 50 fragments with a total mass of about 1 gram to about 1.5 grams. In some embodiments, the plurality of fragments comprises about 4 fragments.

[0478] In some embodiments, the TILs are obtained from tumor fragments. In some embodiments, the tumor fragments are obtained by sharp dissection. In some embodiments, the tumor fragments are approximately 1 mm 3 ~10mm 3 In some embodiments, the tumor fragment is about 1 mm 3 ~8mm 3 In some embodiments, the tumor fragment is about 1 mm 3 In some embodiments, the tumor fragment is about 2 mm 3 In some embodiments, the tumor fragment is about 3 mm 3 In some embodiments, the tumor fragment is about 4 mm 3In some embodiments, the tumor fragment is about 5 mm 3 In some embodiments, the tumor fragment is about 6 mm 3 In some embodiments, the tumor fragment is about 7 mm 3 In some embodiments, the tumor fragment is about 8 mm 3 In some embodiments, the tumor fragment is about 9 mm 3 In some embodiments, the tumor fragment is about 10 mm 3 In some embodiments, the tumor is 1-4 mm x 1-4 mm x 1-4 mm. In some embodiments, the tumor is 1 mm x 1 mm x 1 mm. In some embodiments, the tumor is 2 mm x 2 mm x 2 mm. In some embodiments, the tumor is 3 mm x 3 mm x 3 mm. In some embodiments, the tumor is 4 mm x 4 mm x 4 mm.

[0479] In some embodiments, the tumor is resected to minimize the amount of hemorrhagic, necrotic, and / or fatty tissue on each piece. In some embodiments, the tumor is resected to minimize the amount of hemorrhagic tissue on each piece. In some embodiments, the tumor is resected to minimize the amount of necrotic tissue on each piece. In some embodiments, the tumor is resected to minimize the amount of fatty tissue on each piece.

[0480] In some embodiments, tumor fragmentation is performed to maintain the internal structure of the tumor. In some embodiments, tumor fragmentation is performed without sawing with a scalpel. In some embodiments, TILs are obtained from tumor digests. In some embodiments, tumor digests are generated by incubation in an enzyme medium, such as, but not limited to, RPMI 1640 (2 mM GlutaMAX, 10 mg / mL gentamicin, 30 U / mL DNase, and 1.0 mg / mL collagenase), followed by mechanical dissociation (GentleMACS, Miltenyi Biotec, Auburn, CA). After placing the tumor in the enzyme medium, the tumor may be mechanically dissociated for approximately 1 minute. The solution may then be incubated at 37°C in 5% CO2 for 30 minutes, after which it may be mechanically disrupted again for approximately 1 minute. After again incubating at 37°C in 5% CO2 for 30 minutes, the tumor may be mechanically disrupted a third time for approximately 1 minute. In some embodiments, if large tissue debris was present, one or two additional rounds of mechanical dissociation were applied to the sample after the third mechanical disruption, with incubation for an additional 30 minutes at 37° C. in 5% CO In some embodiments, if the cell suspension contains a large number of red blood cells or dead cells, density gradient separation using Ficoll can be performed at the end of the final incubation to remove these cells.

[0481] In some embodiments, the cell suspension harvested before the first expansion step is referred to as a "primary cell population" or a "freshly harvested" cell population.

[0482] In some embodiments, the cells may be optionally frozen after sampling and cryopreserved before undergoing expansion as described in step B, which is described in more detail below and also illustrated in Figures 1 and 8.

[0483] 1. Pleural fluid T cells and TILs In some embodiments, the sample is a pleural fluid sample. In some embodiments, the source of T cells or TILs for expansion by the processes described herein is a pleural fluid sample. In some embodiments, the sample is a pleural fluid-derived sample. In some embodiments, the source of TILs for expansion by the processes described herein is a pleural fluid-derived sample. See, e.g., the methods described in U.S. Patent Publication No. US2014 / 0295426, which is incorporated herein by reference in its entirety for all purposes.

[0484] In some embodiments, any pleural fluid or pleural effusion that appears to be and / or contains TILs can be utilized. Such samples can be derived from primary or metastatic lung cancer, such as NSCLC or SCLC. In some embodiments, samples can be derived from secondary metastatic cancer cells from another organ, such as the breast, ovary, colon, or prostate. In some embodiments, the sample used in the expansion methods described herein is a pleural effusion. In some embodiments, the sample used in the expansion methods described herein is a pleural transudate. Other biological samples can include other serous fluids containing TILs, including, for example, ascites from the abdomen or pancreatic cyst fluid. Ascites and pleural fluids have very similar chemical systems, and both the abdomen and lungs have mesothelial lines and fluid forms in the pleural and abdominal spaces of the same material in malignant tumors; in some embodiments, such fluids contain TILs. In some embodiments in which the disclosed methods utilize pleural effusion, the same method can be performed with similar results using other cyst fluids containing TILs.

[0485] In some embodiments, the pleural fluid is in an unprocessed form removed directly from the patient. In some embodiments, the unprocessed pleural fluid is placed in a standard collection tube, such as an EDTA or heparin tube, before further processing steps. In some embodiments, the unprocessed pleural fluid is placed in a standard CellSave® tube (Veridex) before further processing steps. In some embodiments, the sample is placed in a CellSave tube immediately after collection from the patient to avoid a decrease in the number of viable TILs. The number of viable TILs can decrease significantly within 24 hours if left in unprocessed pleural fluid, even at 4°C. In some embodiments, the sample is placed in an appropriate collection tube within 1 hour, 5 hours, 10 hours, 15 hours, or up to 24 hours after removal from the patient. In some embodiments, the sample is placed in an appropriate collection tube at 4°C within 1 hour, 5 hours, 10 hours, 15 hours, or up to 24 hours after removal from the patient.

[0486] In some embodiments, the pleural fluid sample from a selected subject may be diluted. In some embodiments, the dilution is 1:10 pleural fluid to diluent. In other embodiments, the dilution is 1:9 pleural fluid to diluent. In other embodiments, the dilution is 1:8 pleural fluid to diluent. In other embodiments, the dilution is 1:5 pleural fluid to diluent. In other embodiments, the dilution is 1:2 pleural fluid to diluent. In other embodiments, the dilution is 1:1 pleural fluid to diluent. In some embodiments, the diluent includes saline, phosphate-buffered saline, another buffer, or a physiologically acceptable diluent. In some embodiments, the sample is placed into a CellSave tube immediately after collection and dilution from the patient to avoid a significant loss of viable TILs, which can occur within 24 to 48 hours if left in unprocessed pleural fluid, even at 4°C. In some embodiments, the pleural fluid sample is placed into a suitable collection tube within 1 hour, 5 hours, 10 hours, 15 hours, 24 hours, 36 hours, or up to 48 hours after removal from the patient and dilution. In some embodiments, the pleural fluid sample is placed into a suitable collection tube within 1 hour, 5 hours, 10 hours, 15 hours, 24 hours, 36 hours, or up to 48 hours after removal from the patient and dilution and diluted at 4°C.

[0487] In yet other embodiments, the pleural fluid sample is concentrated by conventional means prior to further processing steps. In some embodiments, this pretreatment of the pleural fluid is preferred in situations where the pleural fluid must be frozen for transport to the laboratory where the method will be performed or for subsequent analysis (e.g., more than 24-48 hours after collection). In some embodiments, the pleural fluid sample is prepared by centrifuging the pleural fluid sample after it is collected from the subject and resuspending the centrate or pellet in a buffer. In some embodiments, the pleural fluid sample is subjected to multiple centrifugations and resuspensions before being frozen for transport or subsequent analysis and / or processing.

[0488] In some embodiments, the pleural fluid sample is concentrated prior to further processing steps by using a filtration method. In some embodiments, the pleural fluid sample used in further processing is prepared by filtering the fluid through a filter containing a known, essentially uniform pore size that allows the pleural fluid to pass through the membrane but retains tumor cells. In some embodiments, the pore diameter of the membrane can be at least 4 μM. In other embodiments, the pore diameter can be 5 μM or greater, and in other embodiments, any of 6, 7, 8, 9, or 10 μM. After filtration, the TIL-containing cells retained by the membrane can be rinsed from the membrane into an appropriate physiologically acceptable buffer. The thus-enriched TIL-containing cells can then be used in further processing steps of the method.

[0489] In some embodiments, a pleural fluid sample (e.g., including unprocessed pleural fluid), diluted pleural fluid, or resuspended cell pellet is contacted with a lytic reagent that specifically lyses nonnucleated red blood cells present in the sample. In some embodiments, this step is performed before further processing steps in situations where the pleural fluid contains a significant number of RBCs. Suitable lytic reagents include a single lytic reagent or a lytic reagent and a quenching reagent, or a lysing agent, a quenching reagent, and a fixation reagent. Suitable lysis systems are commercially available and include the BD Pharm Lyse™ system (Becton Dickenson). Other lysis systems include the Versalyse™ system, the FACSlyse™ system (Becton Dickenson), the Immunoprep™ system, or the Erythrolyse II system (Beckman Coulter, Inc.), or an ammonium chloride system. In some embodiments, the lytic reagent can vary depending on key requirements, such as efficient lysis of red blood cells and preservation of TILs and their phenotypic characteristics in the pleural fluid. In addition to utilizing a single reagent for lysis, lysis systems useful in the methods described herein can include a second reagent, e.g., one that quenches or delays the effect of the lysis reagent during the remaining steps of the method, such as Stabilyse™ reagent (Beckman Coulter, Inc.) Depending on the choice of lysis reagent or the preferred implementation of the method, conventional fixative reagents can also be used.

[0490] In some embodiments, pleural fluid samples that have been unprocessed, diluted, or multiple centrifuged or processed as described herein are stored frozen at a temperature of about -140°C before being further processed and / or expanded as provided herein.

[0491] B. Step B: First Expansion In some embodiments, the methods provide for obtaining young TILs that are capable of increasing their replication cycle upon administration to a subject / patient and thus may provide additional therapeutic benefit over older TILs (i.e., TILs that have undergone more rounds of replication prior to administration to a subject / patient). The characteristics of young TILs are described in the literature, for example, Donia, et al., Scand. J. Immunol. 2012, 75, 157-167, Dudley, et al., Clin. al.,Clin.Cancer Res.2013,19,OF1-OF9, Besser,et al.,J.Immunother.2009,32:415-423,Robbins,et al.,J.Immunol.2004,173,7125-7130,Shen,et al. al.,J.Immunother.,2007,30,123-129, Zhou,et al., J. Immunother. 2005, 28, 53-62, and Tran, et al., J. Immunother., 2008, 31, 742-751, the disclosures of each of which are incorporated herein by reference.

[0492] The diverse antigen receptors of T and B lymphocytes are generated by somatic recombination of a limited number of gene segments. These gene segments: V (variable), D (diversity), J (joining), and C (constant) determine the binding specificity and downstream applications of immunoglobulins and T cell receptors (TCRs). The present invention provides methods for generating TILs that exhibit and increase T cell repertoire diversity. In some embodiments, TILs obtained by the present methods exhibit increased T cell repertoire diversity. In some embodiments, TILs obtained by the present methods exhibit increased T cell repertoire diversity compared to freshly harvested TILs and / or TILs prepared using methods other than those provided herein, including, for example, methods other than those embodied in FIG. 1. In some embodiments, TILs obtained by the present methods exhibit increased T cell repertoire diversity compared to freshly harvested TILs and / or TILs prepared using a method designated Process 1C as illustrated in FIG. 5 and / or FIG. 6. In some embodiments, the TILs obtained in the first expansion exhibit increased T cell repertoire diversity. In some embodiments, the increased diversity is increased immunoglobulin diversity and / or T cell receptor diversity. In some embodiments, the diversity is in immunoglobulins and in immunoglobulin heavy chains. In some embodiments, the diversity is in immunoglobulins and in immunoglobulin light chains. In some embodiments, the diversity is in T cell receptors. In some embodiments, the diversity is in one of the T cell receptors selected from the group consisting of alpha, beta, gamma, and delta receptors. In some embodiments, expression of T cell receptor (TCR) alpha and / or beta is increased. In some embodiments, expression of T cell receptor (TCR) alpha is increased. In some embodiments, expression of T cell receptor (TCR) beta is increased. In some embodiments, expression of TCRab (i.e., TCRα / β) is increased.

[0493] For example, after dissection or digestion of tumor fragments, such as those described in step A of Figure 1, the resulting cells are cultured in serum containing IL-2 under conditions that favor the growth of TILs over tumor and other cells. In some embodiments, tumor digests are incubated in 2 mL wells in medium containing inactivated human AB serum with 6000 IU / mL of IL-2. This primary cell population is cultured for several days, generally 3-14 days, resulting in a bulk TIL population, generally about 1 x 10 8 In some embodiments, this primary cell population is cultured for 7-14 days, resulting in a bulk TIL population, generally about 1 x 10 8 In some embodiments, this primary cell population is cultured for 10-14 days, resulting in a bulk TIL population, generally about 1 x 10 8 In some embodiments, this primary cell population is cultured for about 11 days, resulting in a bulk TIL population, generally about 1 x 10 8 yielding bulk TIL cells.

[0494] In a preferred embodiment, TIL expansion can be performed using an initial bulk TIL expansion step (such as that described in step B of FIG. 1, which may include a process referred to as pre-REP), as described below and herein, followed by step D below and a second expansion as described herein (step D, which includes a process referred to as the Rapid Expansion Protocol (REP) step), followed by optional cryopreservation, followed by a second step D below and described herein (which includes a process referred to as the restimulation REP step). TILs obtained from this process can optionally be characterized for phenotypic characteristics and metabolic parameters as described herein.

[0495] In embodiments in which TIL cultures are initiated in 24-well plates, e.g., using Costar 24-well cell culture clusters, flat bottom (Corning Incorporated, Corning, NY), 1 x 10 cells are cultured in 2 mL of complete medium (CM) containing IL-2 (6000 IU / mL, Chiron Corp., Emeryville, CA). 6 Tumor digested cells or one tumor fragment can be seeded into each well. In some embodiments, the tumor fragment is approximately 1 mm 3 ~10mm 3 is.

[0496] In some embodiments, the first expansion culture medium is referred to as "CM," an abbreviation for culture medium. In some embodiments, the CM in step B consists of RPMI 1640 with GlutaMAX, supplemented with 10% human AB serum, 25 mM Hepes, and 10 mg / mL gentamicin. The cultures are grown in a 40 mL volume and 10 cm 2 In embodiments initiated in gas-permeable flasks with gas-permeable silicon bottoms (e.g., G-REX10, Wilson Wolf Manufacturing, New Brighton, MN), each flask contains 10-40 x 10 cells in 10-40 mL of CM with IL-2. 6 Live tumor cells or 5–30 tumor fragments were loaded. Both G-REX10 and 24-well plates were incubated in a humidified incubator at 37°C with 5% CO2. Five days after the start of culture, half of the medium was removed and replaced with fresh CM and IL-2. After five days, half of the medium was replaced every 2–3 days.

[0497] After preparation of tumor fragments, the resulting cells (i.e., fragments) are cultured in serum containing IL-2 under conditions that favor the growth of TILs over tumor and other cells. In some embodiments, tumor digests are incubated in 2 mL wells (or in some cases, in the presence of an APC cell population, as outlined herein) in medium containing inactivated human AB serum with 6000 IU / mL of IL-2. This primary cell population is cultured for several days, generally 10-14 days, resulting in a bulk TIL population, generally about 1 x 10 8 In some embodiments, the growth medium during the first expansion contains IL-2 or a variant thereof. In some embodiments, the IL is recombinant human IL-2 (rhIL-2). In some embodiments, the IL-2 stock solution contains 20-30 x 10 cells per 1 mg vial. 6 IU / mg specific activity. In some embodiments, the IL-2 stock solution contains 20×10 6 IU / mg specific activity. In some embodiments, the IL-2 stock solution contains 25×10 6 IU / mg specific activity. In some embodiments, the IL-2 stock solution contains 30×10 6 IU / mg specific activity. In some embodiments, the IL-2 stock solution contains 4-8 x 10 6 In some embodiments, the IL-2 stock solution has a final concentration of 5-7 x 10 IU / mg IL-2. 6 In some embodiments, the IL-2 stock solution has a final concentration of 6×10 IU / mg of IL-2. 6IU / mg IL-2. In some embodiments, the IL-2 stock solution is prepared as described in Example 5. In some embodiments, the first expansion culture medium contains about 10,000 IU / mL IL-2, about 9,000 IU / mL IL-2, about 8,000 IU / mL IL-2, about 7,000 IU / mL IL-2, about 6,000 IU / mL IL-2, or about 5,000 IU / mL IL-2. In some embodiments, the first expansion culture medium contains about 9,000 IU / mL to about 5,000 IU / mL IL-2. In some embodiments, the first expansion culture medium contains about 8,000 IU / mL to about 6,000 IU / mL IL-2. In some embodiments, the first expansion culture medium comprises about 7,000 IU / mL to about 6,000 IU / mL of IL-2. In some embodiments, the first expansion culture medium comprises about 6,000 IU / mL of IL-2. In some embodiments, the cell culture medium further comprises IL-2. In some embodiments, the cell culture medium comprises about 3000 IU / mL of IL-2. In some embodiments, the cell culture medium further comprises IL-2. In some embodiments, the cell culture medium comprises about 3000 IU / mL of IL-2. In some embodiments, the cell culture medium comprises about 1000 IU / mL, about 1500 IU / mL, about 2000 IU / mL, about 2500 IU / mL, about 3000 IU / mL, about 3500 IU / mL, about 4000 IU / mL, about 4500 IU / mL, about 5000 IU / mL, about 5500 IU / mL, about 6000 IU / mL, about 6500 IU / mL, about 7000 IU / mL, about 7500 IU / mL, or about 8000 IU / mL of IL-2. In some embodiments, the cell culture medium contains 1000-2000 IU / mL, 2000-3000 IU / mL, 3000-4000 IU / mL, 4000-5000 IU / mL, 5000-6000 IU / mL, 6000-7000 IU / mL, 7000-8000 IU / mL, or about 8000 IU / mL of IL-2.

[0498] In some embodiments, the first expansion culture medium contains about 500 IU / mL IL-15, about 400 IU / mL IL-15, about 300 IU / mL IL-15, about 200 IU / mL IL-15, about 180 IU / mL IL-15, about 160 IU / mL IL-15, about 140 IU / mL IL-15, about 120 IU / mL IL-15, or about 100 IU / mL IL-15. In some embodiments, the first expansion culture medium contains about 500 IU / mL to about 100 IU / mL IL-15. In some embodiments, the first expansion culture medium contains about 400 IU / mL to about 100 IU / mL IL-15. In some embodiments, the first expansion culture medium comprises about 300 IU / mL to about 100 IU / mL of IL-15. In some embodiments, the first expansion culture medium comprises about 200 IU / mL of IL-15. In some embodiments, the cell culture medium comprises about 180 IU / mL of IL-15. In some embodiments, the cell culture medium further comprises IL-15. In some embodiments, the cell culture medium comprises about 180 IU / mL of IL-15.

[0499] In some embodiments, the first expansion culture medium contains about 20 IU / mL of IL-21, about 15 IU / mL of IL-21, about 12 IU / mL of IL-21, about 10 IU / mL of IL-21, about 5 IU / mL of IL-21, about 4 IU / mL of IL-21, about 3 IU / mL of IL-21, about 2 IU / mL of IL-21, about 1 IU / mL of IL-21, or about 0.5 IU / mL of IL-21. In some embodiments, the first expansion culture medium contains about 20 IU / mL to about 0.5 IU / mL of IL-21. In some embodiments, the first expansion culture medium contains about 15 IU / mL to about 0.5 IU / mL of IL-21. In some embodiments, the first expansion culture medium comprises about 12 IU / mL to about 0.5 IU / mL of IL-21. In some embodiments, the first expansion culture medium comprises about 10 IU / mL to about 0.5 IU / mL of IL-21. In some embodiments, the first expansion culture medium comprises about 5 IU / mL to about 1 IU / mL of IL-21. In some embodiments, the first expansion culture medium comprises about 2 IU / mL of IL-21. In some embodiments, the cell culture medium comprises about 1 IU / mL of IL-21. In some embodiments, the cell culture medium comprises about 0.5 IU / mL of IL-21. In some embodiments, the cell culture medium further comprises IL-21. In some embodiments, the cell culture medium comprises about 1 IU / mL of IL-21.

[0500] In some embodiments, the cell culture medium comprises an anti-CD3 agonist antibody, such as an OKT-3 antibody. In some embodiments, the cell culture medium comprises about 30 ng / mL of the OKT-3 antibody. In some embodiments, the cell culture medium comprises about 0.1 ng / mL, about 0.5 ng / mL, about 1 ng / mL, about 2.5 ng / mL, about 5 ng / mL, about 7.5 ng / mL, about 10 ng / mL, about 15 ng / mL, about 20 ng / mL, about 25 ng / mL, about 30 ng / mL, about 35 ng / mL, about 40 ng / mL, about 50 ng / mL, about 60 ng / mL, about 70 ng / mL, about 80 ng / mL, about 90 ng / mL, about 100 ng / mL, about 200 ng / mL, about 500 ng / mL, or about 1 μg / mL of the OKT-3 antibody. In some embodiments, the cell culture medium comprises OKT-3 antibody at 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 antibody. In some embodiments, the OKT-3 antibody is muromonab. See, for example, Table 1.

[0501] In some embodiments, the cell culture medium comprises one or more TNFRSF agonists in the cell culture medium. In some embodiments, the TNFRSF agonist comprises a 4-1BB agonist. In some embodiments, the TNFRSF agonist is a 4-1BB agonist, and the 4-1BB agonist is selected from the group consisting of urelumab, utomilumab, EU-101, fusion proteins, and fragments, derivatives, variants, biosimilars, and combinations thereof. In some embodiments, the TNFRSF agonist is added at a concentration sufficient to achieve a cell culture medium concentration of 0.1 μg / mL to 100 μg / mL. In some embodiments, the TNFRSF agonist is added at a concentration sufficient to achieve a cell culture medium concentration of 20 μg / mL to 40 μg / mL.

[0502] In some embodiments, in addition to the one or more TNFRSF agonists, the cell culture medium further comprises IL-2 at an initial concentration of about 3000 IU / mL and OKT-3 at an initial concentration of about 30 ng / mL, and the one or more TNFRSF agonists comprise a 4-1BB agonist.

[0503] In some embodiments, the first expansion culture medium is referred to as "CM," an abbreviation for culture medium. In some embodiments, it is referred to as CM1 (culture medium 1). In some embodiments, the CM consists of RPMI 1640 with GlutaMAX, supplemented with 10% human AB serum, 25 mM Hepes, and 10 mg / mL gentamicin. The cultures are grown in a 40 mL volume and 10 cm 2 In embodiments initiated in gas-permeable flasks with gas-permeable silicon bottoms (e.g., G-REX10, Wilson Wolf Manufacturing, New Brighton, MN), 10-40 x 10 cells in 10-40 mL of CM containing IL-2 are cultured. 6 Viable tumor digested cells or 5-30 tumor fragments were placed in each flask. Both the G-REX10 and 24-well plates were incubated in a humidified incubator at 37°C with 5% CO2. Five days after the start of culture, half of the medium was removed and replaced with fresh CM and IL-2. After five days, half of the medium was replaced every 2-3 days. In some embodiments, the CM is CM1 as described in the Examples (see Example 1). In some embodiments, the first expansion occurs in the 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.

[0504] In some embodiments, as discussed in the Examples and Figures, the first expansion (including, for example, a process such as that described in step B of FIG. 1, which may include what is sometimes referred to as pre-REP) process is shortened to 3-14 days. In some embodiments, as discussed in the Examples and shown in FIGS. 4 and 5, the first expansion (including, for example, a process such as that described in step B of FIG. 1, which may include what is sometimes referred to as pre-REP), also including, for example, the expansion described in step B of FIG. 1, is shortened to 7-14 days. In some embodiments, the first expansion in step B is shortened to 10-14 days. In some embodiments, the first expansion is shortened to 11 days, for example, as discussed in the expansion described in step B of FIG. 1.

[0505] In some embodiments, the first TIL expansion can continue for 1 day, 2 days, 3 days, 4 days, 5 days, 6 days, 7 days, 8 days, 9 days, 10 days, 11 days, 12 days, 13 days, or 14 days. In some embodiments, the first TIL expansion can continue for 1 day to 14 days. In some embodiments, the first TIL expansion can continue for 2 days to 14 days. In some embodiments, the first TIL expansion can continue for 3 days to 14 days. In some embodiments, the first TIL expansion can continue for 4 days to 14 days. In some embodiments, the first TIL expansion can continue for 5 days to 14 days. In some embodiments, the first TIL expansion can continue for 6 days to 14 days. In some embodiments, the first TIL expansion can continue for 7 days to 14 days. In some embodiments, the first TIL expansion can continue for 8 days to 14 days. In some embodiments, the first TIL expansion can continue for 9 days to 14 days. In some embodiments, the first TIL expansion can continue for 10 to 14 days. In some embodiments, the first TIL expansion can continue for 11 to 14 days. In some embodiments, the first TIL expansion can continue for 12 to 14 days. In some embodiments, the first TIL expansion can continue for 13 to 14 days. In some embodiments, the first TIL expansion can continue for 14 days. In some embodiments, the first TIL expansion can continue for 1 to 11 days. In some embodiments, the first TIL expansion can continue for 2 to 11 days. In some embodiments, the first TIL expansion can continue for 3 to 11 days. In some embodiments, the first TIL expansion can continue for 4 to 11 days. In some embodiments, the first TIL expansion can continue for 5 to 11 days. In some embodiments, the first TIL expansion can continue for 6 to 11 days. In some embodiments, the first TIL expansion can continue for 7 to 11 days. In some embodiments, the first TIL expansion can continue for 8 to 11 days.In some embodiments, the first TIL expansion can continue for 9 to 11 days. In some embodiments, the first TIL expansion can continue for 10 to 11 days. In some embodiments, the first TIL expansion can continue for 11 days.

[0506] In some embodiments, a combination of IL-2, IL-7, IL-15, and / or IL-21 is used as a combination during the first expansion. In some embodiments, IL-2, IL-7, IL-15, and / or IL-21, and any combination thereof, may be included during the first expansion, including, for example, during step B of the process according to Figure 1 and described herein. In some embodiments, a combination of IL-2, IL-15, and IL-21 is used as a combination during the first expansion. In some embodiments, IL-2, IL-15, and IL-21, and any combination thereof, may be included during the process according to Figure 1 and step B of the process described herein.

[0507] In some embodiments, the first expansion (including the process referred to as pre-REP, e.g., step B according to FIG. 1) process is shortened to 3-14 days, as discussed in the Examples and Figures. In some embodiments, the first expansion of step B is shortened to 7-14 days. In some embodiments, the first expansion of step B is shortened to 10-14 days. In some embodiments, the first expansion is shortened to 11 days.

[0508] In some embodiments, the first expansion, e.g., step B according to FIG. 1, is performed in a closed system bioreactor. In some embodiments, a closed system is used for TIL expansion as described herein. In some embodiments, a single bioreactor is used. In some embodiments, the single bioreactor used is, for example, a G-REX-10 or G-REX-100. In some embodiments, the closed system bioreactor is a single bioreactor.

[0509] 1. Cytokines and other additives The expansion methods described herein generally use culture media containing high doses of cytokines, particularly IL-2, as known in the art. Alternatively, the use of cytokine combinations for rapid and / or secondary expansion of TILs is additionally possible using combinations of two or more of IL-2, IL-15, and IL-21, as described in U.S. Patent Application Publication No. US2017 / 0107490A1 (the disclosure of which is incorporated herein by reference). Thus, possible combinations include IL-2 and IL-15, IL-2 and IL-21, IL-15 and IL-21, and IL-2 or IL-15 and IL-21, the latter finding particular use in many embodiments. The use of cytokine combinations is particularly advantageous for the generation of lymphocytes, particularly T cells as described therein.

[0510] In some embodiments, step B may also include the addition of an OKT-3 antibody or muromonab to the culture medium, as described elsewhere herein. In some embodiments, step B may also include the addition of a 4-1BB agonist to the culture medium, as described elsewhere herein. In some embodiments, step B may also include the addition of an OX-40 agonist to the culture medium, as described elsewhere herein. In other embodiments, additives such as peroxisome proliferator-activated receptor gamma coactivator I-alpha agonists, including proliferator-activated receptor (PPAR)-gamma agonists such as thiazolidinedione compounds, may be used in the culture medium during step B, as described in U.S. Patent Application Publication No. US2019 / 0307796A1 (the disclosure of which is incorporated herein by reference).

[0511] C. Step C: Moving from the first expansion to the second expansion In some cases, the bulk TIL population obtained from the first expansion, including, for example, the TIL population obtained from step B shown in Figure 1, can be immediately cryopreserved using the protocols discussed below. Alternatively, the TIL population obtained from the first expansion, referred to as the second TIL population, can be subjected to a second expansion (which may include an expansion sometimes referred to as REP), as discussed below, and then cryopreserved. Similarly, when genetically modified TILs are used for therapy, the first TIL population (which may be 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 the appropriate therapy before expansion or after the first expansion and before the second expansion.

[0512] In some embodiments, TILs obtained from the first expansion (e.g., from step B shown in FIG. 1 ) are stored until phenotyping for selection. In some embodiments, TILs obtained from the first expansion (e.g., from step B shown in FIG. 1 ) are not stored and proceed directly to the second expansion. In some embodiments, TILs obtained from the first expansion are not cryopreserved after the first expansion and before the second expansion. In some embodiments, the transition from the first expansion to the second expansion occurs about 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, or 14 days after fragmentation occurs. In some embodiments, the transition from the first expansion to the second expansion occurs about 3 to 14 days after fragmentation occurs. In some embodiments, the transition from the first expansion to the second expansion occurs about 4 to 14 days after fragmentation occurs. In some embodiments, the transition from the first expansion to the second expansion occurs about 4 to 10 days after fragmentation occurs. In some embodiments, the transition from the first expansion to the second expansion occurs about 7 to 14 days after fragmentation occurs. In some embodiments, the transition from the first expansion to the second expansion occurs about 14 days after fragmentation occurs.

[0513] In some embodiments, the transition from the first expansion to the second expansion occurs 1 day, 2 days, 3 days, 4 days, 5 days, 6 days, 7 days, 8 days, 9 days, 10 days, 11 days, 12 days, 13 days, or 14 days after fragmentation occurs. In some embodiments, the transition from the first expansion to the second expansion occurs 1 to 14 days after fragmentation occurs. In some embodiments, the first TIL expansion can continue for 2 to 14 days. In some embodiments, the transition from the first expansion to the second expansion occurs 3 to 14 days after fragmentation occurs. In some embodiments, the transition from the first expansion to the second expansion occurs 4 to 14 days after fragmentation occurs. In some embodiments, the transition from the first expansion to the second expansion occurs 5 to 14 days after fragmentation occurs. In some embodiments, the transition from the first expansion to the second expansion occurs 6 to 14 days after fragmentation occurs. In some embodiments, the transition from the first expansion to the second expansion occurs 7 to 14 days after fragmentation occurs. In some embodiments, the transition from the first expansion to the second expansion occurs 8 to 14 days after fragmentation occurs. In some embodiments, the transition from the first expansion to the second expansion occurs 9 to 14 days after fragmentation occurs. In some embodiments, the transition from the first expansion to the second expansion occurs 10 to 14 days after fragmentation occurs. In some embodiments, the transition from the first expansion to the second expansion occurs 11 to 14 days after fragmentation occurs. In some embodiments, the transition from the first expansion to the second expansion occurs 12 to 14 days after fragmentation occurs. In some embodiments, the transition from the first expansion to the second expansion occurs 13 to 14 days after fragmentation occurs. In some embodiments, the transition from the first expansion to the second expansion occurs 14 days after fragmentation occurs. In some embodiments, the transition from the first expansion to the second expansion occurs 1 to 11 days after fragmentation occurs. In some embodiments, the transition from the first expansion to the second expansion occurs 2 to 11 days after fragmentation occurs. In some embodiments, the transition from the first expansion to the second expansion occurs 3 to 11 days after fragmentation occurs.In some embodiments, the transition from the first expansion to the second expansion occurs 4 to 11 days after fragmentation occurs. In some embodiments, the transition from the first expansion to the second expansion occurs 5 to 11 days after fragmentation occurs. In some embodiments, the transition from the first expansion to the second expansion occurs 6 to 11 days after fragmentation occurs. In some embodiments, the transition from the first expansion to the second expansion occurs 7 to 11 days after fragmentation occurs. In some embodiments, the transition from the first expansion to the second expansion occurs 8 to 11 days after fragmentation occurs. In some embodiments, the transition from the first expansion to the second expansion occurs 9 to 11 days after fragmentation occurs. In some embodiments, the transition from the first expansion to the second expansion occurs 10 to 11 days after fragmentation occurs. In some embodiments, the transition from the first expansion to the second expansion occurs 11 days after fragmentation occurs.

[0514] In some embodiments, the TILs are not preserved after the first expansion and before the second expansion, and the TILs proceed directly to the second expansion (e.g., in some embodiments, they are not preserved during the transition from step B to step D shown in FIG. 1). In some embodiments, the transition occurs in a closed system as described herein. In some embodiments, the second TIL population, the TILs from the first expansion, proceed directly to the second expansion without a transition period.

[0515] In some embodiments, the transition from the first expansion to the second expansion, e.g., step C according to FIG. 1, is performed in a closed system bioreactor. In some embodiments, a closed system is used for TIL expansion as described herein. In some embodiments, a single bioreactor is used. In some embodiments, the single bioreactor used is, for example, a G-REX-10 or G-REX-100 bioreactor. In some embodiments, the closed system bioreactor is a single bioreactor.

[0516] D. Step D: Second Enlargement In some embodiments, the TIL cell population is expanded in number after harvesting and initial bulk processing, e.g., after steps A and B, and a transition referred to as step C, as shown in Figure 1. This further expansion is referred to herein as secondary expansion, which may include an expansion process commonly referred to in the art as a rapid expansion process (REP), as well as the process shown in Figure 1 at step D. Secondary expansion is generally accomplished in a gas-permeable container using culture medium containing several components, including feeder cells, a cytokine source, and an anti-CD3 antibody.

[0517] In some embodiments, the second expansion or second TIL expansion (expansion sometimes referred to as REP, and which may include the process shown in step D of FIG. 1) may be performed using any TIL flask or vessel known to one of skill in the art. In some embodiments, the second TIL expansion may continue for 7, 8, 9, 10, 11, 12, 13, or 14 days. In some embodiments, the second TIL expansion may continue for about 7 days to about 14 days. In some embodiments, the second TIL expansion may continue for about 8 days to about 14 days. In some embodiments, the second TIL expansion may continue for about 9 days to about 14 days. In some embodiments, the second TIL expansion may continue for about 10 days to about 14 days. In some embodiments, the second TIL expansion may continue for about 11 days to about 14 days. In some embodiments, the second TIL expansion may continue for about 12 days to about 14 days. In some embodiments, the second TIL expansion can proceed for about 13 to about 14 days. In some embodiments, the second TIL expansion can proceed for about 14 days.

[0518] In some embodiments, the second expansion can be performed in a gas-permeable container using the methods of the present disclosure (e.g., expansion referred to as REP, including the process shown in step D of FIG. 1). 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). Nonspecific T cell receptor stimulation can include, for example, an anti-CD3 antibody, such as about 30 ng / mL OKT3, a mouse monoclonal anti-CD3 antibody (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 antigens during the second expansion, including an antigenic portion, such as an epitope(s) of the cancer, which can be expressed from a vector, e.g., a human leukocyte antigen A2 (HLA-A2)-binding peptide, e.g., 0.3 μM MART-1:26-35(27L) or gpl 00:209-217(210M), optionally in the presence of a T cell growth factor, such as 300 IU / mL IL-2 or IL-15. Other suitable antigens can include, e.g., NY-ESO-1, TRP-1, TRP-2, tyrosinase cancer antigen, MAGE-A3, SSX-2, and VEGFR2, or antigenic portions thereof. TILs can also be rapidly expanded by restimulating with the same antigen(s) of the cancer pulsed onto antigen-presenting cells expressing HLA-A2. Alternatively, the TILs can be further restimulated, for example, with irradiated autologous lymphocytes or irradiated HLA-A2+ allogeneic lymphocytes and IL-2. In some embodiments, the restimulation occurs as part of a second expansion. In some embodiments, the second expansion occurs in the presence of irradiated autologous lymphocytes or with irradiated HLA-A2+ allogeneic lymphocytes and IL-2.

[0519] In some embodiments, the cell culture medium further comprises IL-2. In some embodiments, the cell culture medium comprises about 3000 IU / mL of IL-2. In some embodiments, the cell culture medium comprises about 1000 IU / mL, about 1500 IU / mL, about 2000 IU / mL, about 2500 IU / mL, about 3000 IU / mL, about 3500 IU / mL, about 4000 IU / mL, about 4500 IU / mL, about 5000 IU / mL, about 5500 IU / mL, about 6000 IU / mL, about 6500 IU / mL, about 7000 IU / mL, about 7500 IU / mL, or about 8000 IU / mL of IL-2. In some embodiments, the cell culture medium contains 1000-2000 IU / mL, 2000-3000 IU / mL, 3000-4000 IU / mL, 4000-5000 IU / mL, 5000-6000 IU / mL, 6000-7000 IU / mL, 7000-8000 IU / mL, or 8000 IU / mL of IL-2.

[0520] In some embodiments, the cell culture medium comprises an OKT-3 antibody. In some embodiments, the cell culture medium comprises about 30 ng / mL of the OKT-3 antibody. In some embodiments, the cell culture medium comprises about 0.1 ng / mL, about 0.5 ng / mL, about 1 ng / mL, about 2.5 ng / mL, about 5 ng / mL, about 7.5 ng / mL, about 10 ng / mL, about 15 ng / mL, about 20 ng / mL, about 25 ng / mL, about 30 ng / mL, about 35 ng / mL, about 40 ng / mL, about 50 ng / mL, about 60 ng / mL, about 70 ng / mL, about 80 ng / mL, about 90 ng / mL, about 100 ng / mL, about 200 ng / mL, about 500 ng / mL, or about 1 μg / mL of the OKT-3 antibody. In some embodiments, the cell culture medium comprises 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, or 50 ng / mL to 100 ng / mL of OKT-3 antibody. In some embodiments, the cell culture medium does not contain OKT-3 antibody. In some embodiments, the OKT-3 antibody is muromonab.

[0521] In some embodiments, the cell culture medium comprises one or more TNFRSF agonists in the cell culture medium. In some embodiments, the TNFRSF agonist comprises a 4-1BB agonist. In some embodiments, the TNFRSF agonist is a 4-1BB agonist, and the 4-1BB agonist is selected from the group consisting of urelumab, utomilumab, EU-101, fusion proteins, and fragments, derivatives, variants, biosimilars, and combinations thereof. In some embodiments, the TNFRSF agonist is added at a concentration sufficient to achieve a cell culture medium concentration of 0.1 μg / mL to 100 μg / mL. In some embodiments, the TNFRSF agonist is added at a concentration sufficient to achieve a cell culture medium concentration of 20 μg / mL to 40 μg / mL.

[0522] In some embodiments, in addition to the one or more TNFRSF agonists, the cell culture medium further comprises IL-2 at an initial concentration of about 3000 IU / mL and OKT-3 at an initial concentration of about 30 ng / mL, and the one or more TNFRSF agonists comprise a 4-1BB agonist.

[0523] In some embodiments, a combination of IL-2, IL-7, IL-15, and / or IL-21 is used as a combination during the second expansion. In some embodiments, IL-2, IL-7, IL-15, and / or IL-21, and any combination thereof, may be included during the second expansion, including, for example, during step D of the process according to FIG. 1 and described herein. In some embodiments, a combination of IL-2, IL-15, and IL-21 is used as a combination during the second expansion. In some embodiments, IL-2, IL-15, and IL-21, and any combination thereof, may be included during the process according to FIG. 1 and step D of the process described herein.

[0524] In some embodiments, the second expansion may be performed in a supplemented cell culture medium comprising IL-2, OKT-3, antigen-presenting feeder cells, and optionally a TNFRSF agonist. In some embodiments, the second expansion occurs in a supplemented cell culture medium. In some embodiments, the supplemented cell culture medium comprises IL-2, OKT-3, and antigen-presenting feeder cells. In some embodiments, the second cell culture medium comprises IL-2, OKT-3, and antigen-presenting cells (APCs, also referred to as antigen-presenting feeder cells). In some embodiments, the second expansion occurs in a cell culture medium comprising IL-2, OKT-3, and antigen-presenting feeder cells (i.e., antigen-presenting cells).

[0525] In some embodiments, the second expansion culture medium contains about 500 IU / mL IL-15, about 400 IU / mL IL-15, about 300 IU / mL IL-15, about 200 IU / mL IL-15, about 180 IU / mL IL-15, about 160 IU / mL IL-15, about 140 IU / mL IL-15, about 120 IU / mL IL-15, or about 100 IU / mL IL-15. In some embodiments, the second expansion culture medium contains about 500 IU / mL to about 100 IU / mL IL-15. In some embodiments, the second expansion culture medium contains about 400 IU / mL to about 100 IU / mL IL-15. In some embodiments, the second expansion culture medium comprises about 300 IU / mL to about 100 IU / mL of IL-15. In some embodiments, the second expansion culture medium comprises about 200 IU / mL of IL-15. In some embodiments, the cell culture medium comprises about 180 IU / mL of IL-15. In some embodiments, the cell culture medium further comprises IL-15. In some embodiments, the cell culture medium comprises about 180 IU / mL of IL-15.

[0526] In some embodiments, the second expansion culture medium comprises about 20 IU / mL of IL-21, about 15 IU / mL of IL-21, about 12 IU / mL of IL-21, about 10 IU / mL of IL-21, about 5 IU / mL of IL-21, about 4 IU / mL of IL-21, about 3 IU / mL of IL-21, about 2 IU / mL of IL-21, about 1 IU / mL of IL-21, or about 0.5 IU / mL of IL-21. In some embodiments, the second expansion culture medium comprises about 20 IU / mL to about 0.5 IU / mL of IL-21. In some embodiments, the second expansion culture medium comprises about 15 IU / mL to about 0.5 IU / mL of IL-21. In some embodiments, the second expansion culture medium comprises about 12 IU / mL to about 0.5 IU / mL of IL-21. In some embodiments, the second expansion culture medium comprises about 10 IU / mL to about 0.5 IU / mL of IL-21. In some embodiments, the second expansion culture medium comprises about 5 IU / mL to about 1 IU / mL of IL-21. In some embodiments, the second expansion culture medium comprises about 2 IU / mL of IL-21. In some embodiments, the cell culture medium comprises about 1 IU / mL of IL-21. In some embodiments, the cell culture medium comprises about 0.5 IU / mL of IL-21. In some embodiments, the cell culture medium further comprises IL-21. In some embodiments, the cell culture medium comprises about 1 IU / mL of IL-21.

[0527] In some embodiments, the antigen-presenting feeder cells (APCs) are PBMCs. In some embodiments, the ratio of TILs to PBMCs and / or antigen-presenting cells in the rapid expansion and / or secondary expansion is about 1:25, about 1:50, about 1:100, about 1:125, about 1:150, about 1:175, about 1:200, about 1:225, about 1:250, about 1:275, about 1:300, about 1:325, about 1:350, about 1:375, about 1:400, or about 1:500. In some embodiments, the ratio of TILs to PBMCs in the rapid expansion and / or secondary expansion is 1:50 to 1:300. In some embodiments, the ratio of TILs to PBMCs in the rapid expansion and / or secondary expansion is 1:100 to 1:200.

[0528] In some embodiments, REP and / or secondary expansion is performed in flasks in which bulk TILs are mixed with a 100-fold or 200-fold excess of inactivated feeder cells, 30 mg / mL OKT3 anti-CD3 antibody, and 3000 IU / mL IL-2 in 150 mL of medium. Medium changes are performed (typically a two-thirds medium change by breathing in fresh medium) until the cells are transferred to an alternative growth chamber. Alternative growth chambers include G-REX flasks and gas-permeable vessels, as discussed more fully below.

[0529] In some embodiments, as discussed in the Examples and Figures, the second expansion (which may include a process referred to as the REP process) is shortened to 7-14 days, hi some embodiments, the second expansion is shortened to 11 days.

[0530] In some embodiments, REP and / or second expansion may be performed using previously described T-175 flasks and gas-permeable bags (Tran, et al., J. Immunother. 2008, 31, 742-51; Dudley, et al., J. Immunother. 2003, 26, 332-42) or gas-permeable cultureware (e.g., G-REX flasks). In some embodiments, second expansion (including expansion referred to as rapid expansion) is performed in a T-175 flask, containing approximately 1 x 10 cells suspended in 150 mL of medium. 6 TILs can be added to each T-175 flask. TILs can be cultured in a 1:1 mixture of CM and AIM-V medium supplemented with 3000 IU / mL IL-2 and 30 ng / mL anti-CD3. T-175 flasks can be incubated at 37°C in 5% CO2. Half of the medium can be replaced on day 5 using 50 / 50 medium containing 3000 IU / mL IL-2. In some embodiments, on day 7, cells from two T-175 flasks can be combined in a 3L bag, and 300 mL of AIM-V containing 5% human AB serum and 3000 IU / mL IL-2 was added to the 300 mL TIL suspension. The number of cells in each bag can be counted daily or every other day, and fresh medium can be added to increase the number of cells to 0.5-2.0 x 10. 6 The cell count was maintained at cells / mL.

[0531] In some embodiments, the second expansion (which may include the expansion referred to as REP, as well as the expansion referred to in step D of FIG. 1) may be performed in a 500 mL gas-permeable flask with a 100 cm gas-permeable silicon bottom (G-REX-100, commercially available from Wilson Wolf Manufacturing Corporation, New Brighton, MN, USA). 6 or 10 x 10 6TILs can be cultured with PBMCs in 400 mL of 50 / 50 medium supplemented with 5% human AB serum, 3000 IU / mL IL-2, and 30 ng / mL anti-CD3 (OKT3). The G-REX-100 flask can be incubated at 37°C in 5% CO2. On day 5, 250 mL of supernatant can be removed, placed in a centrifuge bottle, and centrifuged at 1500 rpm (491 x g) for 10 minutes. The TIL pellet can be resuspended in 150 mL of fresh medium containing 5% human AB serum, 3000 IU / mL IL-2, and added back to the original G-REX-100 flask. If TILs are continuously expanded in G-REX-100 flasks, on day 7, the TILs in each G-REX-100 flask may be suspended in 300 mL of medium present in each flask, and the cell suspension may be divided into three 100 mL aliquots, which may be used to seed three G-REX-100 flasks. Then, 150 mL of AIM-V containing 5% human AB serum and 3000 IU / mL of IL-2 may be added to each flask. The G-REX-100 flasks may be incubated at 37°C in 5% CO2, and after 4 days, 150 mL of AIM-V containing 3000 IU / mL of IL-2 may be added to each G-REX-100 flask. On day 14 of culture, the cells may be harvested.

[0532] In some embodiments, the second expansion (including expansion referred to as REP) is performed in flasks where bulk TILs are mixed with a 100-fold or 200-fold excess of inactivated feeder cells, 30 mg / mL OKT3 anti-CD3 antibody, and 3000 IU / mL IL-2 in 150 mL of medium. In some embodiments, medium changes are performed until the cells are transferred to an alternative growth chamber. In some embodiments, two-thirds of the medium is exchanged with fresh medium by respiration. In some embodiments, alternative growth chambers include G-REX flasks and gas-permeable vessels, as discussed more fully below.

[0533] In some embodiments, a second expansion (including expansion referred to as REP) is performed, further comprising selecting TILs for superior tumor reactivity. Any selection method known in the art may be used. For example, the method described in U.S. Patent Application Publication No. 2016 / 0010058A1 (the disclosure of which is incorporated herein by reference) may be used to select TILs for superior tumor reactivity.

[0534] Optionally, a cell viability assay can be performed after the second expansion (including expansion referred to as REP expansion) using standard assays known in the art. For example, a trypan blue exclusion assay, which selectively labels dead cells and allows for assessment of viability, can be performed on a sample of bulk TILs. In some embodiments, TIL samples can be counted and viability determined using a Cellometer K2 Automatic Cell Counter (Nexcelom Bioscience, Lawrence, MA). In some embodiments, viability is determined according to a standard Cellometer K2 Image Cytometer Automatic Cell Counter protocol.

[0535] In some embodiments, secondary expansion of TILs (including expansion referred to as REP) can be performed using T-175 flasks and gas-permeable bags as previously described (Tran, et al., 2008, J Immunother., 31:742-751 and Dudley, et al. 2003, J Immunother., 26:332-342) or gas-permeable G-REX flasks. In some embodiments, secondary expansion is performed using flasks. In some embodiments, secondary expansion is performed using gas-permeable G-REX flasks. In some embodiments, secondary expansion is performed in T-175 flasks and can reach approximately 1 x 10 cells. 6TILs are suspended in approximately 150 mL of medium, which is added to each T-175 flask. The TILs are cultured with irradiated (50 Gy) allogeneic PBMCs at a 1:100 ratio as "feeder" cells, and the cells are cultured in a 1:1 mixture of CM and AIM-V medium (50 / 50 medium) supplemented with 3000 IU / mL IL-2 and 30 ng / mL anti-CD3. The T-175 flasks are incubated at 37°C in 5% CO. In some embodiments, half of the medium is replaced on day 5 using 50 / 50 medium containing 3000 IU / mL 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 IL-2 is added to the 300 mL TIL suspension. The number of cells in each bag can be counted daily or every other day, and fresh medium can be added to obtain a cell count of about 0.5 to about 2.0 x 10 6 The cell count can be maintained at 100 cells / mL.

[0536] In some embodiments, the second expansion (including the expansion referred to as REP) is 100 cm 2 The experiment was carried out in a 500 mL flask with a gas-permeable silicon bottom (G-REX-100, Wilson Wolf), and approximately 5 × 10 6 or 10 x 10 6TILs are cultured with irradiated allogeneic PBMCs at a 1:100 ratio in 400 mL of 50 / 50 medium supplemented with 3000 IU / mL IL-2 and 30 ng / mL anti-CD3. The G-REX-100 flask is incubated at 37°C in 5% CO2. In some embodiments, on day 5, 250 mL of supernatant is removed, placed in a centrifuge bottle, and centrifuged at 1500 rpm (491 g) for 10 minutes. The TIL pellet can then be resuspended in 150 mL of fresh 50 / 50 medium containing 3000 IU / mL IL-2 and added back to the original G-REX-100 flask. In an embodiment in which TILs are serially expanded in G-REX-100 flasks, on day 7, the TILs in each G-REX-100 flask are suspended in 300 mL of medium present in each flask, and the cell suspension is divided into three 100 mL aliquots, which are used to seed three G-REX-100 flasks. Then, 150 mL of AIM-V containing 5% human AB serum and 3000 IU / mL of IL-2 is added to each flask. The G-REX-100 flasks are incubated at 37°C in 5% CO2, and after four days, 150 mL of AIM-V containing 3000 IU / mL of IL-2 is added to each G-REX-100 flask. On day 14 of culture, the cells are harvested.

[0537] Diverse antigen receptors for T and B lymphocytes are generated by somatic recombination of a limited number of gene segments. These gene segments: V (variable), D (diversity), J (joining), and C (constant) determine the binding specificity and downstream applications of immunoglobulins and T cell receptors (TCRs). The present invention provides methods for generating TILs that exhibit and increase T cell repertoire diversity. In some embodiments, TILs obtained by the methods exhibit increased T cell repertoire diversity. In some embodiments, TILs obtained in the second expansion exhibit increased T cell repertoire diversity. In some embodiments, the increased diversity is increased immunoglobulin diversity and / or T cell receptor diversity. In some embodiments, the diversity is in immunoglobulins and in immunoglobulin heavy chains. In some embodiments, the diversity is in immunoglobulins and in immunoglobulin light chains. In some embodiments, the diversity is in T cell receptors. In some embodiments, the diversity is in one of the T cell receptors selected from the group consisting of alpha, beta, gamma, and delta receptors. In some embodiments, expression of T cell receptor (TCR) alpha and / or beta is increased. In some embodiments, expression of T cell receptor (TCR) alpha is increased. In some embodiments, expression of T cell receptor (TCR) beta is increased. In some embodiments, expression of TCRab (i.e., TCRα / β) is increased.

[0538] In some embodiments, the second expansion culture medium (e.g., also referred to as CM2 or second cell culture medium) comprises IL-2, OKT-3, and antigen-presenting feeder cells (APCs), as discussed in further detail below.

[0539] In some embodiments, the second expansion, e.g., step D according to FIG. 1, is performed in a closed system bioreactor. In some embodiments, a closed system is used for TIL expansion as described herein. In some embodiments, a single bioreactor is used. In some embodiments, the single bioreactor used is, for example, a G-REX-10 or G-REX-100. In some embodiments, the closed system bioreactor is a single bioreactor.

[0540] In some embodiments, the rapid or secondary expansion step is divided into multiple steps, and the scale-up of the culture is achieved by (a) culturing the TILs in a small-scale culture in a first vessel, e.g., a G-REX-100MCS vessel, for about 3-7 days, thereby performing the rapid or secondary expansion; and then (b) transferring the TILs in the small-scale culture to a second vessel, e.g., a G-REX-500-MCS vessel, that is larger than the first vessel, and culturing the TILs from the small-scale culture in a larger culture in the second vessel for about 4-7 days.

[0541] In some embodiments, the rapid or second expansion step is divided into multiple steps, and the rapid or second expansion step is achieved by (a) culturing the TILs in a first small-scale culture in a first container, e.g., a G-REX-100MCS container, for about 3-7 days, and then (b) transferring and distributing the TILs from the first small-scale culture into at least 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, or 20 second containers equal in size to the first container, whereby a portion of the TILs from the first small-scale culture transferred to each second container are cultured in a second small-scale culture for about 4-7 days.

[0542] In some embodiments, the first small-scale TIL culture is distributed into a plurality of about 2-5 subpopulations of TILs.

[0543] In some embodiments, the rapid or secondary expansion step is divided into multiple steps, and the scale-out and scale-up of the culture is achieved by (a) culturing the TILs in a small-scale culture in a first vessel, e.g., a G-REX-100MCS vessel, for about 3-7 days, and then (b) transferring and distributing the TILs from the small-scale culture into at least 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, or 20 second vessels, e.g., a G-REX-500MCS vessel, that are larger in size than the first vessel, and culturing a portion of the TILs from the small-scale culture transferred to each second vessel in a larger culture for about 4-7 days.

[0544] In some embodiments, the rapid or secondary expansion step is divided into multiple steps, and the rapid or secondary expansion step is performed by (a) culturing the TILs in a small-scale culture in a first vessel, e.g., a G-REX-100MCS vessel, for about 5 days, and then (b) transferring and distributing the TILs from the small-scale culture into two, three, or four second vessels, e.g., a G-REX-500MCS vessel, that are larger in size than the first vessel, thereby achieving scale-out and scale-up of the culture, and in each second vessel, a portion of the TILs from the small-scale culture transferred to such second vessel are cultured in a larger culture for about 6 days.

[0545] In some embodiments, upon splitting of the rapid or second expansion, each second container contains at least 10 8 In some embodiments, upon rapid or second expansion split, each second vessel contains at least 10 TILs. 8 TIL, at least 10 9 TIL, or at least 10 10 In one exemplary embodiment, each second container contains at least 10 TILs. 10 Contains TILs.

[0546] In some embodiments, the first small-scale TIL culture is allocated into a plurality of subpopulations. In some embodiments, the first small-scale TIL culture is allocated into a plurality of about 2-5 subpopulations. In some embodiments, the first small-scale TIL culture is allocated into a plurality of about 2, 3, 4, or 5 subpopulations.

[0547] In some embodiments, after completion of rapid or secondary expansion, the plurality of subpopulations comprises a therapeutically effective amount of TILs. In some embodiments, after completion of rapid or secondary expansion, one or more subpopulations of TILs are pooled together to generate a therapeutically effective amount of TILs. In some embodiments, after completion of rapid expansion, each subpopulation of TILs comprises a therapeutically effective amount of TILs.

[0548] In some embodiments, rapid or secondary expansion is carried out for about 3-7 days before being split into multiple steps. In some embodiments, the splitting of rapid or secondary expansion occurs on about day 3, day 4, day 5, day 6, or day 7 after the initiation of rapid or secondary expansion.

[0549] In some embodiments, the rapid or secondary expansion division occurs at about day 7, 8, 9, 10, 11, 12, 13, 14, 15, or 16, 17, or 18 after the initiation of the first expansion (i.e., pre-REP expansion). In one exemplary embodiment, the rapid or secondary expansion division occurs at about day 16 after the initiation of the first expansion.

[0550] In some embodiments, the rapid secondary expansion is carried out for about 7-11 days after division, hi some embodiments, the rapid or secondary expansion is carried out for about 5, 6, 7, 8, 9, 10, or 11 days after division.

[0551] In some embodiments, the cell culture medium used for rapid or secondary expansion before division contains the same components as the cell culture medium used for rapid or secondary expansion after division. In some embodiments, the cell culture medium used for rapid or secondary expansion before division contains different components than the cell culture medium used for rapid or secondary expansion after division.

[0552] In some embodiments, the cell culture medium used for rapid or secondary expansion before division comprises IL-2, optionally OKT-3, and further optionally APC. In some embodiments, the cell culture medium used for rapid or secondary expansion before division comprises IL-2, OKT-3, and further optionally APC. In some embodiments, the cell culture medium used for rapid or secondary expansion before division comprises IL-2, OKT-3, and further optionally APC.

[0553] In some embodiments, cell culture medium used for rapid or second expansion before division is produced by supplementing cell culture medium during first expansion with fresh culture medium comprising IL-2, optionally OKT-3, and further optionally APCs. In some embodiments, cell culture medium used for rapid or second expansion before division is produced by supplementing cell culture medium during first expansion with fresh culture medium comprising IL-2, OKT-3, and APCs. In some embodiments, cell culture medium used for rapid or second expansion before division is produced by replacing cell culture medium during first expansion with fresh culture medium comprising IL-2, optionally OKT-3, and further optionally APCs. In some embodiments, cell culture medium used for rapid or second expansion before division is produced by replacing cell culture medium during first expansion with fresh cell culture medium comprising IL-2, OKT-3, and further optionally APCs.

[0554] In some embodiments, the cell culture medium used for rapid or second expansion after division comprises IL-2, and optionally OKT-3. In some embodiments, the cell culture medium used for rapid or second expansion after division comprises IL-2 and OKT-3. In some embodiments, the cell culture medium used for rapid or second expansion after division is generated by replacing the cell culture medium used for rapid or second expansion before division with fresh culture medium comprising IL-2 and optionally OKT-3. In some embodiments, the cell culture medium used for rapid or second expansion after division is generated by replacing the cell culture medium used for rapid or second expansion before division with fresh culture medium comprising IL-2 and OKT-3.

[0555] In some embodiments, the rapid expansion disruption occurs in a closed system.

[0556] In some embodiments, scaling up TIL cultures during rapid or secondary expansion involves adding fresh cell culture medium to the TIL culture (also referred to as feeding the TILs). In some embodiments, feeding involves frequently adding fresh cell culture medium to the TIL culture. In some embodiments, feeding involves adding fresh cell culture medium to the TIL culture at regular intervals. In some embodiments, fresh cell culture medium is supplied to the TILs via a constant flow. In some embodiments, an automated cell expansion system, such as the Xuri W25, is used for rapid expansion and feeding.

[0557] 1. Feeder cells and antigen-presenting cells In some embodiments, the second expansion procedures described herein (including, for example, expansion as described in step D of FIG. 1 and referred to as REP) require excess feeder cells during REP TIL expansion and / or during second expansion. In many embodiments, the feeder cells are peripheral blood mononuclear cells (PBMCs) obtained from a standard whole blood unit from a healthy blood donor. PBMCs are obtained using standard methods, such as Ficoll-Paque gradient separation.

[0558] Generally, allogeneic PBMCs are inactivated by either irradiation or heat treatment and used in the REP procedure, as described in the Examples, which provide an exemplary protocol for assessing the replicative incompetence of irradiated allogeneic PBMCs.

[0559] In some embodiments, if the total viable cell count on day 14 is less than the initial viable cell count placed in culture on day 0 of REP and / or day 0 of secondary expansion (i.e., the start day of secondary expansion), the PBMCs are considered replication-incompetent and are approved for use in the TIL expansion procedures described herein.

[0560] In some embodiments, if the total viable cell count cultured in the presence of OKT3 and IL-2 on days 7 and 14 does not increase from the initial viable cell count placed in culture on REP day 0 and / or secondary expansion day 0 (i.e., the start day of secondary expansion), the PBMCs are considered replication-incompetent and are approved for use in the TIL expansion procedures described herein. In some embodiments, PBMCs are cultured in the presence of 30 ng / mL OKT3 antibody and 3000 IU / mL IL-2.

[0561] In some embodiments, if the total viable cell counts cultured in the presence of OKT3 and IL-2 on days 7 and 14 do not increase from the initial viable cell counts placed in culture on day 0 of REP and / or day 0 of secondary expansion (i.e., the start day of secondary expansion), PBMCs are considered replication-incompetent and are approved for use in the TIL expansion procedures described herein. In some embodiments, PBMCs are cultured in the presence of 5-60 ng / mL OKT3 antibody and 1000-6000 IU / mL IL-2. In some embodiments, PBMCs are cultured in the presence of 10-50 ng / mL OKT3 antibody and 2000-5000 IU / mL IL-2. In some embodiments, PBMCs are cultured in the presence of 20-40 ng / mL OKT3 antibody and 2000-4000 IU / mL IL-2. In some embodiments, PBMCs are cultured in the presence of 25 to 35 ng / mL of OKT3 antibody and 2500 to 3500 IU / mL of IL-2.

[0562] 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 is about 1:25, about 1:50, about 1:100, about 1:125, about 1:150, about 1:175, about 1:200, about 1:225, about 1:250, about 1:275, about 1:300, about 1:325, about 1:350, about 1:375, about 1:400, or about 1:500. In some embodiments, the ratio of TILs to antigen-presenting feeder cells in the second expansion is 1:50 to 1:300. In some embodiments, the ratio of TILs to antigen-presenting feeder cells in the second expansion is 1:100 to 1:200.

[0563] In some embodiments, the second expansion procedure described herein comprises approximately 2.5 x 10 9 Feeder cells: approximately 100 x 10 6 In other embodiments, the second expansion procedure described herein requires a ratio of about 2.5 x 10 TILs. 9Feeder cells: approximately 50 x 10 6 In yet other embodiments, the second expansion procedure described herein requires a ratio of about 2.5 x 10 TILs. 9 Feeder cells: approximately 25 x 10 6 TILs are required.

[0564] In some embodiments, the second expansion procedure described herein requires excess feeder cells during the second expansion. In many embodiments, the feeder cells are peripheral blood mononuclear cells (PBMCs) obtained from a standard whole blood unit from a healthy blood donor. PBMCs are obtained using standard methods, such as Ficoll-Paque gradient separation. In some embodiments, artificial antigen-presenting (aAPC) cells are used in place of PBMCs.

[0565] Generally, allogeneic PBMCs are inactivated by either irradiation or heat treatment and used in the TIL expansion procedures described herein, including the exemplary procedures described in the figures and examples.

[0566] In some embodiments, artificial antigen-presenting cells are used in the second expansion as a replacement for or in combination with PBMCs.

[0567] 2. Cytokines and other additives The expansion methods described herein generally use culture media containing high doses of cytokines, particularly IL-2, as known in the art.

[0568] Alternatively, the use of combinations of cytokines for rapid and / or secondary expansion of TILs is additionally possible using combinations of two or more of IL-2, IL-15, and IL-21, as described in U.S. Patent Application Publication No. US2017 / 0107490A1 (the disclosure of which is incorporated herein by reference). Thus, possible combinations include IL-2 and IL-15, IL-2 and IL-21, IL-15 and IL-21, and IL-2, IL-15, and IL-21, the latter being particularly useful in many embodiments. The use of combinations of cytokines is particularly advantageous for the generation of lymphocytes, particularly T cells as described therein.

[0569] In some embodiments, step D may also include the addition of an OKT-3 antibody or muromonab to the culture medium, as described elsewhere herein. In some embodiments, step D may also include the addition of a 4-1BB agonist to the culture medium, as described elsewhere herein. In some embodiments, step D may also include the addition of an OX-40 agonist to the culture medium, as described elsewhere herein. In addition, additives such as peroxisome proliferator-activated receptor gamma coactivator I-alpha agonists, including proliferator-activated receptor (PPAR)-gamma agonists such as thiazolidinedione compounds, may be used in the culture medium during step D, as described in U.S. Patent Application Publication No. US2019 / 0307796A1 (the disclosure of which is incorporated herein by reference).

[0570] E. Step E: Harvesting TILs After the second expansion step, the cells may be harvested. In some embodiments, the TILs are harvested after one, two, three, four, or more expansion steps, for example, as provided in Figure 1. In some embodiments, the TILs are harvested after two expansion steps, for example, as provided in Figure 1.

[0571] TIL can be collected in any suitable sterilization manner, for example, by centrifugation.The method for collecting TIL is well known in the art, and any such known method can be used in this process.In some embodiments, TIL is collected using an automated system.

[0572] Cell harvesters and / or cell processing systems are commercially available from a variety of sources, including, for example, Fresenius Kabi, Tomtec Life Science, Perkin Elmer, and Inotech Biosystems International, Inc. Any cell-based harvester can be used with the present methods. In some embodiments, the cell harvester and / or cell processing system is a membrane-based cell harvester. In some embodiments, cell harvesting is by a cell processing system such as the LOVO system (manufactured by Fresenius Kabi). The term "LOVO cell processing system" also refers to any device or apparatus manufactured by any vendor that pumps a cell-containing solution through a membrane or filter, such as a spinning membrane or spinning filter, in a sterile and / or closed system environment, allowing continuous flow, processes the cells, and removes the supernatant or cell culture medium without pelleting. In some embodiments, the cell harvester and / or cell processing system can perform cell separation, washing, fluid exchange, concentration, and / or other cell processing steps within a sterile, closed system.

[0573] In some embodiments, harvesting, e.g., step E according to FIG. 1, is performed from a closed system bioreactor. In some embodiments, a closed system is used for TIL expansion as described herein. In some embodiments, a single bioreactor is used. In some embodiments, the single bioreactor used is, for example, a G-REX-10 or G-REX-100. In some embodiments, the closed system bioreactor is a single bioreactor.

[0574] In some embodiments, step E according to Figure 1 is performed according to a process described herein. In some embodiments, the closed system is accessed via a syringe under sterile conditions to maintain the sterility and closure of the closed system. In some embodiments, the closed system described in the Examples is used.

[0575] In some embodiments, TILs are harvested according to the methods described in the Examples. In some embodiments, TILs between days 1 and 11 are harvested using the methods described in the steps referred to herein, such as harvesting TILs on day 11 in the Examples. In some embodiments, TILs between days 12 and 24 are harvested using the methods described in the steps referred to herein, such as harvesting TILs on day 22 in the Examples. In some embodiments, TILs between days 12 and 22 are harvested using the methods described in the steps referred to herein, such as harvesting TILs on day 22 in the Examples.

[0576] F. Step F: Final Blend and Transfer to Injection Vessel After completing steps A-E, provided in exemplary order in Figure 1 and outlined in detail above and herein, the cells are transferred to a container for use in patient administration, such as an infusion bag or sterile vial. In some embodiments, once therapeutically sufficient numbers of TILs are obtained using the expansion methods described above, they are transferred to a container for use in patient administration.

[0577] In some embodiments, TILs expanded using the APCs of the present disclosure are administered to a patient as a pharmaceutical composition. In some embodiments, the pharmaceutical composition is a TIL suspension in a sterile buffer. TILs expanded using the PBMCs of the present disclosure can be administered by any suitable route known in the art. In some embodiments, T cells are administered as a single intra-arterial or intravenous infusion, preferably lasting approximately 30-60 minutes. Other suitable routes of administration include intraperitoneal, intrathecal, and intralymphatic administration.

[0578] III. Gen3 TIL Manufacturing Process Without being limited to any particular theory, it is believed that the first expansion by priming, which primes T cells for activation, followed by a rapid second expansion that promotes T cell activation, as described in the methods of the present invention, allows for the preparation of expanded T cells that retain a "younger" phenotype, and therefore, it is expected that the expanded T cells of the present invention will exhibit greater cytotoxicity against cancer cells than T cells expanded by other methods. In particular, it is believed that the activation of T cells primed by exposure to an anti-CD3 antibody (e.g., OKT-3), IL-2, and optionally antigen-presenting cells (APCs), followed by subsequent exposure to additional anti-CD3 antibody (e.g., OKT-3), IL-2, and APCs, as taught by the methods of the present invention, limits or avoids T cell maturation in culture, generating a T cell population with a less mature phenotype that is less exhausted by expansion in culture and exhibits greater cytotoxicity against cancer cells. In some embodiments, the rapid second expansion step is divided into multiple steps, and the scale-up of the culture is achieved by (a) performing a rapid second expansion by culturing T cells in a small-scale culture in a first vessel, e.g., a G-REX-100MCS vessel, for about 3-4 days, and then (b) transferring the T cells in the small-scale culture to a second vessel, e.g., a G-REX-500MCS vessel, that is larger than the first vessel, and culturing the T cells from the small-scale culture in a larger culture in the second vessel for about 4-7 days. In some embodiments, the rapid expansion step is divided into multiple steps to achieve scale-out of the culture by (a) performing a rapid second expansion by culturing T cells in a first small-scale culture in a first container, e.g., a G-REX-100MCS container, for 3-4 days, and then (b) transferring and distributing T cells from the first small-scale culture into at least 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, or 20 second containers equal in size to the first container, wherein a portion of the T cells from the first small-scale culture transferred to such second container are cultured in a second small-scale culture for about 4-7 days.In some embodiments, the rapid expansion step is divided into multiple steps, and the scale-out and scale-up of the culture is achieved by (a) performing a rapid secondary expansion by culturing T cells in a small-scale culture in a first vessel, e.g., a G-REX-100MCS vessel, for about 3-4 days, and then (b) transferring and distributing the T cells from the small-scale culture into at least 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, or 20 second vessels, e.g., G-REX-500MCS vessels, that are larger in size than the first vessel, wherein a portion of the T cells from the small-scale culture transferred to each second vessel are cultured in a larger culture for about 4-7 days. In some embodiments, the rapid expansion step is divided into multiple steps, and the scale-out and scale-up of the culture is achieved by (a) performing a rapid secondary expansion by culturing T cells in a small-scale culture in a first vessel, e.g., a G-REX-100MCS vessel, for about 4 days, and then (b) transferring and distributing the T cells from the small-scale culture into two, three, or four second vessels, e.g., a G-REX-500MCS vessel, that are larger in size than the first vessel, and culturing a portion of the T cells from the small-scale culture transferred to each second vessel in a larger culture for about 5 days.

[0579] In some embodiments, upon division of the rapid expansion, each second container contains at least 10 8 In some embodiments, upon splitting of the rapid expansion, each second vessel contains at least 10 TILs. 8 TIL, at least 10 9 TIL, or at least 10 10 In one exemplary embodiment, each second container contains at least 10 TILs. 10 Contains TILs.

[0580] In some embodiments, the first small-scale TIL culture is allocated into a plurality of subpopulations. In some embodiments, the first small-scale TIL culture is allocated into a plurality of about 2-5 subpopulations. In some embodiments, the first small-scale TIL culture is allocated into a plurality of about 2, 3, 4, or 5 subpopulations.

[0581] In some embodiments, after completion of rapid expansion, the plurality of subpopulations comprises a therapeutically effective amount of TILs. In some embodiments, after completion of rapid expansion, one or more subpopulations of TILs are pooled together to generate a therapeutically effective amount of TILs. In some embodiments, after completion of rapid expansion, each subpopulation of TILs comprises a therapeutically effective amount of TILs.

[0582] In some embodiments, rapid expansion is carried out for about 1-5 days before being divided into multiple steps, hi some embodiments, the division of rapid expansion occurs on about day 1, day 2, day 3, day 4, or day 5 after the initiation of rapid expansion.

[0583] In some embodiments, the rapid expansion split occurs at about day 8, 9, 10, 11, 12, or 13 after the initiation of the primary expansion (i.e., pre-REP expansion). In one exemplary embodiment, the rapid expansion split occurs at about day 10 after the initiation of the primary expansion by priming. In another exemplary embodiment, the rapid expansion split occurs at about day 11 after the initiation of the primary expansion.

[0584] In some embodiments, rapid expansion is carried out for an additional 4-11 days after division, hi some embodiments, rapid expansion is carried out for an additional 3, 4, 5, 6, 7, 8, 9, 10, or 11 days after division.

[0585] In some embodiments, the cell culture medium used for pre-mitotic rapid expansion comprises the same components as the cell culture medium used for post-mitotic rapid expansion, hi some embodiments, the cell culture medium used for pre-mitotic rapid expansion comprises different components than the cell culture medium used for post-mitotic rapid expansion.

[0586] In some embodiments, the cell culture medium used for pre-mitotic rapid expansion comprises IL-2, optionally OKT-3, and further optionally APC. In some embodiments, the cell culture medium used for pre-mitotic rapid expansion comprises IL-2, OKT-3, and further optionally APC. In some embodiments, the cell culture medium used for pre-mitotic rapid expansion comprises IL-2, OKT-3, and further optionally APC.

[0587] In some embodiments, the cell culture medium used for pre-division rapid expansion is produced by supplementing the cell culture medium during the first expansion with fresh culture medium comprising IL-2, optionally OKT-3, and further optionally APCs. In some embodiments, the cell culture medium used for pre-division rapid expansion is produced by supplementing the cell culture medium during the first expansion with fresh culture medium comprising IL-2, OKT-3, and APCs. In some embodiments, the cell culture medium used for pre-division rapid expansion is produced by replacing the cell culture medium during the first expansion with fresh culture medium comprising IL-2, optionally OKT-3, and further optionally APCs. In some embodiments, the cell culture medium used for pre-division rapid expansion is produced by replacing the cell culture medium during the first expansion with fresh cell culture medium comprising IL-2, OKT-3, and further optionally APCs.

[0588] In some embodiments, the cell culture medium used for post-division rapid expansion comprises IL-2, and optionally OKT-3. In some embodiments, the cell culture medium used for post-division rapid expansion comprises IL-2 and OKT-3. In some embodiments, the cell culture medium used for post-division rapid expansion is generated by replacing the cell culture medium used for pre-division rapid expansion with fresh culture medium comprising IL-2 and optionally OKT-3. In some embodiments, the cell culture medium used for post-division rapid expansion is generated by replacing the cell culture medium used for pre-division rapid expansion with fresh culture medium comprising IL-2 and OKT-3.

[0589] In some embodiments, the rapid expansion disruption occurs in a closed system.

[0590] In some embodiments, scaling up TIL cultures during rapid expansion involves adding fresh cell culture medium to the TIL culture (also referred to as feeding the TILs). In some embodiments, feeding involves frequently adding fresh cell culture medium to the TIL culture. In some embodiments, feeding involves adding fresh cell culture medium to the TIL culture at regular intervals. In some embodiments, fresh cell culture medium is supplied to the TILs via a constant flow. In some embodiments, an automated cell expansion system, such as the Xuri W25, is used for rapid expansion and feeding.

[0591] In some embodiments, the rapid second expansion occurs after the T cell activation resulting from the first expansion by priming has begun to diminish, reduce, wane, or subside.

[0592] In some embodiments, the rapid second expansion occurs after the activation of T cells resulting from the first expansion by priming has occurred at or about 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44 , 45, 46, 47, 48, 49, 50, 51, 52, 53, 54, 55, 56, 57, 58, 59, 60, 61, 62, 63, 64, 65, 66, 67, 68, 69, 70, 71, 72, 73, 74, 75, 76, 77, 78, 79, 80, 81, 82, 83, 84, 85, 86, 87, 88, 89, 90, 91, 92, 93, 94, 95, 96, 97, 98, 99, or after a 100% reduction.

[0593] In some embodiments, the rapid second expansion is performed after the activation of T cells resulting from the first expansion by priming has decreased by a percentage ranging from exactly or about 1% to 100%.

[0594] In some embodiments, the rapid second expansion is performed after the activation of T cells resulting from the first expansion by priming has decreased by a percentage in the range of exactly or about 1% to 10%, 10% to 20%, 20% to 30%, 30% to 40%, 40% to 50%, 50% to 60%, 60% to 70%, 70% to 80%, 80% to 90%, or 90% to 100%.

[0595] In some embodiments, the rapid second expansion is performed by priming the T cells to a concentration of at least about 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50, 51, 52, 53, 54, 55, 56, 57, 58, 59, 60, 61, 62, 63, 64, 65, 66, 67, 68, 69, 70, 71, 72, 73, 74, 75, 76, 77, 78, 79, 80, 81, 82, 83, 84, 85, 86, 87, 88, 89, 90, 91, 92, 93, 94, 95, 96, 97, 98, 99, 100, 101, 102, 103, 104, 105, 106, 107, 108, 109, 110, 111, 112, 113, 114, 115, 116, 117, 118, 119, 43, 44, 45, 46, 47, 48, 49, 50, 51, 52, 53, 54, 55, 56, 57, 58, 59, 60, 61, 62, 63, 64, 65, 66, 67, 68, 69, 70, 71, 72, 73, 74, 75, 76, 77, 78, 79, 80, 81, 82, 83, 84, 85, 86, 87, 88, 89, 90, 91, 92, 93, 94, 95, 96, 97, 98, or 99% reduction.

[0596] In some embodiments, the rapid second expansion is achieved by priming the activation of T cells resulting from the first expansion, but not by more than about 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50, 51, 52, 53, 54, 55, 56, 57, 58, 59, 60, 61, 62, 63, 64, 65, 66, 67, 68, 69, 70, 71, 72, 73, 74, 75, 76, 77, 78, 79, 80, 81, 82, 83, 84, 85, 86, 87, 88, 89, 90, 91, 92, 93, 94, 95, 96, 97, 98, 99, or after a 100% reduction.

[0597] In some embodiments, the decreased activation of T cells resulting from the first expansion by priming is determined by a decrease in the amount of interferon gamma released by the T cells in response to stimulation with an antigen.

[0598] In some embodiments, the first expansion by priming of T cells occurs over a period of up to or about 7 days or about 8 days.

[0599] In some embodiments, the first expansion by priming of T cells occurs over a period of at most about 1 day, 2 days, 3 days, 4 days, 5 days, 6 days, 7 days, or 8 days.

[0600] In some embodiments, the first expansion by priming of T cells occurs over a period of 1, 2, 3, 4, 5, 6, 7, or 8 days.

[0601] In some embodiments, the rapid secondary expansion of T cells occurs over a period of up to exactly or about 11 days.

[0602] In some embodiments, the rapid secondary expansion of T cells occurs over a period of at most about 1 day, 2 days, 3 days, 4 days, 5 days, 6 days, 7 days, 8 days, 9 days, 10 days, or 11 days.

[0603] In some embodiments, the rapid secondary expansion of T cells occurs over a period of 1 day, 2 days, 3 days, 4 days, 5 days, 6 days, 7 days, 8 days, 9 days, 10 days, or 11 days.

[0604] In some embodiments, the first expansion by priming of T cells occurs over a period of exactly or about 1 day to exactly or about 7 days, and the second rapid expansion of T cells occurs over a period of exactly or about 1 day to exactly or about 11 days.

[0605] In some embodiments, the priming first expansion of T cells occurs over a period of up to exactly or about 1 day, 2 days, 3 days, 4 days, 5 days, 6 days, 7 days, or 8 days, and the rapid second expansion of T cells occurs over a period of up to exactly or about 1 day, 2 days, 3 days, 4 days, 5 days, 6 days, 7 days, 8 days, 9 days, 10 days, or 11 days.

[0606] In some embodiments, the first expansion by priming of T cells occurs over a period of exactly or about 1 day to exactly or about 8 days, and the second rapid expansion of T cells occurs over a period of exactly or about 1 day to exactly or about 9 days.

[0607] In some embodiments, the priming first expansion of T cells occurs over an 8 day period, and the rapid second expansion of T cells occurs over a 9 day period.

[0608] In some embodiments, the first expansion by priming of T cells occurs over a period of exactly or about 1 day to exactly or about 7 days, and the second rapid expansion of T cells occurs over a period of exactly or about 1 day to exactly or about 9 days.

[0609] In some embodiments, the first priming expansion of T cells occurs over a 7 day period, and the second rapid expansion of T cells occurs over a 9 day period.

[0610] In some embodiments, the T cells are tumor infiltrating lymphocytes (TILs).

[0611] In some embodiments, the T cells are bone marrow infiltrating lymphocytes (MIL).

[0612] In some embodiments, the T cells are peripheral blood lymphocytes (PBLs).

[0613] In some embodiments, the T cells are obtained from a donor suffering from cancer.

[0614] In some embodiments, the T cells are TILs obtained from a tumor resected from a patient suffering from cancer.

[0615] In some embodiments, the T cells are MILs obtained from the bone marrow of a patient suffering from a hematological malignancy.

[0616] In some embodiments, the T cells are PBLs obtained from peripheral blood mononuclear cells (PBMCs) from a donor. In some embodiments, the donor is afflicted with cancer. In some embodiments, the cancer is a cancer selected from the group consisting of melanoma, ovarian cancer, endometrial cancer, thyroid 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)), glioblastoma (including GBM), gastrointestinal cancer, renal cancer, and renal cell carcinoma. In some embodiments, the cancer is selected from the group consisting of melanoma, ovarian cancer, endometrial cancer, thyroid 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)), glioblastoma (including GBM), gastrointestinal cancer, renal cancer, and renal cell carcinoma. In some embodiments, the donor is afflicted with a tumor. In some embodiments, the tumor is a liquid tumor. In some embodiments, the tumor is a solid tumor. In some embodiments, the donor is afflicted with a hematological malignancy.

[0617] In certain aspects of the present disclosure, immune effector cells, e.g., T cells, can be obtained from a unit of blood collected from a subject using any number of techniques known to those skilled in the art, such as FICOLL separation. In a preferred aspect, cells from an individual's circulating blood are obtained by apheresis. The apheresis product typically includes lymphocytes, e.g., T cells, monocytes, granulocytes, B cells, other nucleated white blood cells, red blood cells, and platelets. In one aspect, cells collected by apheresis are washed to remove the plasma fraction, and optionally, the cells can be placed in an appropriate buffer or medium for subsequent processing steps. In some embodiments, the cells are washed with phosphate-buffered saline (PBS). In alternative embodiments, the wash solution may lack calcium, magnesium, or many, but not all, divalent cations. In one embodiment, T cells are isolated from peripheral blood lymphocytes by lysing red blood cells and depleting monocytes, for example, by centrifugation through a PERCOLL gradient or by counterflow centrifugal elutriation.

[0618] In some embodiments, the T cells are PBLs isolated from lymphocyte-enriched whole blood or apheresis products from a donor. In some embodiments, the donor is afflicted with cancer. In some embodiments, the cancer is selected from the group consisting of melanoma, ovarian cancer, endometrial cancer, thyroid 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)), glioblastoma (including GBM), gastrointestinal cancer, renal cancer, and renal cell carcinoma. In some embodiments, the cancer is selected from the group consisting of melanoma, ovarian cancer, endometrial cancer, thyroid 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)), glioblastoma (including GBM), gastrointestinal cancer, renal cancer, and renal cell carcinoma. In some embodiments, the donor is afflicted with a tumor. In some embodiments, the tumor is a liquid tumor. In some embodiments, the tumor is a solid tumor. In some embodiments, the donor is afflicted with a hematological malignancy. In some embodiments, PBLs are isolated from lymphocyte-enriched whole blood or apheresis products by using positive or negative selection methods, i.e., by depleting PBLs using marker(s) for T cell phenotype, e.g., CD3+CD45+, or by removing cells of non-T cell phenotype, leaving PBLs. In other embodiments, PBLs are isolated by gradient centrifugation. Upon isolation of PBLs from donor tissue, the first primed expansion of PBLs may be performed according to any of the first primed expansion steps of the methods described herein to place a suitable number of isolated PBLs (in some embodiments, approximately 1 x 10 cells / ml) under first primed expansion culture. 7 The cells can be initiated by seeding with pluripotent stem (PBLs).

[0619] An exemplary TIL process known as Process 3 (also referred to herein as Gen3), including some of these features, is shown in FIG. 8 (particularly, e.g., FIG. 8B and / or FIG. 8C and / or FIG. 8D), and some of the advantages of this embodiment of the invention over Gen2 are described in FIGS. 1, 2, 8, 30, and 31 (particularly, e.g., FIG. 8A and / or FIG. 8B and / or FIG. 8C and / or FIG. 8D). A Gen3 embodiment is shown in FIGS. 1, 8, and 30 (particularly, e.g., FIG. 8A and / or FIG. 8B and / or FIG. 8C and / or FIG. 8D). Process 2A or Gen2 or Gen2A is also described in U.S. Patent Publication No. 2018 / 0280436, which is incorporated herein by reference in its entirety. The Gen3 process is also described in International Patent Publication No. WO2020 / 096988.

[0620] As discussed herein and generally outlined, TILs are harvested from patient samples and engineered to expand their numbers before transplantation into the patient using the TIL expansion process described herein and referred to as Gen3. In some embodiments, TILs may optionally be genetically engineered, as discussed below. In some embodiments, TILs may be cryopreserved before or after expansion. Upon thawing, they may be restimulated to enhance their metabolism before infusion into the patient.

[0621] In some embodiments, as discussed in detail below and in the Examples and Figures, the priming first expansion (including the process referred to herein as Pre-Rapid Expansion (Pre-REP) and shown as step B in FIG. 8 (particularly, e.g., FIG. 8A and / or FIG. 8B and / or FIG. 8C and / or FIG. 8D)) is shortened to 1-8 days, and the rapid second expansion (including the process referred to herein as Rapid Expansion Protocol (REP) and shown as step D in FIG. 8 (particularly, e.g., FIG. 8A and / or FIG. 8B and / or FIG. 8C and / or FIG. 8D)) is shortened to 1-9 days. In some embodi...

Claims

A population of tumor-infiltrating lymphocytes (TILs) and at least one KRAS inhibitor for use in the treatment of cancer in a patient or subject in need thereof, optionally wherein the patient or subject has received at least one prior therapy, and the at least one prior therapy optionally comprises an anti-PD1 antibody, a population of tumor-infiltrating lymphocytes (TILs) and at least one KRAS inhibitor for use. The method for treating cancer according to claim 2, (a) obtaining and / or receiving a first TIL population from a tumor excised from the subject or patient by processing a tumor sample obtained from the subject into a plurality of tumor fragments; (b) adding the first TIL population to a closed system; (c) performing a first expansion to generate a second TIL population by culturing the first TIL population in a cell culture medium containing IL-2, wherein the first expansion is performed in a closed container providing a first gas-permeable surface area, the first expansion is performed for about 3 to 14 days to obtain the second TIL population, and the transition from step (b) to step (c) occurs without opening the system, the step of generating the second TIL population; (d) performing a second expansion to generate a third TIL population by supplementing the cell culture medium of the second TIL population with additional IL-2, OKT-3, and antigen-presenting cells (APCs), wherein the second expansion is performed for about 7 to 14 days to obtain the third TIL population, the third TIL population is a therapeutic TIL population, the second expansion 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, the step of generating the third TIL population; (e) collecting the therapeutic TIL population obtained from step (d), wherein the transition from step (d) to step (e) occurs without opening the system, the step of collecting; (f) transferring the collected TIL population from step (e) to an infusion bag, wherein the transfer from step (e) to (f) occurs without opening the system, the step of transferring; (g) cryopreserving the infusion bag containing the collected TIL population from step (f) using a cryopreservation process; (h) administering a therapeutically effective dose of the third TIL population from the infusion bag of step (g) to the subject; (i) administering at least one KRAS inhibitor to the subject, A population of tumor infiltrating lymphocytes (TIL) and at least one KRAS inhibitor for use according to claim 1.

3. The method for treating cancer is (a) obtaining and / or receiving a first TIL population from surgical resection, needle biopsy, core biopsy, fine biopsy, or other means for obtaining a sample containing a mixture of tumor cells and TIL cells from the patient or the subject; (b) adding the first TIL population to a closed system; (c) performing a first expansion by culturing the first TIL population in a cell culture medium containing IL-2 to generate a second TIL population, wherein the first expansion is performed in a closed container providing a first gas permeable surface area, the first expansion is performed for about 3 to 11 days to obtain the second TIL population, and the transition from step (b) to step (c) occurs without opening the system, the step of generating the second TIL population; (d) performing a second expansion by supplementing the cell culture medium of the second TIL population with additional IL-2, OKT-3, and antigen presenting cells (APC) to generate a third TIL population, wherein the second expansion is performed for about 7 to 11 days to obtain the third TIL population, the second expansion 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, the step of generating the third TIL population; (e) collecting the third TIL population obtained from step (d), wherein the transition from step (d) to step (e) occurs without opening the system, the step of collecting; (f) transferring the collected third TIL population from step (e) to an infusion bag, wherein the transfer from step (e) to (f) occurs without opening the system, the step of transferring; Step (g): cryopreserving the infusion bag containing the collected TIL population from step (f) using a cryopreservation process; Step (h): administering a therapeutically effective dose of the third TIL population from the infusion bag of step (g) to the subject; Step (i): administering at least one KRAS inhibitor to the subject, comprising: A population of tumor-infiltrating lymphocytes (TILs) and at least one KRAS inhibitor for use according to claim 1. **Claim 4** (i) the patient or the subject has a KRAS gene mutation; (ii) the patient or the subject has a cancer exhibiting a p.G12C mutation; (iii) the cancer has been previously treated with a KRAS inhibitor or the cancer has not been previously treated with a KRAS inhibitor and / or (iv) The KRAS inhibitor is AMG-510 (sotorasib), AMG-510 racemate (sotorasib racemate), MRTX849 (adagrasib), JNJ-74699157 / ARS-3248, JDQ443, LY3499446, LY3537982, RLY-1971 / GDC-6036, BBP-454, BI 1701963, BI 1823911, mRNA-5671 / V941, D-1553, BI-2852, BI-3406, ARS-1620, BAY-293, MRTX-1257, PROTAC K-Ras Degrader-1, LC-2, ARS-853, ARS-1323, ARS-1323-alkyne, ARS-1630, K-Ras G12C-IN-2, KRAS inhibitor-6, KRAS inhibitor 8, KRAS inhibitor 7, KRAS G12C inhibitor 15, KRAS G12C inhibitor 5, KRAS G12C inhibitor 13, KRAS G12C inhibitor 17, KRAS G12C inhibitor 16, KRAS G12C inhibitor 14, K-Ras G12C inhibitor 4, K-Ras G12C inhibitor 1, K-Ras G12C inhibitor 3, K-Ras G12C inhibitor 2, 6H05, SAH-SOS1ASelected from the group consisting of TFA, KRAS inhibitor 10, SAH-SOS1A, atorvastatin-PEG3-FITC, C6ME, CS-0115617, HY-130260, HY-135864, HY-135866, Cmpd2, CS-0115618, CS-0115620, EX-A4387, CS-0106134, HY-135865, 2241719-75-3, HY-125873, CS-0046138, CS-0046137, CS-0101474, HY-125875, CS-0102608, CS-0102610, CS-0102606, HY-112493, CS-0046139, 1-{4-[6-chloro-8-fluoro-7-(5-methyl-1h-indazol-4-yl)quinazolin-4-yl]piperazin-1-yl}propan-1-one, HY-126292, HY-112491, CS-0046136, HY-114168, HY-125874, HY-112494, CS-0102607, BCP29475|2206735-61-5, HY-112492, CS-0078097, 2158296-45-6, HY-125872, and 2158297-63-1, and pharmaceutically acceptable salts thereof, and optionally, the KRAS inhibitor is selected from the group consisting of AMG-510 (sotorasib), AMG-510 racemate (sotorasib racemate), and MRTX849 (adagrasib). A population of tumor-infiltrating lymphocytes (TILs) and at least one KRAS inhibitor for use according to any one of claims 1 to 3. **Claim 5** (i) the cancer has been previously treated with a PD-1 inhibitor and / or a PD-L1 inhibitor or a biosimilar thereof, optionally the cancer has been previously treated with a PD-1 inhibitor or a biosimilar thereof, and further optionally the PD-1 inhibitor is selected from the group consisting of nivolumab, pembrolizumab, and biosimilars thereof, or the patient has been further previously treated with a PD-L1 inhibitor or a biosimilar thereof, optionally the PD-L1 inhibitor is selected from the group consisting of avelumab, atezolizumab, durvalumab, and biosimilars thereof; (ii) the cancer has not been previously treated with a PD-1 inhibitor and / or a PD-L1 inhibitor or a biosimilar thereof; (iii) the cancer has been previously treated with a CTLA-4 inhibitor or a biosimilar thereof, optionally the CTLA-4 inhibitor is selected from the group consisting of ipilimumab, tremelimumab, and biosimilars thereof, or (iv) the cancer has been previously treated with a chemotherapy regimen, optionally the chemotherapy regimen comprises dacarbazine or temozolomide; A population of tumor-infiltrating lymphocytes (TILs) and at least one KRAS inhibitor for use according to any one of claims 1 to 3. Claim 6 (i) wherein said first expansion is carried out over a period of about 11 days, (ii) wherein said IL-2 is present in said cell culture medium at an initial concentration of 1000 IU / mL to 6000 IU / mL in said first expansion, (iii) wherein in said second expansion step, said IL-2 is present at an initial concentration of 1000 IU / mL to 6000 IU / mL and said OKT-3 antibody is present at an initial concentration of about 30 ng / mL, (iv) wherein said first expansion is carried out using a gas-permeable container, (v) wherein said second expansion is carried out using a gas-permeable container, (vi) wherein said first cell culture medium further comprises a cytokine selected from the group consisting of IL-4, IL-7, IL-15, IL-21, and combinations thereof, or (vii) wherein said second cell culture medium further comprises a cytokine selected from the group consisting of IL-4, IL-7, IL-15, IL-21, and combinations thereof, A population of tumor-infiltrating lymphocytes (TIL) and at least one KRAS inhibitor for use according to claim 2 or 3. Claim 7 Further comprising the step of treating the patient with a non-myeloablative lymphocyte depletion regimen prior to administering said TIL to the patient, and optionally, (i) The non-myeloablative lymphocyte depletion regimen comprises administering cyclophosphamide at a dose of 60 mg / m ２ / day for 2 days, followed by administering fludarabine at a dose of 25 mg / m ２ / day for 5 days, or (ii) The myelosuppressive lymphocyte depletion regimen comprises administering cyclophosphamide at a dose of 60 mg / m ２ / day, fludarabine at a dose of 25 mg / m ２ / day for 2 days, and subsequently administering fludarabine at a dose of 25 mg / m ２ / day for 3 days, and further optionally, the cyclophosphamide is administered together with mesna. A population of tumor-infiltrating lymphocytes (TIL) and at least one KRAS inhibitor for use according to any one of claims 1 to 3. Claim 8 Further comprising the step of treating the patient with an IL-2 regimen starting on the day following administration of said third TIL population to the patient, or Further comprising the step of treating the patient with an IL-2 regimen starting on the same day as administration of said third TIL population to the patient, Optionally, said IL-2 regimen is a high-dose IL-2 regimen comprising aldesleukin at 600,000 or 720,000 IU / kg administered as a 15-minute bolus intravenous infusion every 8 hours up to the tolerated limit, or a biosimilar or variant thereof, A population of tumor-infiltrating lymphocytes (TIL) and at least one KRAS inhibitor for use according to any one of claims 1 to 3. Claim 9 A population of tumor-infiltrating lymphocytes (TILs) and at least one KRAS inhibitor for use according to any one of claims 1 to 3, wherein the effective dosage of the TILs is about 1×10⁹, 2×10⁹, 3×10⁹, 4×10⁹, 5×10⁹, 6×10⁹, 7×10⁹, 8×10⁹, 9×10⁹, 1×10¹⁰, 2×10¹⁰, 3×10¹⁰, 4×10¹⁰, 5×10¹⁰, 6×10¹⁰, 7×10¹⁰, 8×10¹⁰, 9×10¹⁰ and 1×10¹¹.

10. (i) The first expansion in step (c) and the second expansion in step (d) are each performed individually within a period of 11 days, or (ii) steps (a) to (f) are performed in about 10 days to about 22 days, A population of tumor-infiltrating lymphocytes (TILs) and at least one KRAS inhibitor for use according to any one of claims 2 or 3.

11. The subject has received a previous treatment comprising administration of a KRAS inhibitor prior to step (a), optionally wherein the previous treatment comprises administration of sotorasib or adagrasib, or a pharmaceutically acceptable salt thereof, at a dosage of about 500 - 1500 mg, further optionally wherein the sotorasib is administered at a dosage of about 960 mg, or the adagrasib is administered at a dosage of about 600 mg, and / or wherein the previous treatment comprises administration of sotorasib or adagrasib, or a pharmaceutically acceptable salt thereof, twice a day. A population of tumor-infiltrating lymphocytes (TILs) and at least one KRAS inhibitor for use according to claim 2 or 3.

12. (i) The at least one KRAS inhibitor is administered simultaneously with the therapeutically effective dosage of the third TIL population, optionally wherein the administration of the at least one KRAS inhibitor is maintained after the administration of the therapeutically effective dosage of the third TIL population, or (ii) The at least one KRAS inhibitor is administered after the administration of the therapeutically effective dose of the third TIL population, and optionally, the subject is administered the at least one KRAS inhibitor at least one week after the administration of the therapeutically effective dose of the third TIL population, or the patient is also administered the at least one KRAS inhibitor before the administration of the therapeutically effective dose of the third TIL population, and optionally, the at least one KRAS inhibitor is not administered simultaneously with the therapeutically effective dose of the third TIL population. A population of tumor-infiltrating lymphocytes (TIL) and at least one KRAS inhibitor for use, Optionally, the at least one KRAS inhibitor comprises sotorasib or adagrasib administered at a dose of about 500 - 1500 mg, or a pharmaceutically acceptable salt thereof, and further optionally, the sotorasib is administered at a dose of about 960 mg, or the adagrasib is administered at a dose of about 600 mg, and optionally, the sotorasib or adagrasib is administered twice daily. A population of tumor-infiltrating lymphocytes (TIL) and at least one KRAS inhibitor for use according to claim 2 or 3.

13. The cancer is (i) selected from the group consisting of glioblastoma (GBM), gastrointestinal cancer, melanoma, ovarian cancer, endometrial cancer, thyroid cancer, colorectal cancer, cervical cancer, non-small cell lung cancer (NSCLC), lung cancer, bladder cancer, breast cancer, endometrial cancer, cholangiocarcinoma, cancer caused by human papillomavirus, head and neck cancer (including head and neck squamous cell carcinoma (HNSCC)), renal cancer, renal cell carcinoma, multiple myeloma, chronic lymphocytic leukemia, acute lymphoblastic leukemia, diffuse large B-cell lymphoma, non-Hodgkin lymphoma, Hodgkin lymphoma, follicular lymphoma, and mantle cell lymphoma; (ii) selected from the group consisting of cutaneous melanoma, ocular melanoma, uveal melanoma, and conjunctival malignant melanoma; (iii) selected from the group consisting of pleomorphic xanthoastrocytoma, embryonal dysplastic neuroepithelial tumor, ganglioglioma, and pilocytic astrocytoma; (iv) endometrial adenocarcinoma having non-small cell lung cancer (NSCLC); (v) endometrial adenocarcinoma having marked mucinous differentiation (ECMD); (vi) papillary thyroid carcinoma; (vii) serous low-grade or borderline ovarian cancer. (viii) being hairy cell leukemia, or (ix) being Langerhans cell histiocytosis, optionally, the cancer is a cancer having a p.G12C mutation in the KRAS protein, or the cancer is non-small cell lung cancer (NSCLC) having a p.G12C mutation, A population of tumor-infiltrating lymphocytes (TIL) and at least one KRAS inhibitor for use according to any one of claims 1 to 3.

14. further comprising the step of treating the patient with an IL-2 regimen after said administration of said third TIL population to the patient, optionally further comprising the step of treating the patient with an IL-2 regimen on the same day as said administration of said third TIL population to the patient, and further optionally, (i) the IL-2 regimen comprises nemvaleukin, (ii) the patient has previously received checkpoint inhibitor therapy, or (iii) the patient has previously received KRAS inhibitor therapy, optionally, the patient has NSCLC, A population of tumor-infiltrating lymphocytes (TIL) and at least one KRAS inhibitor for use according to any one of claims 1 to 3.

15. A population of tumor-infiltrating lymphocytes (TIL) and at least one KRAS inhibitor for use in the treatment of NSCLC in a patient in need thereof, wherein the patient or the subject has received at least one prior therapy, and the at least one prior therapy comprises checkpoint inhibitor therapy.

16. A population of tumor-infiltrating lymphocytes (TIL) and at least one KRAS inhibitor for use in the treatment of cancer in a patient or subject in need thereof, wherein the method of treating the cancer comprises (a) treating the patient with a myeloablative lymphodepleting regimen comprising melphalan, (b) administering a population of tumor-infiltrating lymphocytes (TIL) and one or more KRAS inhibitors, (c) treating the patient with an IL-2 regimen after administration of the population of TIL, wherein the patient or the subject has cancer, A population of tumor-infiltrating lymphocytes (TIL) and at least one KRAS inhibitor for use.

17. The melphalan is intravenously administered continuously for 2 days at a dose of about 100 mg / m ２ or The IL-2 regimen comprises administering low-dose IL-2 daily for up to 14 days after administration of the TIL population. A population of tumor-infiltrating lymphocytes (TILs) and at least one KRAS inhibitor for use according to claim 16.

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