Treatment of nsclc patients refractory to Anti-PD-1 antibody

Abstract

To provide treatment of NSCLC patients refractory to an anti-PD-1 antibody.SOLUTION: The present invention provides improved and / or shortened processes and methods for preparing TILs in order to prepare therapeutic populations of TILs with increased therapeutic efficacy for treatment of non-small cell lung carcinoma (NSCLC), where the NSCLC is refractory to treatment with an anti-PD-1 antibody.SELECTED DRAWING: Figure 1

Description

[Technical Field]

[0001] CROSS-REFERENCE TO RELATED APPLICATIONS This application claims priority to U.S. Provisional Patent Application No. 62 / 725,976, filed August 31, 2018, and U.S. Provisional Patent Application No. 62 / 726,919, filed September 4, 2018, which are incorporated by reference in their entireties.

[0002] Sequence Listing This application contains a Sequence Listing that has been submitted electronically in ASCII format and is incorporated herein by reference in its entirety. The ASCII copy, created on August 23, 2019, is named 116983-5043-WO_ST25.txt and is 168 kilobytes in size. [Background technology]

[0003] The treatment of bulky, refractory cancers using adoptive transfer of tumor-infiltrating lymphocytes (TILs) offers a powerful approach for treating patients with poor prognosis. (Gattinoni, et al., Nat. Rev. Immunol. 2006, 6, 383-393) Successful immunotherapy requires large numbers of TILs, and commercialization requires a robust and reliable process. This has been difficult to achieve due to technical, logistical, and regulatory challenges associated with cell expansion. 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. REP requires a large excess (e.g., 200-fold) of irradiated allogeneic peripheral blood mononuclear cells (PBMCs, also known as mononuclear cells (MNCs)) often derived from multiple donors as feeder cells, as well as anti-CD3 antibodies (OKT3) and high doses of IL-2, but can result in a 1,000-fold expansion of TILs over 14 days. Dudley, et al., J. Immunother. 2003, 26, 332-42. TILs undergoing the REP procedure have been used successfully in adoptive cell therapy following host immunosuppression in patients with melanoma. Current infusion acceptance parameters depend on the composition of the TILs (e.g., CD28, CD8, or CD4 positivity) as well as the fold expansion and survival rate of the REP product.

[0004] Current TIL production and treatment processes are limited by length, cost, sterility concerns, and other factors described herein, severely limiting the ability to treat patients who are refractory to anti-PD-1. 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 viable treatment options remaining. The present invention fulfills this need by providing an abbreviated production process for use in generating TILs that can be used to treat non-small cell lung cancer (NSCLC) patients who are refractory to anti-PD-1 treatment. Summary of the Invention

[0005] The present invention provides improved and / or novel methods for expanding TILs and generating therapeutic TIL populations for use in treating patients with non-small cell lung cancer (NSCLC) who are refractory to anti-PD-1 therapy. Or provide an abbreviated method.

[0006] The present invention provides a method of treating non-small cell lung cancer (NSCLC) with a population of tumor-infiltrating lymphocytes (TILs), 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 and TIL cells from a patient's NSCLC tumor, including from multiple tumor fragments or biopsies; (c) contacting the tumor fragment with a first cell culture medium; (d) performing an initial expansion of the first TIL population in a first cell culture medium to obtain a second TIL population, the second TIL population being at least 5-fold more numerous than the first TIL population, and the first cell culture medium comprising IL-2; (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 days after the start of the rapid expansion, the 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 14 days or less; (f) harvesting a third population of TILs; (g) administering a therapeutically effective portion of the third population of TILs to a patient with NSCLC; Methods are provided wherein the NSCLC is refractory to treatment with an anti-PD-1 antibody.

[0007] In some embodiments, "obtaining" indicates that the TILs used in the method and / or process may be derived directly from a sample (including a surgical resection, needle biopsy, core biopsy, mini biopsy, or other sample) as part of a method and / or process step. In some embodiments, "receiving" indicates that the TILs used in the method and / or process may be derived indirectly from a sample (including a surgical resection, needle biopsy, core biopsy, mini biopsy, or other sample) and then used in the method and / or process (e.g., if step (a) begins with TILs that have already been derived from a sample by another process not included in part (a), such TILs may be referred to as "received").

[0008] In some embodiments, obtaining the first population of TILs comprises a multi-lesion sampling method.

[0009] In some embodiments, the refractory NSCLC has been previously treated with an anti-PD-1 and / or anti-PD-L1 and / or anti-PD-L2 antibody.

[0010] In some embodiments, the refractory NSCLC has not been previously treated with an anti-PD-1 and / or anti-PD-L1 antibody.

[0011] In some embodiments, the refractory NSCLC has been treated with a chemotherapeutic agent.

[0012] In some embodiments, the refractory NSCLC has been previously treated with an anti-PD-1 and / or anti-PD-L1 antibody and has been previously treated with a chemotherapeutic agent.

[0013] In some embodiments, the refractory NSCLC has not been previously treated with an anti-PD-1 and / or anti-PD-L1 antibody and has been previously treated with a chemotherapeutic agent.

[0014] In some embodiments, the refractory NSCLC is currently being treated with a chemotherapeutic agent. has not been treated with chemotherapy.

[0015] In some embodiments, the refractory NSCLC has low PD-L1 expression.

[0016] In some embodiments, the refractory NSCLC has been previously treated with an anti-PD-1 and / or anti-PD-L1 antibody and has low PD-L1 expression.

[0017] In some embodiments, the refractory NSCLC has not been previously treated with an anti-PD-1 and / or anti-PD-L1 antibody and has low PD-L1 expression.

[0018] In some embodiments, the refractory NSCLC has been treated with a chemotherapeutic agent and has low PD-L1 expression.

[0019] In some embodiments, the refractory NSCLC has been treated with a chemotherapeutic agent, but is not currently being treated with a chemotherapeutic agent and has low PD-L1 expression.

[0020] In some embodiments, the refractory NSCLC has not been previously treated with an anti-PD-1 and / or anti-PD-L1 antibody and has bulky disease at baseline.

[0021] In some embodiments, the refractory NSCLC has been previously treated with an anti-PD-1 and / or anti-PD-L1 antibody and has bulky disease at baseline.

[0022] In some embodiments, the refractory NSCLC has been treated with a chemotherapy agent and has bulky disease at baseline.

[0023] In some embodiments, the refractory NSCLC has been treated with a chemotherapeutic agent, but is not currently being treated with a chemotherapeutic agent and has bulky disease at baseline.

[0024] In some embodiments, a bulky mass lesion is indicated when the maximum tumor diameter is greater than 7 cm measured in either the transverse or coronal plane, or when an enlarged lymph node has a short-axis diameter of 20 mm or greater.

[0025] In some embodiments, the refractory NSCLC is refractory to at least two prior courses of systemic therapy, not including neoadjuvant or adjuvant therapy.

[0026] In some embodiments, the refractory NSCLC is refractory to an anti-PD-1 antibody selected from the group consisting of nivolumab, pembrolizumab, ipilimumab, JS001, TSR-042, pidilizumab, (BGB-A317, SHR-1210, REGN2810, MDX-1106, PDR001, clonally derived anti-PD-1: RMP1-14, and the anti-PD-1 antibodies disclosed in U.S. Pat. No. 8,008,449, durvalumab, atezolizumab, avelumab, and fragments, derivatives, variants, and biosimilars thereof.

[0027] In some embodiments, the refractory NSCLC is refractory to pembrolizumab or a biosimilar thereof.

[0028] In some embodiments, the refractory NSCLC is refractory to nivolumab or a biosimilar thereof.

[0029] In some embodiments, the refractory NSCLC is treated with ipilimumab or a biosimilar thereof. - refractory.

[0030] In some embodiments, the refractory NSCLC is refractory to ipilimumab or a biosimilar thereof and pembrolizumab or a biosimilar thereof.

[0031] In some embodiments, the refractory NSCLC is refractory to ipilimumab or a biosimilar thereof and nivolumab or a biosimilar thereof.

[0032] In some embodiments, the refractory NSCLC is refractory to durvalumab or a biosimilar thereof.

[0033] In some embodiments, the refractory NSCLC is refractory to atezolizumab or a biosimilar thereof.

[0034] In some embodiments, the refractory NSCLC is refractory to avelumab or a biosimilar thereof.

[0035] In some embodiments, the initial expansion is performed over a period of 21 days or less.

[0036] In some embodiments, the initial expansion is performed over a period of 14 days or less.

[0037] In some embodiments, the initial expansion occurs over a period of about 11 days and the rapid expansion occurs over a period of about 11 days.

[0038] In some embodiments, IL-2 is present in the first cell culture medium at an initial concentration of between 1000 IU / mL and 6000 IU / mL.

[0039] In some embodiments, IL-2 is present at an initial concentration of between 1000 IU / mL and 6000 IU / mL, and the OKT-3 antibody is present in the second cell culture medium at an initial concentration of about 30 ng / mL.

[0040] In some embodiments, the initial expansion is performed using a gas-permeable container.

[0041] In some embodiments, the rapid expansion is achieved using a gas-permeable container.

[0042] 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.

[0043] 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.

[0044] In some embodiments, the method further comprises treating the patient with a non-myeloablative lymphodepleting regimen prior to administering the third population of TILs to the patient.

[0045] In some embodiments, the non-myeloablative lymphodepleting regimen is 60 mg / m 2 / day for 2 days followed by cyclophosphamide at a dose of 25 mg / m 2 10 mg / day for 5 days.

[0046] 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.

[0047] 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.

[0048] In some embodiments, the present invention provides a method of treating non-small cell lung cancer (NSCLC) with a population of tumor-infiltrating lymphocytes (TILs), comprising: (a) resecting one or more tumors from a patient, wherein the one or more tumors comprise a first population of TILs; (b) fragmenting one or more tumors into tumor fragments; (c) contacting the tumor fragment with a first cell culture medium; (d) performing an initial expansion of the first TIL population in a first cell culture medium to obtain a second TIL population, the second TIL population being at least 5-fold more numerous than the first TIL population, and the first cell culture medium comprising IL-2; (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 days after the start of the rapid expansion, the 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 14 days or less; (f) harvesting a third population of TILs; (g) administering a therapeutically effective portion of the third population of TILs to a patient with cancer; The method is provided wherein the cancer is refractory to treatment with an anti-PD-1 antibody.

[0049] In some embodiments, the refractory NSCLC has been previously treated with an anti-PD-1 and / or anti-PD-L1 antibody.

[0050] In some embodiments, the refractory NSCLC has not been previously treated with an anti-PD-1 and / or anti-PD-L1 antibody.

[0051] In some embodiments, the refractory NSCLC has been treated with a chemotherapeutic agent.

[0052] In some embodiments, the refractory NSCLC has been previously treated with an anti-PD-1 and / or anti-PD-L1 antibody and has been previously treated with a chemotherapeutic agent.

[0053] In some embodiments, the refractory NSCLC has not been previously treated with an anti-PD-1 and / or anti-PD-L1 antibody and has been previously treated with a chemotherapeutic agent.

[0054] In some embodiments, the refractory NSCLC has been treated with a chemotherapeutic agent, but is not currently being treated with a chemotherapeutic agent.

[0055] In some embodiments, the refractory NSCLC has low PD-L1 expression.

[0056] In some embodiments, the refractory NSCLC has been previously treated with an anti-PD-1 and / or anti-PD-L1 antibody and has low PD-L1 expression.

[0057] In some embodiments, the refractory NSCLC is treated with anti-PD-1 and / or anti-PD-L 1 antibody and have low PD-L1 expression.

[0058] In some embodiments, the refractory NSCLC has been treated with a chemotherapeutic agent and has low PD-L1 expression.

[0059] In some embodiments, the refractory NSCLC has been treated with a chemotherapeutic agent, but is not currently being treated with a chemotherapeutic agent and has low PD-L1 expression.

[0060] In some embodiments, the refractory NSCLC has not been previously treated with an anti-PD-1 and / or anti-PD-L1 antibody and has bulky disease at baseline.

[0061] In some embodiments, the refractory NSCLC has been previously treated with an anti-PD-1 and / or anti-PD-L1 antibody and has bulky disease at baseline.

[0062] In some embodiments, the refractory NSCLC has been treated with a chemotherapy agent and has bulky disease at baseline.

[0063] In some embodiments, the refractory NSCLC has been treated with a chemotherapeutic agent, but is not currently being treated with a chemotherapeutic agent and has bulky disease at baseline.

[0064] In some embodiments, a bulky mass lesion is indicated when the maximum tumor diameter is greater than 7 cm measured in either the transverse or coronal plane, or when an enlarged lymph node has a short-axis diameter of 20 mm or greater.

[0065] In some embodiments, the refractory NSCLC is refractory to at least two prior courses of systemic therapy, not including neoadjuvant or adjuvant therapy.

[0066] In some embodiments, the refractory NSCLC is refractory to an anti-PD-1 antibody selected from the group consisting of nivolumab, pembrolizumab, ipilimumab, JS001, TSR-042, pidilizumab, (BGB-A317, SHR-1210, REGN2810, MDX-1106, PDR001, clonally derived anti-PD-1: RMP1-14, and the anti-PD-1 antibodies disclosed in U.S. Pat. No. 8,008,449, durvalumab, atezolizumab, avelumab, and fragments, derivatives, variants, and biosimilars thereof.

[0067] In some embodiments, the refractory NSCLC is refractory to pembrolizumab or a biosimilar thereof.

[0068] In some embodiments, the refractory NSCLC is refractory to nivolumab or a biosimilar thereof.

[0069] In some embodiments, the refractory NSCLC is refractory to ipilimumab or a biosimilar thereof.

[0070] In some embodiments, the refractory NSCLC is refractory to ipilimumab or a biosimilar thereof and pembrolizumab or a biosimilar thereof.

[0071] In some embodiments, the refractory NSCLC is refractory to ipilimumab or a biosimilar thereof and nivolumab or a biosimilar thereof.

[0072] In some embodiments, the refractory NSCLC is refractory to durvalumab or a biosimilar thereof.

[0073] In some embodiments, the refractory NSCLC is refractory to atezolizumab or a biosimilar thereof.

[0074] In some embodiments, the refractory NSCLC is refractory to avelumab or a biosimilar thereof.

[0075] In some embodiments, the initial expansion is performed over a period of 21 days or less.

[0076] In some embodiments, the initial expansion is performed over a period of 14 days or less.

[0077] In some embodiments, the initial expansion occurs over a period of about 11 days and the rapid expansion occurs over a period of about 11 days.

[0078] In some embodiments, IL-2 is present in the first cell culture medium at an initial concentration of between 1000 IU / mL and 6000 IU / mL.

[0079] In some embodiments, IL-2 is present at an initial concentration of between 1000 IU / mL and 6000 IU / mL, and the OKT-3 antibody is present in the second cell culture medium at an initial concentration of about 30 ng / mL.

[0080] In some embodiments, the initial expansion is performed using a gas-permeable container.

[0081] In some embodiments, the rapid expansion is achieved using a gas-permeable container.

[0082] 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.

[0083] 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.

[0084] In some embodiments, the method further comprises treating the patient with a non-myeloablative lymphodepleting regimen prior to administering the third population of TILs to the patient.

[0085] In some embodiments, the non-myeloablative lymphodepleting regimen is 60 mg / m 2 / day for 2 days followed by cyclophosphamide at a dose of 25 mg / m 2 10 mg / day for 5 days.

[0086] 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.

[0087] 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.

[0088] In some embodiments, the present invention provides a method for treating a subject having non-small cell lung cancer (NSCLC), wherein the cancer is refractory to treatment with an anti-PD-1 antibody, the method comprising administering expanded tumor-infiltrating lymphocytes (TILs): (a) obtaining and / or receiving a first population of TILs from one or more tumors resected from a subject by processing one or more tumors 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 produce a second TIL population, wherein the first expansion is performed in a sealed container providing a first gas permeable surface area, and wherein the first expansion is performed for about 3~11 performing a first expansion, which is carried out for 3 to 14 days, to obtain a second TIL population, the second TIL population being at least 50-fold more numerous than the first TIL population, and the transition from step (b) to step (c) occurring 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 produce a third TIL population, wherein the second expansion is 7~11 performing a second expansion for 7 to 14 days to obtain a third TIL population, the third TIL population being a therapeutic TIL population that includes an increased subpopulation of effector T cells and / or central memory T cells compared to the second TIL population, the second expansion being performed in a sealed container that provides a second gas permeable surface area, and the transition from step (c) to step (d) occurring 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 to the subject from the infusion bag of step (g).

[0089] In some embodiments, "obtaining" indicates that the TILs used in the method and / or process may be derived directly from a sample (including a surgical resection, needle biopsy, core biopsy, mini biopsy, or other sample) as part of a method and / or process step. In some embodiments, "receiving" indicates that the TILs used in the method and / or process may be derived indirectly from a sample (including a surgical resection, needle biopsy, core biopsy, mini biopsy, or other sample) and then used in the method and / or process (e.g., if step (a) begins with TILs that have already been derived from a sample by another process not included in part (a), such TILs may be referred to as "received").

[0090] In some embodiments, the refractory NSCLC has been previously treated with an anti-PD-1 and / or anti-PD-L1 antibody.

[0091] In some embodiments, the refractory NSCLC has not been previously treated with an anti-PD-1 and / or anti-PD-L1 antibody.

[0092] In some embodiments, the refractory NSCLC has been treated with a chemotherapeutic agent.

[0093] In some embodiments, the refractory NSCLC has been previously treated with an anti-PD-1 and / or anti-PD-L1 antibody and has been previously treated with a chemotherapeutic agent.

[0094] In some embodiments, the refractory NSCLC has not been previously treated with an anti-PD-1 and / or anti-PD-L1 antibody and has been previously treated with a chemotherapeutic agent.

[0095] In some embodiments, the refractory NSCLC has been treated with a chemotherapeutic agent, but is not currently being treated with a chemotherapeutic agent.

[0096] In some embodiments, the refractory NSCLC has low PD-L1 expression.

[0097] In some embodiments, the refractory NSCLC has been previously treated with an anti-PD-1 and / or anti-PD-L1 antibody and has low PD-L1 expression.

[0098] In some embodiments, the refractory NSCLC has not been previously treated with an anti-PD-1 and / or anti-PD-L1 antibody and has low PD-L1 expression.

[0099] In some embodiments, the refractory NSCLC has been treated with a chemotherapeutic agent and has low PD-L1 expression.

[0100] In some embodiments, the refractory NSCLC has been treated with a chemotherapeutic agent, but is not currently being treated with a chemotherapeutic agent and has low PD-L1 expression.

[0101] In some embodiments, the refractory NSCLC has not been previously treated with an anti-PD-1 and / or anti-PD-L1 antibody and has bulky disease at baseline.

[0102] In some embodiments, the refractory NSCLC has been previously treated with an anti-PD-1 and / or anti-PD-L1 antibody and has bulky disease at baseline.

[0103] In some embodiments, the refractory NSCLC has been treated with a chemotherapy agent and has bulky disease at baseline.

[0104] In some embodiments, the refractory NSCLC has been treated with a chemotherapeutic agent, but is not currently being treated with a chemotherapeutic agent and has bulky disease at baseline.

[0105] In some embodiments, a bulky mass lesion is indicated when the maximum tumor diameter is greater than 7 cm measured in either the transverse or coronal plane, or when an enlarged lymph node has a short-axis diameter of 20 mm or greater.

[0106] In some embodiments, the refractory NSCLC is refractory to at least two prior courses of systemic therapy, not including neoadjuvant or adjuvant therapy.

[0107] In some embodiments, the refractory NSCLC is refractory to an anti-PD-1 antibody selected from the group consisting of nivolumab, pembrolizumab, ipilimumab, JS001, TSR-042, pidilizumab, (BGB-A317, SHR-1210, REGN2810, MDX-1106, PDR001, clonally derived anti-PD-1: RMP1-14, and the anti-PD-1 antibodies disclosed in U.S. Pat. No. 8,008,449, durvalumab, atezolizumab, avelumab, and fragments, derivatives, variants, and biosimilars thereof.

[0108] In some embodiments, the refractory NSCLC is refractory to pembrolizumab or a biosimilar thereof.

[0109] In some embodiments, the refractory NSCLC is refractory to nivolumab or a biosimilar thereof.

[0110] In some embodiments, the refractory NSCLC is refractory to ipilimumab or a biosimilar thereof.

[0111] In some embodiments, the refractory NSCLC is refractory to ipilimumab or a biosimilar thereof and pembrolizumab or a biosimilar thereof.

[0112] In some embodiments, the refractory NSCLC is refractory to ipilimumab or a biosimilar thereof and nivolumab or a biosimilar thereof.

[0113] In some embodiments, the refractory NSCLC is refractory to durvalumab or a biosimilar thereof.

[0114] In some embodiments, the refractory NSCLC is refractory to atezolizumab or a biosimilar thereof.

[0115] In some embodiments, the refractory NSCLC is refractory to avelumab or a biosimilar thereof.

[0116] In some embodiments, the initial expansion is performed over a period of 21 days or less.

[0117] In some embodiments, the initial expansion is performed over a period of 14 days or less.

[0118] In some embodiments, the initial expansion is performed over a period of about 3 to 11 days, and the second expansion is performed over a period of about 7 to 11 days.

[0119] In some embodiments, the initial expansion occurs over a period of about 11 days and the rapid expansion occurs over a period of about 11 days.

[0120] In some embodiments, IL-2 is present in the first cell culture medium at an initial concentration of between 1000 IU / mL and 6000 IU / mL.

[0121] In some embodiments, IL-2 is present at an initial concentration of between 1000 IU / mL and 6000 IU / mL, and the OKT-3 antibody is present in the second cell culture medium at an initial concentration of about 30 ng / mL.

[0122] In some embodiments, the initial expansion is performed using a gas-permeable container.

[0123] In some embodiments, the rapid expansion is achieved using a gas-permeable container.

[0124] 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.

[0125] 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.

[0126] In some embodiments, the method further comprises treating the patient with a non-myeloablative lymphodepleting regimen prior to administering the third population of TILs to the patient.

[0127] In some embodiments, the non-myeloablative lymphodepleting regimen is 60 mg / m 2 / day for 2 days followed by cyclophosphamide at a dose of 25 mg / m 2 10 mg / day for 5 days.

[0128] 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.

[0129] 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. [Brief explanation of the drawings]

[0130] [Figure 1] Illustrative Process 2A chart providing an overview of steps A-F. [Figure 2A] Process flow chart for Process 2A. [Figure 2B] Process flow chart for Process 2A. [Figure 2C] Process flow chart for Process 2A. [Figure 3] FIG. 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 Process 2A, a 22-day TIL manufacturing process. [Figure 5] 1 is a comparison table of steps A-F from exemplary embodiments of Process 1C and Process 2A. [Figure 6] Detailed comparison of Process 1C embodiment and Process 2A embodiment. [Figure 7]Study flowchart for the combination cohorts: Cohort 1A (MM), Cohort 2A (HNSCC), and Cohort 3A (NSCLC). Abbreviations: Cy = cyclophosphamide, EOA = end of evaluation, EOS = end of study, EOT = end of treatment, Flu = fludarabine, IL-2 = interleukin-2, NMA-LD = nonmyeloablative lymphodepletion, Q3W = every 3 weeks, TIL = tumor-infiltrating lymphocytes. Patients in Cohorts 1A, 2A, and 3A will receive a single infusion of pembrolizumab after completion of tumor resection for TIL production and undergo a baseline scan before initiating the NMA-LD regimen. In this particular study, the next dose of pembrolizumab will be administered no sooner than after completion of IL-2 and will continue Q3W ± 3 days thereafter until 2 years (24 months) or disease progression or unacceptable toxicity, whichever occurs first. [Figure 8] Study flowchart for single-agent cohort: Cohort 3B (NSCLC). Abbreviations: Cy = cyclophosphamide, EOA = end of evaluation, EOS = end of study, EOT = end of treatment, Flu = fludarabine, IL-2 = interleukin-2, NMA-LD = nonmyeloablative lymphodepletion, TIL = tumor-infiltrating lymphocytes. [Figure 9] 1 shows a diagram of an embodiment of Process 2A, a 22-day TIL manufacturing process. [Figure 10]Structures IA and IB are provided, with the cylinders representing individual polypeptide binding domains. Structures IA and IB comprise three linearly linked TNFRSF-binding domains, derived from antibodies that bind, for example, to 4-1BBL or 4-1BB, that fold to form a trivalent protein, which is then linked to a second trivalent protein via an IgG1-Fc (comprising the CH3 and CH2 domains), which then uses disulfide bonds (small oblongs) to link two of the trivalent proteins together, stabilizing the structure and providing an agonist capable of linking the six receptor and signaling protein intracellular signaling domains 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.

[0131] Brief description of the sequence listing SEQ ID NO: 1 is the amino acid sequence of the heavy chain of muromonab.

[0132] SEQ ID NO: 2 is the amino acid sequence of the light chain of muromonab.

[0133] SEQ ID NO: 3 is the amino acid sequence of recombinant human IL-2 protein.

[0134] SEQ ID NO: 4 is the amino acid sequence of aldesleukin.

[0135] SEQ ID NO: 5 is the amino acid sequence of recombinant human IL-4 protein.

[0136] SEQ ID NO: 6 is the amino acid sequence of recombinant human IL-7 protein.

[0137] SEQ ID NO: 7 is the amino acid sequence of recombinant human IL-15 protein.

[0138] SEQ ID NO: 8 is the amino acid sequence of recombinant human IL-21 protein.

[0139] SEQ ID NO: 9 is the amino acid sequence of human 4-1BB.

[0140] SEQ ID NO: 10 is the amino acid sequence of mouse 4-1BB.

[0141] SEQ ID NO: 11 is the heavy chain of the 4-1BB agonist monoclonal antibody utomilumab (PF-05082566).

[0142] SEQ ID NO: 12 is the light chain of the 4-1BB agonist monoclonal antibody utomilumab (PF-05082566).

[0143] SEQ ID NO: 13 is the heavy chain variable region (VH) of the 4-1BB agonist monoclonal antibody utomilumab (PF-05082566).

[0144] SEQ ID NO: 14 is the light chain variable region (VL) of the 4-1BB agonist monoclonal antibody utomilumab (PF-05082566).

[0145] SEQ ID NO: 15 is the heavy chain CDR1 of the 4-1BB agonist monoclonal antibody utomilumab (PF-05082566).

[0146] SEQ ID NO: 16 is the heavy chain CDR2 of the 4-1BB agonist monoclonal antibody utomilumab (PF-05082566).

[0147] SEQ ID NO: 17 is the heavy chain CDR3 of the 4-1BB agonist monoclonal antibody utomilumab (PF-05082566).

[0148] SEQ ID NO: 18 is the light chain CDR1 of the 4-1BB agonist monoclonal antibody utomilumab (PF-05082566).

[0149] SEQ ID NO: 19 is the 4-1BB agonist monoclonal antibody utomilumab (PF-05 082566) light chain CDR2.

[0150] SEQ ID NO: 20 is the light chain CDR3 of the 4-1BB agonist monoclonal antibody utomilumab (PF-05082566).

[0151] SEQ ID NO: 21 is the heavy chain of the 4-1BB agonist monoclonal antibody urelumab (BMS-663513).

[0152] SEQ ID NO: 22 is the light chain of the 4-1BB agonist monoclonal antibody urelumab (BMS-663513).

[0153] SEQ ID NO: 23 is the heavy chain variable region (VH) of the 4-1BB agonist monoclonal antibody urelumab (BMS-663513).

[0154] SEQ ID NO: 24 is the light chain variable region (VL) of the 4-1BB agonist monoclonal antibody urelumab (BMS-663513).

[0155] SEQ ID NO: 25 is the heavy chain CDR1 of the 4-1BB agonist monoclonal antibody urelumab (BMS-663513).

[0156] SEQ ID NO: 26 is the heavy chain CDR2 of the 4-1BB agonist monoclonal antibody urelumab (BMS-663513).

[0157] SEQ ID NO: 27 is the heavy chain CDR3 of the 4-1BB agonist monoclonal antibody urelumab (BMS-663513).

[0158] SEQ ID NO: 28 is the light chain CDR1 of the 4-1BB agonist monoclonal antibody urelumab (BMS-663513).

[0159] SEQ ID NO: 29 is the light chain CDR2 of the 4-1BB agonist monoclonal antibody urelumab (BMS-663513).

[0160] SEQ ID NO: 30 is the light chain CDR3 of the 4-1BB agonist monoclonal antibody urelumab (BMS-663513).

[0161] SEQ ID NO: 31 is the Fc domain of the TNFRSF agonist fusion protein.

[0162] SEQ ID NO: 32 is the linker of the TNFRSF agonist fusion protein.

[0163] SEQ ID NO: 33 is the linker of the TNFRSF agonist fusion protein.

[0164] SEQ ID NO: 34 is the linker of the TNFRSF agonist fusion protein.

[0165] SEQ ID NO: 35 is the linker of the TNFRSF agonist fusion protein.

[0166] SEQ ID NO: 36 is the linker of the TNFRSF agonist fusion protein.

[0167] SEQ ID NO: 37 is the linker of the TNFRSF agonist fusion protein.

[0168] SEQ ID NO: 38 is the linker of the TNFRSF agonist fusion protein.

[0169] SEQ ID NO: 39 is the linker of the TNFRSF agonist fusion protein.

[0170] SEQ ID NO: 40 is the linker of the TNFRSF agonist fusion protein.

[0171] SEQ ID NO: 41 is the linker of the TNFRSF agonist fusion protein.

[0172] SEQ ID NO: 42 is the Fc domain of the TNFRSF agonist fusion protein.

[0173] SEQ ID NO: 43 is the linker of the TNFRSF agonist fusion protein.

[0174] SEQ ID NO: 44 is the linker of the TNFRSF agonist fusion protein.

[0175] SEQ ID NO: 45 is the linker of the TNFRSF agonist fusion protein.

[0176] SEQ ID NO: 46 is the 4-1BB ligand (4-1BBL) amino acid sequence.

[0177] SEQ ID NO:47 is the soluble portion of the 4-1BBL polypeptide.

[0178] SEQ ID NO: 48 is the heavy chain variable region (VH) of 4-1BB agonist antibody 4B4-1-1 version 1.

[0179] SEQ ID NO: 49 is the light chain variable region (VL) of 4-1BB agonist antibody 4B4-1-1 version 1.

[0180] SEQ ID NO: 50 is the heavy chain variable region (VH) of the 4-1BB agonist antibody 4B4-1-1 version 2.

[0181] SEQ ID NO: 51 is the light chain variable region (VL) of the 4-1BB agonist antibody 4B4-1-1 version 2.

[0182] SEQ ID NO: 52 is the heavy chain variable region (VH) of the 4-1BB agonist antibody H39E3-2.

[0183] SEQ ID NO: 53 is the light chain variable region (VL) of the 4-1BB agonist antibody H39E3-2.

[0184] SEQ ID NO: 54 is the amino acid sequence of human OX40.

[0185] SEQ ID NO: 55 is the amino acid sequence of mouse OX40.

[0186] SEQ ID NO: 56 is the heavy chain of the OX40 agonist monoclonal antibody tabolixizumab (MEDI-0562).

[0187] SEQ ID NO: 57 is the light chain of the OX40 agonist monoclonal antibody tabolixizumab (MEDI-0562).

[0188] SEQ ID NO: 58 is the heavy chain variable region (VH) of the OX40 agonist monoclonal antibody tabolixizumab (MEDI-0562).

[0189] SEQ ID NO: 59 is the light chain variable region (VL) of the OX40 agonist monoclonal antibody tabolixizumab (MEDI-0562).

[0190] SEQ ID NO: 60 is the heavy chain CDR1 of the OX40 agonist monoclonal antibody tabolixizumab (MEDI-0562).

[0191] SEQ ID NO: 61 is the heavy chain CDR2 of the OX40 agonist monoclonal antibody tabolixizumab (MEDI-0562).

[0192] SEQ ID NO: 62 is the heavy chain CDR3 of the OX40 agonist monoclonal antibody tabolixizumab (MEDI-0562).

[0193] SEQ ID NO: 63 is the light chain CDR1 of the OX40 agonist monoclonal antibody tabolixizumab (MEDI-0562).

[0194] SEQ ID NO: 64 is the light chain CDR2 of the OX40 agonist monoclonal antibody tabolixizumab (MEDI-0562).

[0195] SEQ ID NO: 65 is the light chain CDR3 of the OX40 agonist monoclonal antibody tabolixizumab (MEDI-0562).

[0196] SEQ ID NO: 66 is the heavy chain of the OX40 agonist monoclonal antibody 11D4.

[0197] SEQ ID NO: 67 is the light chain of the OX40 agonist monoclonal antibody 11D4.

[0198] SEQ ID NO: 68 is the heavy chain variable region (VH) of the OX40 agonist monoclonal antibody 11D4.

[0199] SEQ ID NO: 69 is the light chain variable region (VL) of the OX40 agonist monoclonal antibody 11D4.

[0200] SEQ ID NO: 70 is the heavy chain CDR1 of the OX40 agonist monoclonal antibody 11D4.

[0201] SEQ ID NO: 71 is the heavy chain CDR2 of the OX40 agonist monoclonal antibody 11D4.

[0202] SEQ ID NO: 72 is the heavy chain CDR3 of the OX40 agonist monoclonal antibody 11D4.

[0203] SEQ ID NO: 73 is the light chain CDR1 of the OX40 agonist monoclonal antibody 11D4.

[0204] SEQ ID NO: 74 is the light chain CDR2 of the OX40 agonist monoclonal antibody 11D4.

[0205] SEQ ID NO: 75 is the light chain CDR3 of the OX40 agonist monoclonal antibody 11D4.

[0206] SEQ ID NO: 76 is the heavy chain of the OX40 agonist monoclonal antibody 18D8.

[0207] SEQ ID NO: 77 is the light chain of the OX40 agonist monoclonal antibody 18D8.

[0208] SEQ ID NO: 78 is the heavy chain variable region (VH) of the OX40 agonist monoclonal antibody 18D8.

[0209] SEQ ID NO: 79 is the light chain variable region (VL) of the OX40 agonist monoclonal antibody 18D8.

[0210] SEQ ID NO: 80 is the heavy chain CDR1 of the OX40 agonist monoclonal antibody 18D8.

[0211] SEQ ID NO: 81 is the heavy chain CDR2 of the OX40 agonist monoclonal antibody 18D8.

[0212] SEQ ID NO: 82 is the heavy chain CDR3 of the OX40 agonist monoclonal antibody 18D8.

[0213] SEQ ID NO: 83 is the light chain CDR1 of the OX40 agonist monoclonal antibody 18D8.

[0214] SEQ ID NO: 84 is the light chain CDR2 of the OX40 agonist monoclonal antibody 18D8.

[0215] SEQ ID NO: 85 is the light chain CDR3 of the OX40 agonist monoclonal antibody 18D8.

[0216] SEQ ID NO: 86 is the heavy chain variable region (VH) of the OX40 agonist monoclonal antibody Hu119-122.

[0217] SEQ ID NO: 87 is the light chain variable region (VL) of the OX40 agonist monoclonal antibody Hu119-122.

[0218] SEQ ID NO: 88 is the heavy chain CDR1 of the OX40 agonist monoclonal antibody Hu119-122.

[0219] SEQ ID NO: 89 is the heavy chain CDR2 of the OX40 agonist monoclonal antibody Hu119-122.

[0220] SEQ ID NO: 90 is the heavy chain CDR3 of the OX40 agonist monoclonal antibody Hu119-122.

[0221] SEQ ID NO: 91 is the light chain CDR1 of the OX40 agonist monoclonal antibody Hu119-122.

[0222] SEQ ID NO: 92 is the light chain CDR2 of the OX40 agonist monoclonal antibody Hu119-122.

[0223] SEQ ID NO: 93 is the light chain CDR3 of the OX40 agonist monoclonal antibody Hu119-122.

[0224] SEQ ID NO: 94 is the heavy chain variable region (VH) of the OX40 agonist monoclonal antibody Hu106-222.

[0225] SEQ ID NO: 95 is the light chain variable region (VL) of the OX40 agonist monoclonal antibody Hu106-222.

[0226] SEQ ID NO: 96 is the heavy chain CDR1 of the OX40 agonist monoclonal antibody Hu106-222.

[0227] SEQ ID NO: 97 is the heavy chain CDR2 of the OX40 agonist monoclonal antibody Hu106-222.

[0228] SEQ ID NO: 98 is the heavy chain CDR3 of the OX40 agonist monoclonal antibody Hu106-222.

[0229] SEQ ID NO: 99 is the light chain CDR1 of the OX40 agonist monoclonal antibody Hu106-222.

[0230] SEQ ID NO: 100 is the light chain CDR2 of the OX40 agonist monoclonal antibody Hu106-222.

[0231] SEQ ID NO: 101 is the light chain CDR3 of the OX40 agonist monoclonal antibody Hu106-222.

[0232] SEQ ID NO: 102 is the OX40 ligand (OX40L) amino acid sequence.

[0233] SEQ ID NO: 103 is the soluble portion of the OX40L polypeptide.

[0234] SEQ ID NO: 104 is an alternative soluble portion of the OX40L polypeptide.

[0235] SEQ ID NO: 105 is the heavy chain variable region (VH) of OX40 agonist monoclonal antibody 008.

[0236] SEQ ID NO: 106 is the light chain variable region (VL) of OX40 agonist monoclonal antibody 008.

[0237] SEQ ID NO: 107 is the heavy chain variable region (VH) of OX40 agonist monoclonal antibody 011.

[0238] SEQ ID NO: 108 is the light chain variable region (VL) of OX40 agonist monoclonal antibody 011.

[0239] SEQ ID NO: 109 is the heavy chain variable region (VH) of OX40 agonist monoclonal antibody 021.

[0240] SEQ ID NO: 110 is the light chain variable region (VL) of OX40 agonist monoclonal antibody 021.

[0241] SEQ ID NO: 111 is the heavy chain variable region (V) of the OX40 agonist monoclonal antibody 023. H).

[0242] SEQ ID NO: 112 is the light chain variable region (VL) of OX40 agonist monoclonal antibody 023.

[0243] SEQ ID NO: 113 is the heavy chain variable region (VH) of an OX40 agonist monoclonal antibody.

[0244] SEQ ID NO: 114 is the light chain variable region (VL) of an OX40 agonist monoclonal antibody.

[0245] SEQ ID NO: 115 is the heavy chain variable region (VH) of an OX40 agonist monoclonal antibody.

[0246] SEQ ID NO: 116 is the light chain variable region (VL) of the OX40 agonist monoclonal antibody.

[0247] SEQ ID NO: 117 is the heavy chain variable region (VH) of a humanized OX40 agonist monoclonal antibody.

[0248] SEQ ID NO: 118 is the heavy chain variable region (VH) of a humanized OX40 agonist monoclonal antibody.

[0249] SEQ ID NO: 119 is the light chain variable region (VL) of a humanized OX40 agonist monoclonal antibody.

[0250] SEQ ID NO: 120 is the light chain variable region (VL) of a humanized OX40 agonist monoclonal antibody.

[0251] SEQ ID NO: 121 is the heavy chain variable region (VH) of a humanized OX40 agonist monoclonal antibody.

[0252] SEQ ID NO: 122 is the heavy chain variable region (VH) of a humanized OX40 agonist monoclonal antibody.

[0253] SEQ ID NO: 123 is the light chain variable region (VL) of a humanized OX40 agonist monoclonal antibody.

[0254] SEQ ID NO: 124 is the light chain variable region (VL) of a humanized OX40 agonist monoclonal antibody.

[0255] SEQ ID NO: 125 is the heavy chain variable region (VH) of an OX40 agonist monoclonal antibody.

[0256] SEQ ID NO: 126 is the light chain variable region (VL) of an OX40 agonist monoclonal antibody.

[0257] SEQ ID NO: 127 is the heavy chain amino acid sequence of the PD-1 inhibitor nivolumab.

[0258] SEQ ID NO: 128 is the light chain amino acid sequence of the PD-1 inhibitor nivolumab.

[0259] SEQ ID NO: 129 represents the heavy chain variable region (V) of the PD-1 inhibitor nivolumab H ) amino acid sequence.

[0260] SEQ ID NO: 130 represents the light chain variable region (V) of the PD-1 inhibitor nivolumab L ) amino acid sequence.

[0261] SEQ ID NO: 131 is the heavy chain CDR1 amino acid sequence of the PD-1 inhibitor nivolumab.

[0262] SEQ ID NO: 132 is the heavy chain CDR2 amino acid sequence of the PD-1 inhibitor nivolumab.

[0263] SEQ ID NO: 133 is the heavy chain CDR3 amino acid sequence of the PD-1 inhibitor nivolumab.

[0264] SEQ ID NO: 134 is the light chain CDR1 amino acid sequence of the PD-1 inhibitor nivolumab.

[0265] SEQ ID NO: 135 is the light chain CDR2 amino acid sequence of the PD-1 inhibitor nivolumab.

[0266] SEQ ID NO: 136 is the light chain CDR3 amino acid sequence of the PD-1 inhibitor nivolumab.

[0267] SEQ ID NO: 137 is the heavy chain amino acid sequence of the PD-1 inhibitor pembrolizumab.

[0268] SEQ ID NO: 138 is the light chain amino acid sequence of the PD-1 inhibitor pembrolizumab.

[0269] SEQ ID NO: 139 is the heavy chain variable region (V) of the PD-1 inhibitor pembrolizumab H ) amino acid sequence.

[0270] SEQ ID NO: 140 represents the light chain variable region (V) of the PD-1 inhibitor pembrolizumab L ) amino acid sequence.

[0271] SEQ ID NO: 141 is the heavy chain CDR1 amino acid sequence of the PD-1 inhibitor pembrolizumab.

[0272] SEQ ID NO: 142 is the heavy chain CDR2 amino acid sequence of the PD-1 inhibitor pembrolizumab.

[0273] SEQ ID NO: 143 is the heavy chain CDR3 amino acid sequence of the PD-1 inhibitor pembrolizumab.

[0274] SEQ ID NO: 144 is the light chain CDR1 amino acid sequence of the PD-1 inhibitor pembrolizumab.

[0275] SEQ ID NO: 145 is the light chain CDR2 amino acid sequence of the PD-1 inhibitor pembrolizumab.

[0276] SEQ ID NO: 146 is the light chain CDR3 amino acid sequence of the PD-1 inhibitor pembrolizumab.

[0277] SEQ ID NO: 147 is the heavy chain amino acid sequence of the PD-L1 inhibitor durvalumab.

[0278] SEQ ID NO: 148 is the light chain amino acid sequence of the PD-L1 inhibitor durvalumab.

[0279] SEQ ID NO: 149 represents the heavy chain variable region (V) of the PD-L1 inhibitor durvalumab H ) amino acid sequence.

[0280] SEQ ID NO: 150 represents the light chain variable region (V) of the PD-L1 inhibitor durvalumab L) amino acid sequence.

[0281] SEQ ID NO: 151 is the heavy chain CDR1 amino acid sequence of the PD-L1 inhibitor durvalumab.

[0282] SEQ ID NO: 152 is the heavy chain CDR2 amino acid sequence of the PD-L1 inhibitor durvalumab.

[0283] SEQ ID NO: 153 is the heavy chain CDR3 amino acid sequence of the PD-L1 inhibitor durvalumab.

[0284] SEQ ID NO: 154 is the light chain CDR1 amino acid sequence of the PD-L1 inhibitor durvalumab.

[0285] SEQ ID NO: 155 is the light chain CDR2 amino acid sequence of the PD-L1 inhibitor durvalumab.

[0286] SEQ ID NO: 156 is the light chain CDR3 amino acid sequence of the PD-L1 inhibitor durvalumab.

[0287] SEQ ID NO: 157 is the heavy chain amino acid sequence of the PD-L1 inhibitor avelumab.

[0288] SEQ ID NO: 158 is the light chain amino acid sequence of the PD-L1 inhibitor avelumab.

[0289] SEQ ID NO: 159 is the heavy chain variable region (V) of the PD-L1 inhibitor avelumab H ) amino acid sequence.

[0290] SEQ ID NO: 160 represents the light chain variable region (V) of the PD-L1 inhibitor avelumab L ) amino acid sequence.

[0291] SEQ ID NO: 161 is the heavy chain CDR1 amino acid sequence of the PD-L1 inhibitor avelumab.

[0292] SEQ ID NO: 162 is the heavy chain CDR2 amino acid sequence of the PD-L1 inhibitor avelumab.

[0293] SEQ ID NO: 163 is the heavy chain CDR3 amino acid sequence of the PD-L1 inhibitor avelumab.

[0294] SEQ ID NO: 164 is the light chain CDR1 amino acid sequence of the PD-L1 inhibitor avelumab.

[0295] SEQ ID NO: 165 is the light chain CDR2 amino acid sequence of the PD-L1 inhibitor avelumab.

[0296] SEQ ID NO: 166 is the light chain CDR3 amino acid sequence of the PD-L1 inhibitor avelumab.

[0297] SEQ ID NO: 167 is the heavy chain amino acid sequence of the PD-L1 inhibitor atezolizumab.

[0298] SEQ ID NO: 168 is the light chain amino acid sequence of the PD-L1 inhibitor atezolizumab.

[0299] SEQ ID NO: 169 is the heavy chain variable region (V H ) amino acid sequence.

[0300] SEQ ID NO: 170 represents the light chain variable region (V) of the PD-L1 inhibitor atezolizumab L ) amino acid sequence.

[0301] SEQ ID NO: 171 is the heavy chain CDR1 amino acid sequence of the PD-L1 inhibitor atezolizumab.

[0302] SEQ ID NO: 172 is the heavy chain CDR2 amino acid sequence of the PD-L1 inhibitor atezolizumab.

[0303] SEQ ID NO: 173 is the heavy chain CDR3 amino acid sequence of the PD-L1 inhibitor atezolizumab.

[0304] SEQ ID NO: 174 is the light chain CDR1 amino acid sequence of the PD-L1 inhibitor atezolizumab.

[0305] SEQ ID NO: 175 is the light chain CDR2 amino acid sequence of the PD-L1 inhibitor atezolizumab.

[0306] SEQ ID NO: 176 is the light chain CDR3 amino acid sequence of the PD-L1 inhibitor atezolizumab. DETAILED DESCRIPTION OF THE INVENTION

[0307] I. Introduction Adoptive cell therapy utilizing TILs cultured ex vivo by rapid expansion protocol (REP) has been successful in patients with cancers such as melanoma after host immunosuppression. Current infusion acceptance parameters rely on the readout of TIL composition (e.g., CD28, CD8, or CD4 positivity) as well as the numerical fold expansion and viability of the REP product.

[0308] Current REP protocols offer little insight into the health of TILs infused into patients. T cells undergo significant metabolic shifts during their maturation from naive to effector T cells (see Chang, et al., Nat. Immunol. 2016, 17, 364, expressly incorporated herein in its entirety, specifically for discussion, as well as markers of anaerobic and aerobic metabolism). For example, naive T cells rely on mitochondrial respiration to produce ATP, whereas mature, healthy effector T cells, such as TILs, are highly glycolytic and rely on aerobic glycolysis to provide the bioenergetic substrates necessary for proliferation, migration, activation, and antitumor efficacy.

[0309] Current TIL production and treatment processes are limited by length, cost, sterility concerns, and other factors described herein, severely limiting the ability to treat patients who are refractory to anti-PD-1. 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 viable treatment options remaining. The present invention fulfills this need by providing an abbreviated production process for use in generating TILs that can be used to treat non-small cell lung cancer (NSCLC) patients who are refractory to anti-PD-1 treatment.

[0310] II. 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.

[0311] 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) 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.

[0312] The term "in vivo" refers to events that take place inside a subject's body.

[0313] 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.

[0314] The term "ex vivo" refers to events involving treating or performing procedures on 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.

[0315] The term "rapid expansion" refers to an increase in the number of antigen-specific TILs by at least about 3-fold (or 4, 5, 6, 7, 8, or 9-fold) over a one-week period, more preferably by at least about 10-fold (or 20, 30, 40, 50, 60, 70, 80, or 90-fold) over a one-week period, or most preferably by at least about 100-fold over a one-week period. Several rapid expansion protocols are described herein.

[0316]

[0317] 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 patient tissue samples as outlined herein (sometimes referred to as "freshly harvested"), and "secondary TILs" are any TIL cell populations that have been expanded or propagated as discussed herein, including, but not limited to, bulk TILs and expanded TILs ("REP TILs" or "post-REP TILs"). TIL cell populations may include genetically modified TILs.

[0318] As used herein, a "population of cells" (including TILs) refers to several cells that share a common trait. Generally, a population is roughly 1 x 10 6 ~1×10 10 The number of TILs ranges from approximately 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.

[0319] "Cryopreserved TILs" as used herein refer to primary, bulk, or expanded TILs. Cryopreservation refers to the processing and storage of TILs (TILs) 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.

[0320] 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.

[0321] TILs can generally be biochemically defined using cell surface markers, or functionally defined by their ability to infiltrate tumors and carry out treatment.TILs can generally be classified by expressing one or more biomarkers: CD4, CD8, TCRαβ, CD27, CD28, CD56, CCR7, CD45Ra, CD95, PD-1, and CD25.In addition and alternatively, TILs can be functionally defined by their ability to infiltrate solid tumors when reintroduced into patients.

[0322] 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.

[0323] The term "central memory T cells" refers to cells that are CD45R0+ and CCR7 (CCR7 hi ) and CD62L (CD62 hi Central memory T cells are a subset of T cells that constitutively express CD4+, CD3+, CD127 (IL-7R), and IL-15R. 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.

[0324] 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 lo ), heterogeneous or low CD62L expression (CD62L lo), 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 (including interferon-γ, IL-4, and IL-5) after antigen stimulation. 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.

[0325] 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. Examples of closed systems include, for example, a sealed G vessel. Once the tumor segments are added to the closed system, the system is not opened to the outside environment until the TILs are ready to be administered to the patient.

[0326] 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.

[0327] 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. Preferably, peripheral blood mononuclear cells are irradiated allogeneic peripheral blood mononuclear cells. PBMCs are a type of antigen-presenting cell.

[0328] The term "anti-CD3 antibody" refers to an antibody or a variant thereof, e.g., a monoclonal antibody, including human, humanized, chimeric, or murine antibodies directed against the CD3 receptor in the T cell antigen receptor of mature T cells. Anti-CD3 antibodies include OKT-3, also known as muromonab. Anti-CD3 antibodies also include UHCT1 clones, also known as T3 and CD3ε. Other anti-CD3 antibodies include, for example, otelixizumab, teplizumab, and visilizumab.

[0329] The term "OKT-3" (also referred to herein as "OKT3") refers to a monoclonal antibody or biosimilar or variant thereof, including a human, humanized, chimeric, or murine antibody directed against the CD3 receptor in the T cell antigen receptor of mature T cells, and includes commercially available forms such as OKT-3 (30 ng / mL, MACS GMP CD3 pure, Miltenyi Biotech, Inc., San Diego, CA, USA) and muromonab or 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). Hybridomas capable of producing OKT-3 are listed in the American Type Culture Collection (American Type Culture Collection). The hybridoma capable of producing OKT-3 has also been deposited with the European Collection of Authenticated Cell Cultures (ECACC) and has been assigned ATCC accession number CRL 8001. The hybridoma capable of producing OKT-3 has also been deposited with the European Collection of Authenticated Cell Cultures (ECACC) and has been assigned catalog number 86022706. [Table 1]

[0330] The term "IL-2" (also referred to herein as "IL2") refers to the T-cell growth factor known as interleukin-2, in all forms, including human and mammalian forms, conservative amino acid substitutions, glycoforms, biosimilars, and variants thereof. IL-2 includes IL-2 of the formula (I). 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 forms of IL-2 such as aldesleukin (PROLEUKIN, commercially available from multiple sources at 22 million IU per single-use vial), as well as forms of recombinant IL-2 (catalog number CYT-209-b) commercially available from 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 form of IL-2 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 encompasses pegylated forms of IL-2 described herein, including the pegylated IL2 prodrug NKTR-214, available from Nektar Therapeutics (South San Francisco, CA, USA). NKTR-214 and pegylated IL-2 suitable for use in the present invention are described in U.S. Patent Application Publication No. US2014 / 0328791A1 and International Patent Application Publication No. WO2012 / 065086A1, 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. [Table 2]

[0331] 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 then 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 sources, 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: 5).

[0332] 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 α and common γ chain receptor, which stimulates T cell development in the thymus and T cell proliferation in the periphery. Recombinant human IL-7 suitable for use in the present invention is available from ProSpec-Tany TechnoGene Ltd., East Brunswick, NJ, USA (catalog no. CYT-254) and ThermoFisher. Human IL-15 is commercially available from several sources, including Gibco Scientific, Inc., Waltham, MA, USA (human IL-15 recombinant protein, catalog number Gibco PHC0071). The amino acid sequence of recombinant human IL-7 suitable for use in the present invention is shown in Table 2 (SEQ ID NO: 6).

[0333] 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 thereof. 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 sources, 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: 7).

[0334] 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 thereof. 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 primarily stimulates natural killer T cells and activated human CD4 + It is produced by T cells. Recombinant human IL-21 is a single, non-glycosylated polypeptide chain containing 132 amino acids with a molecular 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: 8).

[0335] 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 age, weight, tumor size, extent of infection or metastasis, and patient (subject) condition. Generally, the 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 of body weight) 5 ~10 6 , 10 5 ~10 10 , 10 5 ~10 11 , 10 6 ~10 10 , 10 6 ~10 11 , 107 ~10 11 , 10 7 ~10 10 , 10 8 ~10 11 , 10 8 ~10 10 , 10 9 ~10 11 , or 10 9 ~10 10 The tumor-infiltrating lymphocyte (optionally including genetically modified cytotoxic lymphocyte) composition may also be administered multiple times at these dosages. The tumor-infiltrating lymphocyte (optionally including genetically modified cytotoxic lymphocyte) composition may also be administered multiple times at these dosages. The tumor-infiltrating lymphocyte (optionally including genetically modified cytotoxic lymphocyte) composition may also be administered multiple times at these dosages. Administration can be by using conventional techniques (see, e.g., Rosenberg et al., New Eng. J. of Med. 319:1676, 1988). Optimal dosages and treatment regimes for a particular patient can be readily determined by one skilled in the art of medicine by monitoring the patient for signs of disease and adjusting treatment accordingly.

[0336] As used herein, the term "microenvironment" 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 mechanistic cues that promote neoplastic transformation, support tumor growth and invasion, protect tumors from host immunity, foster therapeutic resistance, and provide a niche for successful 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.

[0337] In embodiments, the present invention includes a method of treating cancer with a population of TILs, wherein the patient is pre-treated with non-myeloablative chemotherapy prior to infusion of TILs according to the present invention. In some embodiments, a population of TILs can be provided, wherein the patient is pre-treated with non-myeloablative chemotherapy prior to infusion of TILs according to the present invention. In embodiments, the non-myeloablative chemotherapy is cyclophosphamide 60 mg / kg / day for two days (27 and 26 days before TIL infusion) and fludarabine 25 mg / m2 / day for five days (27-23 days before TIL infusion). In embodiments, after non-myeloablative chemotherapy and TIL infusion according to the present invention (day 0), the patient receives an intravenous infusion of IL-2 at 720,000 IU / kg every 8 hours to physiological tolerance.

[0338] Experimental results 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") in patients prior to introducing the rTILs of the present invention.

[0339] 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 mode 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 in which the compound is delivered.

[0340] The terms "treatment," "treating," "treating," and the like refer to obtaining a desired pharmacological and / or physiological effect. The effect may be prophylactic, in that it completely or partially prevents a disease or its symptoms, and / or therapeutic, in that it partially or completely cures a disease and / or side effects resulting from a disease. As used herein, "treatment" encompasses any treatment of a disease in a mammal, particularly a human, and includes (a) preventing a disease from occurring in a subject who may be susceptible to the disease but has not yet been diagnosed as having the disease; (b) inhibiting a disease, i.e., arresting the development or progression of a disease; and (c) palliating a disease, i.e., inducing regression of a disease. "Treatment" includes causing and / or alleviating one or more disease symptoms. "Treatment" is also meant 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.

[0341] 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 essentially the same relationship to each other. For example, nucleic acids are typically produced recombinantly, with two or more sequences from unrelated genes arranged to create a new functional nucleic acid, such as 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 essentially the same relationship to each other (e.g., a fusion protein).

[0342] 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 program suite available from the BLAST website of the U.S. government's National Center for Biotechnology Information. Comparison between two sequences can be performed using either the BLASTN or BLASTP algorithm. BLASTN is used to compare nucleic acid sequences, and BLASTP is used to compare amino acid sequences. ALIGN, available from DNASTAR, ALIGN-2 (Genentech, South San Francisco, California), or MegAlign are additional publicly available software programs that can be used to align sequences. Those skilled in the art can determine appropriate parameters for maximum alignment depending on the particular alignment software. In certain embodiments, the default parameters of the alignment software are used.

[0343] As used herein, the term "variant" includes, but is not limited to, antibodies or fusion proteins that contain 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 certain 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.

[0344] 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 + TILs 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 TIL cell populations that have been expanded or propagated as discussed herein, including bulk TILs, expanded TILs, and the like, as discussed herein. L ("REP TIL"), as well as a "reREP TIL." A reREP TIL may include, for example, a second extended TIL or a second additional extended TIL (e.g., such as those described in step D of FIG. 8, which includes a TIL referred to as a reREP TIL).

[0345] TILs can generally be biochemically defined using cell surface markers, or functionally defined by their ability to infiltrate tumors and carry out treatment.TILs can generally be classified by expressing one or more biomarkers: CD4, CD8, TCRαβ, CD27, CD28, CD56, CCR7, CD45Ra, CD95, PD-1, and CD25.In addition and alternatively, TILs can be functionally defined by their ability to infiltrate solid tumors when reintroduced into patients. A TILS can be further characterized by efficacy, for example, a TILS can be considered efficacious if the release of interferon (IFNγ) 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.

[0346] 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.

[0347] 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 the 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 may 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.

[0348] 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 present invention is contemplated. Additional active pharmaceutical ingredients, such as other drugs, can also be incorporated into the compositions and methods described.

[0349] 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%, more preferably still within 10%, and even more preferably within 5%. The allowable variation encompassed by the terms "about" or "approximately" depends on the particular system under study and can be readily understood by those skilled in the art. Furthermore, as used herein, the terms "about" and "approximately" mean that dimensions, sizes, formulations, parameters, shapes, and other quantities and characteristics are not, and need not be, exact, but may be approximate and / or larger or smaller, and may be within tolerances, as needed. , conversion factors, rounding, measurement error, etc., and other factors known to those skilled in the art. Generally, 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 vastly different sizes, shapes, and dimensions may employ the described configurations.

[0350] 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 with respect to additional unrecited claim elements or steps, if any, excluded from the scope of the claim(s). 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 elements, steps, or materials other than those specified in the claim, and in the latter case, 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 those that do 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."

[0351] 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, termed 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 can 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.

[0352] 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.

[0353] The terms "monoclonal antibody," "mAb," "monoclonal antibody composition," or The monoclonal antibody compositions, or their plurals, refer to preparations 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 monoclonal antibodies 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 monoclonal antibodies in the recombinant host cells. Recombinant production of antibodies is described in more detail below.

[0354] 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 V Land (v) isolated complementarity-determining regions (CDRs). L and V H are encoded by separate genes, which can be recombined using recombinant methods to L and V H The regions can 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.

[0355] 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 a human germline immunoglobulin sequence. The human antibodies of the present 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.

[0356] The term "human monoclonal antibody" refers to antibodies displaying a single binding specificity having variable regions in which both the framework and CDR regions are derived from human germline immunoglobulin sequences. In 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. is produced.

[0357] As used herein, the term "recombinant human antibody" includes all human antibodies prepared, expressed, created, 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, created, 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), thus allowing for the development of recombinant antibody Vs. 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.

[0358] As used herein, "isotype" refers to the antibody class (e.g., IgM or IgG1) that is encoded by heavy chain constant region genes.

[0359] 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."

[0360] 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.

[0361] 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 the recipient's hypervariable region are replaced by residues from a hypervariable region of a non-human species (donor antibody), such as mouse, rat, rabbit, or non-human primate, possessing the desired specificity, affinity, and capacity. In some cases, Fv framework (FR) residues of the human immunoglobulin are replaced by corresponding non-human residues. Furthermore, humanized antibodies may contain residues not found in the recipient or donor antibody. These modifications are made to further improve 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 humanized antibody described herein may comprise any of the following: The antibodies to be used may 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, for example, in International Patent Application Publication Nos. WO1988 / 07089A1, WO1996 / 14339A1, WO1998 / 05787A1, WO1998 / 23289A1, WO1999 / 51642A1, WO99 / 58572A1, WO2000 / 09560A2, WO2000 / 32767A1, WO2000 / 42072A2, WO2002 / 442 15A2, WO2002 / 060919A2, WO2003 / 074569A2, WO2004 / 016750A2, WO2004 / 029207A2, WO2004 / 0357 52A2, WO2004 / 063351A2, WO2004 / 074455A2, WO2004 / 099249A2, WO2005 / 040217A2, WO2005 / 0709 63A1, 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).

[0362] 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.

[0363] A "diabody" is a small antibody fragment that has two antigen-binding sites. The fragments bind to the same polypeptide chain (V H -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 allow pairing of the two domains on the same chain is used, the domains are forced to pair with the complementary domains on another chain, creating two antigen-binding sites. Bispecific antibodies are more fully described, for example, in 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.

[0364] The term "glycosylation" refers to modified derivatives of antibodies. Aglycosylated antibodies lack glycosylation. Glycosylation can be altered to, for example, increase the affinity of an 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 can increase the affinity of an antibody for an 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 invention, thereby producing antibodies with altered glycosylation. For example, the Ms704, Ms705, and Ms709 cell lines Cell lines Ms704, Ms705, and Ms709 lack the fucosyltransferase gene FUT8 (α(1,6) fucosyltransferase) so that the expressed antibodies lack fucose on their carbohydrates. The FUT8- / - cell line was created 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 a cell line with a functionally disrupted FUT8 gene encoding a fucosyltransferase, thereby causing antibodies expressed in such cell lines to exhibit hypofucosylation by reducing or eliminating α1,6-linkage-related enzymes. The document also describes a cell line with reduced or no enzymatic activity for adding fucose to N-acetylglucosamine linked to the Fc region of an antibody, 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, Lec13 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 a glycoprotein-modifying glycosyltransferase (e.g., β(1,4)-N-acetylglucosaminyltransferase III (GnTIII)), such that antibodies expressed in the engineered cell line exhibit increased bisecting GlcNac structures, resulting in increased ADCC activity of the antibody (see also Umana, et al., Nat. Biotech. 1999, 17, 176-180). Alternatively, a fucosidase enzyme can be used to cleave the fucose residues of the antibody, for example, the fucosidase α-L-fucosidase removes fucosyl residues from antibodies as described in Tarentino, et al., Biochem. 1975, 14, 5516-5523.

[0365] "PEGylation" typically 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)-polyethylene glycol (PEG) or a poly(ethylene glycol)-poly ... 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 a non-glycosylated antibody. Methods for pegylation are known in the art and can be applied to the antibodies of the present 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).

[0366] 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 with respect to product safety, purity, and potency. Furthermore, a similar biological or "biosimilar" drug is a biological drug that is similar to another biological drug already approved for use by the European Medicines Agency. The term "biosimilar" is also used interchangeably by regulatory agencies in other countries and regions. Biological products or biologics are drugs made by or derived from living sources, such as bacteria or yeast. They are derived from relatively small molecules, such as human insulin or erythropoietin. A biosimilar may consist of a single molecule, such as a monoclonal antibody, or a complex molecule such as a monoclonal antibody. For example, if the reference IL-2 protein is aldesleukin (PROLEUKIN), a protein approved by a drug regulatory agency for aldesleukin is a "biosimilar" of aldesleukin or a "biosimilar thereof." In Europe, a similar biological or "biosimilar" medicinal product is a biopharmaceutical similar to another biopharmaceutical already approved 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 authorization or licensing applications under Article 6 of Regulation (EC) No. 726 / 2004 and Article 10(4) of Directive 2001 / 83 / EC. An already authorized original biopharmaceutical is sometimes referred to as a "reference medicinal product" in Europe. Some of the requirements for a product to be considered a biosimilar are outlined in the CHMP guidelines on Similar Biological Medicinal Products. Additionally, product-specific guidelines, including those related to monoclonal antibody biosimilars, are provided by the EMA for each product and are published on its website. The 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 to treat the same conditions as the reference medicinal product. Thus, the biosimilars described herein may be considered to have similar or very similar quality characteristics to the reference medicinal product. Alternatively, or in addition, the biosimilars described herein may be considered to have similar or very similar biological activity to the reference medicinal product. Alternatively, or in addition, the biosimilars described herein may be considered to have a similar or very similar safety profile to the reference medicinal product.Alternatively, or in addition, a biosimilar described herein may be considered to have similar or very similar efficacy to a reference drug. As described herein, a biosimilar in Europe is compared to a reference drug authorized by the EMA. However, in some cases, a biosimilar may be compared to a biopharmaceutical authorized outside the European Economic Area (a non-EEA-authorized "comparator") in certain studies. Such studies include, for example, certain clinical studies and in vivo non-clinical studies. As used herein, the term "biosimilar" also relates to a biopharmaceutical that has been or can be compared to a non-EEA-authorized 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 contain an amino acid sequence that has 97% or greater sequence identity to the amino acid sequence of its reference drug, e.g., 97%, 98%, 99%, or 100% sequence identity. A biosimilar may contain 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 drug, provided that the differences do not result in changes in the drug's safety and / or efficacy. A biosimilar may have the same or different glycosylation pattern as the reference drug. In particular, but not exclusively, a biosimilar may have a different glycosylation pattern if the differences address or are intended to address safety concerns associated with the reference drug. In addition, a biosimilar may deviate from the reference drug in, for example, its strength, dosage form, formulation, excipients, and / or presentation, provided that the drug's safety and efficacy are not compromised. A biosimilar may contain differences, for example, in its pharmacokinetic (PK) and / or pharmacodynamic (PD) profile compared to the reference drug, but is still considered sufficiently similar to the reference drug to be approved or considered suitable for approval.In certain circumstances, biosimilars may have different binding characteristics compared to the reference drug. Biosimilars exhibit distinct characteristics, and different binding characteristics are not considered a barrier to approval by regulatory authorities such as the EMA as similar biological products. The term "biosimilar" is also used interchangeably by regulatory bodies in other countries and regions.

[0367] III. TIL Manufacturing Process An exemplary TIL process known as Process 2A that includes some of these features is shown in Figure 2, and some of the advantages of this embodiment of the invention over Process 1C are described in Figures F and G. An embodiment of Process 2A is shown in Figure 1.

[0368] As discussed herein, the present invention may include steps related to restimulating cryopreserved TILs to increase their metabolic activity, and therefore their relative health, before transplantation into a patient, and methods for testing said metabolic health. As generally outlined herein, TILs are generally harvested from patient samples and manipulated to expand their numbers before transplantation into a patient. In some embodiments, TILs may optionally be genetically engineered, as discussed below.

[0369] In some embodiments, TILs can be cryopreserved. Once thawed, they can be restimulated to enhance their metabolism before infusion into patients.

[0370] In some embodiments, as discussed in detail below and in the Examples and Figures, the first expansion (including the process referred to as preREP and shown as step A in FIG. 1 ) is shortened to 3-14 days, and the second expansion (including the process referred to as REP and shown as step B in FIG. 1 ) is shortened to 7-14 days. In some embodiments, the first expansion (e.g., the expansion described as step B in FIG. 1 ) is shortened to 11 days, and the second expansion (e.g., the expansion described as step D in FIG. 1 ) is shortened to 11 days. In some embodiments, the combination of the first and second expansions (e.g., the expansions described as steps B and D in FIG. 1 ) is shortened to 22 days, as discussed in detail below and in the Examples and Figures.

[0371] 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.

[0372] A. Step A: Obtaining a patient tumor sample Generally, TILs are initially obtained from a patient's tumor sample ("primary TILs") and 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.

[0373] 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 involves multi-lesion sampling (i.e., obtaining samples from one or more tumor sites and / or locations of 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. The tumor sample may also be a liquid tumor, such as a tumor obtained from a hematological malignancy. The solid tumor may be of lung tissue. In some embodiments, useful TILs are obtained from non-small cell lung cancer (NSCLC).

[0374] Once obtained, tumor samples are typically cut into sections of 1 to approximately 8 mm using sharp scraping. 3 fragmented into small pieces between about 2-3 mm 3 are particularly useful. TILs are cultured from these fragments using enzymatic tumor digests. Such tumor digests can be cultured in enzymatic media (e.g., Roswell Tumor digests can be produced by incubation in a 5% CO2-containing medium (RPMI) containing 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 produced by placing the tumor in enzyme medium, mechanically dissociating the tumor for approximately 1 minute, followed by incubation at 37°C in 5% CO2 for 30 minutes, and then repeating cycles of mechanical dissociation and incubation under the aforementioned conditions until only small tissue fragments are present. If the cell suspension contains a large number of red blood cells or dead cells at the end of this process, density gradient separation using FICOLL branched hydrophilic polysaccharides can be performed to remove these cells. Alternative methods known in the art, such as those described in U.S. Patent Application Publication No. 2012 / 0244133A1 (the disclosure of which is incorporated herein by reference), can be used. Any of the foregoing methods can be used in any of the embodiments described herein for methods of expanding TILs or treating cancer.

[0375] Generally, the harvested cell suspension is referred to as a "primary cell population" or "freshly harvested" cell population.

[0376] In some embodiments, fragmentation comprises physical fragmentation, including, for example, exfoliation and digestion. In some embodiments, fragmentation is physical fragmentation. In some embodiments, fragmentation is exfoliation. In some embodiments, fragmentation is by digestion. In some embodiments, TILs may be initially cultured from enzymatic tumor digests and tumor fragments obtained from a patient. In embodiments, TILs may be initially cultured from enzymatic tumor digests and tumor fragments obtained from a patient.

[0377] 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 tumor acquisition 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 fragments comprises about 30 to about 60 fragments, and has a total volume of about 1300 mm 3 ~approx. 1500mm 3 In some embodiments, the plurality of fragments comprises about 50 fragments and has a total volume of about 1350 mm 3 In some embodiments, the plurality of fragments comprises about 50 fragments and has a total mass of about 1 gram to about 1.5 grams. In some embodiments, the plurality of fragments comprises about 4 fragments.

[0378] 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 obtained by Approximately 1mm 3 ~10mm 3 In some embodiments, the tumor fragment is between about 1 mm 3 ~8mm 3 In some embodiments, the tumor fragment is between 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 mm3 In some embodiments, the tumor fragment is about 5 mm 3 In some embodiments, the tumor fragment is about 6 mm 3 In some embodiments, the tumor fragment is about 7 mm 3 In some embodiments, the tumor fragment is about 8 mm 3 In some embodiments, the tumor fragment is about 9 mm 3 In some embodiments, the tumor fragment is about 10 mm 3 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.

[0379] 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.

[0380] 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 can be mechanically dissociated for approximately 1 minute. The solution can then be incubated at 37°C in 5% CO2 for 30 minutes, after which it can be mechanically disrupted again for approximately 1 minute. After another 30 minutes of incubation at 37°C in 5% CO2, the tumor can be mechanically disrupted a third time for approximately 1 minute. In some embodiments, after the third mechanical disruption if large tissue fragments were present, the sample was subjected to one or two additional rounds of mechanical dissociation, with or without an additional incubation for 30 minutes at 37° C. in 5% CO. In some embodiments, if the cell suspension contained 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.

[0381] In some embodiments, the cell suspension harvested prior to the first expansion step is referred to as a "primary cell population" or "freshly harvested" cell population.

[0382] 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 illustrated in FIG. 1.

[0383] B. Step B: First Expansion In some embodiments, the methods provide for obtaining young TILs, which upon administration to a subject / patient can increase their replication cycle and thus may provide additional therapeutic benefit over older TILs (i.e., TILs that have undergone more rounds of replication before administration to a subject / patient). Characteristics of young TILs can be found in the literature, e.g., Donia, et al., Scandinavian Journal of Immunology ,75:157-167(2012), Dudley et al.,Clin Cancer Res,16:6122-6131(2010), Huang et al.,J Immunother,28(3):258-267(2005), Besser et al.,Clin Cancer Res,19(17):OF1-OF9(2013), Besser et al.,J Immunother 32:415-423(2009), Robbins,et al.,J Immunol 2004;173:7125-7130, Shen et al.,J Immunother,30:123-129(2007), Zhou,et al.,J Immunother,28:53-62(2005), and Tran,et al.,J Immunother, 31:742-751 (2008), all of which are incorporated herein by reference in their entireties.

[0384] The diverse antigen receptors of T and B lymphocytes are produced by somatic recombination of a limited but numerous 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 α, β, γ, and δ receptors. In some embodiments, there is increased expression of T cell receptor (TCR) α and / or β. In some embodiments, there is increased expression of T cell receptor (TCR) α. In some embodiments, there is increased expression of T cell receptor (TCR) β. In some embodiments, there is increased expression of T cell receptor (TCR) α. In some embodiments, there is increased expression of T cell receptor (TCR) β. In some embodiments, there is increased expression of TCRab (i.e., TCR α / β).

[0385] After detachment or digestion of tumor fragments, for example as 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 containing 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 approximately 1 x 10 8 In some embodiments, this primary cell population is cultured for a period of 7-14 days, resulting in a bulk TIL population, generally about 1 x 10 8 In some embodiments, this primary cell population is cultured for a period of 10-14 days, resulting in a bulk TIL population, generally about 1 x 10 8 In some embodiments, this primary cell population is cultured for a period of about 11 days, resulting in a bulk TIL population, generally about 1 x 10 8 yielding bulk TIL cells.

[0386] In a preferred embodiment, the expansion of TILs is carried out using an initial bulk TIL as described below and herein. A TIL expansion step (which can include, for example, a process referred to as pre-REP, such as that described in step B of FIG. 1), followed by a second expansion as described in step D below and herein (step D, which includes a process referred to as the Rapid Expansion Protocol (REP) step), followed by optional cryopreservation, and 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.

[0387] In embodiments in which TIL cultures are initiated in 24-well plates, e.g., Costar Using 24-well cell culture clusters, flat bottom (Corning Incorporated, Corning, NY), 1 × 10 cells were 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 It is between.

[0388] In some embodiments, the first expansion culture medium is referred to as "CM," an abbreviation for culture medium. In some embodiments, the CM of step B is 10% human AB serum, 25 mM The culture consisted of RPMI 1640 with GlutaMAX, supplemented with Hepes, and 10 mg / mL gentamicin. The culture was 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) (Figure 1), 10-40 x 10 cells in 10-40 mL of CM containing IL-2 are cultured. 6 Live 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.

[0389] 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 in medium containing inactivated human AB serum containing 6000 IU / mL of IL-2 (or, optionally, in the presence of an APC cell population, as outlined herein). This primary cell population is cultured for several days, generally 10-14 days, resulting in a bulk TIL population, generally approximately 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 IL-2 to about 10,000 IU / mL IL-2. The first expansion culture medium contains about 5,000 IU / mL of IL-2. In some embodiments, the first expansion culture medium contains about 8,000 IU / mL to about 6,000 IU / mL of IL-2. In some embodiments, the first expansion culture medium contains about 7,000 IU / mL to about 6,000 IU / mL of IL-2. In some embodiments, the first expansion culture medium contains about 6,000 IU / mL of IL-2. In embodiments, the cell culture medium further contains IL-2. In some embodiments, the cell culture medium contains about 3000 IU / mL of IL-2. In embodiments, the cell culture medium further contains IL-2. In a preferred embodiment, the cell culture medium contains about 3000 IU / mL of IL-2. In 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 embodiments, the cell culture medium comprises between 1000-2000 IU / mL, between 2000-3000 IU / mL, between 3000-4000 IU / mL, between 4000-5000 IU / mL, between 5000-6000 IU / mL, 6000-7000 IU / mL, 7000-8000 IU / mL, or about 8000 IU / mL of IL-2.

[0390] 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 embodiments, the cell culture medium further comprises IL-15. In a preferred embodiment, the cell culture medium comprises about 180 IU / mL of IL-15.

[0391] In some embodiments, the first expansion culture medium comprises about 20 IU / mL IL-21, about 15 IU / mL IL-21, about 12 IU / mL IL-21, about 10 IU / mL IL-21, about 5 IU / mL IL-21, about 4 IU / mL IL-21, about 3 IU / mL IL-21, about 2 IU / mL IL-21, about 1 IU / mL IL-21, or about 0.5 IU / mL IL-21. In some embodiments, the first expansion culture medium comprises about 20 IU / mL to about 0.5 IU / mL IL-21. In some embodiments, the first expansion culture medium comprises about 15 IU / mL to about 0.5 IU / mL IL-21. In some embodiments, the first expansion culture medium contains about 12 IU / mL to about 0.5 IU / mL of IL-21. In some embodiments, the first expansion culture medium contains about 10 IU / mL to about 0.5 IU / mL of IL-21. In some embodiments, the first expansion culture medium contains about 5 IU / mL to about 1 IU / mL of IL-21. In some embodiments, the first expansion culture medium contains about 2 IU / mL of IL-21. In some embodiments, the cell culture medium contains about 1 IU / mL of IL-21. In some embodiments, the cell culture medium contains about 0.5 IU / mL of IL-21. In embodiments, the cell culture medium further contains IL-21. In a preferred embodiment, the cell culture medium contains about 1 IU / mL of IL-21.

[0392] In embodiments, the cell culture medium comprises an OKT-3 antibody. The cell culture medium comprises about 30 ng / mL of OKT-3 antibody. In embodiments, the cell culture medium comprises about 0.1 ng / mL, about 0.5 ng / mL, about 1 ng / mL, about 2.5 ng / mL, about 5 ng / mL, about 7.5 ng / mL, about 10 ng / mL, about 15 ng / mL, about 20 ng / mL, about 25 ng / mL, about 30 ng / mL, about 35 ng / mL, about 40 ng / mL, about 50 ng / mL, about 60 ng / mL, about 70 ng / mL, about 80 ng / mL, about 90 ng / mL, about 100 ng / mL, about 200 ng / mL, about 500 ng / mL, and about 1 μg / mL of OKT-3 antibody. In embodiments, the cell culture medium comprises an OKT-3 antibody at a concentration of between 0.1 ng / mL and 1 ng / mL, between 1 ng / mL and 5 ng / mL, between 5 ng / mL and 10 ng / mL, between 10 ng / mL and 20 ng / mL, between 20 ng / mL and 30 ng / mL, between 30 ng / mL and 40 ng / mL, between 40 ng / mL and 50 ng / mL, and between 50 ng / mL and 100 ng / mL. In some embodiments, the cell culture medium does not comprise an OKT-3 antibody. In some embodiments, the OKT-3 antibody is muromonab. [Table 3]

[0393] 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 concentration in the cell culture medium of between 0.1 μg / mL and 100 μg / mL. In some embodiments, the TNFRSF agonist is added at a concentration sufficient to achieve a concentration in the cell culture medium of between 20 μg / mL and 40 μg / mL.

[0394] 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.

[0395] 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. When the culture is 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) (Figure 1), 10-40 x 10 cells in 10-40 mL of CM containing IL-2 are cultured. 6 5 live tumor digestive cells or 5 Approximately 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 two to three 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.

[0396] In some embodiments, the first expansion (e.g., including 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, as discussed in the Examples and Figures. In some embodiments, the first expansion (e.g., including a process such as that described in step B of FIG. 1, which may include what is sometimes referred to as pre-REP) is shortened to 7-14 days, as discussed in the Examples and shown in FIGS. 4 and 5, as well as the expansion described in step B of FIG. 1, for example. 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, as discussed in the expansion described in step B of FIG. 1, for example.

[0397] In some embodiments, the first TIL expansion can proceed 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 proceed for 1 day to 14 days. In some embodiments, the first TIL expansion can proceed for 2 days to 14 days. In some embodiments, the first TIL expansion can proceed for 3 days to 14 days. In some embodiments, the first TIL expansion can proceed for 4 days to 14 days. In some embodiments, the first TIL expansion can proceed for 5 days to 14 days. In some embodiments, the first TIL expansion can proceed for 6 days to 14 days. In some embodiments, the first TIL expansion can proceed for 7 days to 14 days. In some embodiments, the first TIL expansion can proceed for 8 days to 14 days. In some embodiments, the first TIL expansion can proceed for 9 days to 14 days. In some embodiments, the first TIL expansion is allowed to proceed for 10 to 14 days. In some embodiments, the first TIL expansion is allowed to proceed for 11 to 14 days. In some embodiments, the first TIL expansion is allowed to proceed for 12 to 14 days. In some embodiments, the first TIL expansion is allowed to proceed for 13 to 14 days. In some embodiments, the first TIL expansion is allowed to proceed for 14 days. In some embodiments, the first TIL expansion is allowed to proceed for 1 to 11 days. In some embodiments, the first TIL expansion is allowed to proceed for 2 to 11 days. In some embodiments, the first TIL expansion is allowed to proceed for 3 to 11 days. In some embodiments, the first TIL expansion is allowed to proceed for 4 to 11 days. In some embodiments, the first TIL expansion is allowed to proceed for 5 to 11 days. In some embodiments, the first TIL expansion is allowed to proceed for 6 to 11 days. In some embodiments, the first TIL expansion is allowed to proceed for 7 to 11 days. In some embodiments, the first TIL expansion is allowed to proceed for 8 to 11 days.In some embodiments, the first TIL expansion is allowed to proceed for 9 to 11 days. In some embodiments, the first TIL expansion is allowed to proceed for 10 to 11 days. In some embodiments, the first TIL expansion is allowed to proceed for 11 days.

[0398] 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 of their Any combination may be included in the first expansion, including, for example, in the step B 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 in the step B process according to Figure 1 and described herein.

[0399] In some embodiments, the first expansion (e.g., step B according to FIG. 1 , including the process referred to as pre-REP) 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.

[0400] In some embodiments, the first expansion, e.g., step B according to FIG. 1, is performed in a closed bioreactor. In some embodiments, a closed system is used for TIL expansion as described herein. In some embodiments, a single bioreactor is used. In some embodiments, the single bioreactor used is, for example, a G-REX-10 or G-REX-100. In some embodiments, the closed bioreactor is a single bioreactor.

[0401] C. Step C: Transition from the first extension to the second extension In some cases, the bulk TIL population obtained from the first expansion, including, for example, the TIL population obtained from step B, as shown in Figure 1, can be immediately cryopreserved using the protocol 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) and then cryopreserved as discussed below. 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 treatment before expansion or after the first expansion and before the second expansion.

[0402] In some embodiments, TILs obtained from the first expansion (e.g., from step B as shown in FIG. 1 ) are stored until phenotyping for selection. In some embodiments, TILs obtained from the first expansion (e.g., from step B as 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 4-10 days after fragmentation occurs. In some embodiments, the transition from the first expansion to the second expansion occurs about 7-14 days after fragmentation occurs. In some embodiments, the transition from the first expansion to the second expansion occurs about 14 days after fragmentation occurs.

[0403] 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 day to 14 days after fragmentation occurs. In some embodiments, the transition from the first expansion to the second expansion occurs 1 day to 14 days after fragmentation occurs. TIL expansion can proceed 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.

[0404] 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, there is no preservation during the transition from step B to step D as shown in FIG. 1). In some embodiments, the transition occurs in a closed system, as described herein. In some embodiments, the TILs from the first expansion, the second TIL population, proceed directly to the second expansion without a transition period.

[0405] 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. In some embodiments, the closed system bioreactor is a single bioreactor.

[0406] 1. Cytokines The expansion methods described herein generally use culture media containing high doses of cytokines, particularly IL-2, as known in the art.

[0407] Alternatively, International Publication No. WO2015 / 189356 and International Publication No. WO2015 It is further possible to use combinations of cytokines for rapid and / or secondary expansion of TILs, such as combinations of two or more of IL-2, IL-15, and IL-21, as generally outlined in US Pat. No. 6,299,357, the entire contents of which are expressly 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 of which finds particular use in many embodiments. The use of combinations of cytokines is particularly advantageous for the generation of lymphocytes, particularly T cells as described therein. [Table 4]

[0408] D. Step D: Second Expansion 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 FIG. 1). This further expansion is referred to herein as second expansion, which may include an expansion process commonly referred to in the art as the rapid expansion process (REP, and the process shown in FIG. 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.

[0409] In some embodiments, the second expansion of TILs or secondary TIL expansion (which may include expansion sometimes referred to as REP, and the process shown in step D of FIG. 1) can be performed using any TIL flask or vessel known to those of skill in the art. In some embodiments, the second TIL expansion can proceed for 7, 8, 9, 10, 11, 12, 13, or 14 days. In some embodiments, the second TIL expansion can proceed for about 7 to about 14 days. In some embodiments, the second TIL expansion can proceed for about 7 to about 14 days. The TIL expansion can proceed for about 8 days to about 14 days. In some embodiments, the second TIL expansion can proceed for about 9 days to about 14 days. In some embodiments, the second TIL expansion can proceed for about 10 days to about 14 days. In some embodiments, the second TIL expansion can proceed for about 11 days to about 14 days. In some embodiments, the second TIL expansion can proceed for about 12 days to about 14 days. In some embodiments, the second TIL expansion can proceed for about 13 days to about 14 days. In some embodiments, the second TIL expansion can proceed for about 14 days.

[0410] In 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 thereof, such as a cancer epitope(s), which can be expressed from a vector such as a human leukocyte antigen A2 (HLA-A2)-binding peptide, e.g., 0.3 μM MART-1:26-35(27L) or gpl00:209-217(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, for example, 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 cancer antigen(s) pulsed onto antigen-presenting cells expressing HLA-A2. Alternatively, 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.

[0411] In 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 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 embodiments, the cell culture medium comprises between 1000-2000 IU / mL, between 2000-3000 IU / mL, between 3000-4000 IU / mL, between 4000-5000 IU / mL, between 5000-6000 IU / mL, 6000-7000 IU / mL, 7000-8000 IU / mL, or 8000 IU / mL of IL-2.

[0412] In embodiments, the cell culture medium comprises an OKT-3 antibody. In some embodiments, the cell culture medium comprises about 30 ng / mL of an OKT-3 antibody. In 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, or about 100 ng / mL. ng / mL, about 100 ng / mL, about 200 ng / mL, about 500 ng / mL, and about 1 μg / mL of OKT-3 antibody. In embodiments, the cell culture medium contains between 0.1 ng / mL and 1 ng / mL, between 1 ng / mL and 5 ng / mL, between 5 ng / mL and 10 ng / mL, between 10 ng / mL and 20 ng, between 20 ng / mL and 30 ng / mL, between 30 ng / mL and 40 ng / mL, between 40 ng / mL and 50 ng / mL, and 50 ng / mL and 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.

[0413] 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 concentration in the cell culture medium of between 0.1 μg / mL and 100 μg / mL. In some embodiments, the TNFRSF agonist is added at a concentration sufficient to achieve a concentration in the cell culture medium of between 20 μg / mL and 40 μg / mL.

[0414] 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.

[0415] 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, in step D 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 second expansion. In some embodiments, IL-2, IL-15, and IL-21, and any combination thereof, may be included during the step D process according to Figure 1 and described herein.

[0416] In some embodiments, the second expansion may be performed in a supplemented cell culture medium containing IL-2, OKT-3, antigen-presenting feeder cells, and optionally a TNFRSF agonist. In some embodiments, the second expansion occurs in a supplemented cell culture medium. In some embodiments, the supplemented cell culture medium contains IL-2, OKT-3, and antigen-presenting feeder cells. In some embodiments, the second cell culture medium contains IL-2, OKT-3, and antigen-presenting cells (APCs, also referred to as antigen-presenting feeder cells). In some embodiments, the second expansion occurs in a cell culture medium containing IL-2, OKT-3, and antigen-presenting feeder cells (i.e., antigen-presenting cells).

[0417] In some embodiments, the second expansion culture medium comprises 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 comprises about 500 IU / mL to about 100 IU / mL IL-15. In some embodiments, the second expansion culture medium comprises 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 embodiments, the cell culture medium further comprises IL-15. In a preferred embodiment, the cell culture medium comprises about 180 IU / mL of IL-15.

[0418] In some embodiments, the second expansion culture medium comprises about 20 IU / mL IL-21, about 15 IU / mL IL-21, about 12 IU / mL IL-21, about 10 IU / mL IL-21, about 5 IU / mL IL-21, about 4 IU / mL IL-21, about 3 IU / mL IL-21, about 2 IU / mL IL-21, about 1 IU / mL IL-21, or about 0.5 IU / mL IL-21. In some embodiments, the second expansion culture medium comprises about 20 IU / mL to about 0.5 IU / mL IL-21. In some embodiments, the second expansion culture medium comprises about 15 IU / mL to about 0.5 IU / mL 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 embodiments, the cell culture medium further comprises IL-21. In a preferred embodiment, the cell culture medium comprises about 1 IU / mL of IL-21.

[0419] In some embodiments, the antigen-presenting feeder cells (APCs) are PBMCs. In embodiments, the ratio of TILs to PBMCs and / or antigen-presenting cells during 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 embodiments, the ratio of TILs to PBMCs during rapid expansion and / or secondary expansion is between 1:50 and 1:300. In embodiments, the ratio of TILs to PBMCs during rapid expansion and / or secondary expansion is between 1:100 and 1:200.

[0420] In embodiments, REP and / or secondary expansion are 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 2 / 3 medium changes with respiration with 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.

[0421] In some embodiments, the second expansion (which may include a process referred to as the REP process) is shortened to 7-14 days, as discussed in the Examples and Figures. In some embodiments, the second expansion is shortened to 11 days.

[0422] In embodiments, REP and / or second expansion can 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 (G-Rex flasks). In some embodiments, second expansion (including expansion referred to as rapid expansion) is performed in T-175 flasks, containing approximately 1× 1000 cells suspended in 150 mL of medium. 10 6TILs 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 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 increase the number of cells to 0.5-2.0 x 10. 6 The cell count was maintained between 100 and 150 cells / mL.

[0423] In embodiments, the second expansion (which may include the expansion referred to as REP, as well as the expansion referenced in step D of FIG. 1) can be performed in a 500 mL gas-permeable flask with a 100 cm gas-permeable silicone bottom (G-Rex 100, commercially available from Wilson Wolf Manufacturing Corporation, New Brighton, MN, USA). 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 and placed in a centrifuge bottle and centrifuged at 1500 rpm (491 x g) for 10 minutes. The TIL pellet can be resuspended in 150 mL of fresh medium containing 5% human AB serum, 3000 IU / mL IL-2, and returned 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 can be suspended in the 300 mL of medium present in each flask, and the cell suspension can be divided into three 100 mL aliquots, which can be used to seed three G-Rex 100 flasks. Then, 150 mL of AIM-V containing 5% human AB serum and 3000 IU / mL IL-2 can be added to each flask. The G-Rex 100 flasks are incubated at 37°C in 5% CO2, and after 4 days, 150 mL of AIM-V containing 3000 IU / mL IL-2 can be added to each G-Rex 100 flask. On day 14 of culture, the cells can be harvested.

[0424] In embodiments, the second expansion (including the 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 replaced by respiration with fresh medium. In some embodiments, the alternative growth chamber includes G-REX flasks and gas-permeable vessels, as discussed more fully below.

[0425] In 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 can 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, can be used to select TILs for superior tumor reactivity.

[0426] Optionally, cell viability assays 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 can be performed on a sample of bulk TILs, which selectively labels dead cells and allows for assessment of viability. In some embodiments, TIL samples are cultured in a C Cells may be counted and viability determined using a Cellometer K2 automated cell counter (Nexcelom Bioscience, Lawrence, MA). In some embodiments, viability is determined according to standard Cellometer K2 Image Cytometer automated cell counter protocols.

[0427] In some embodiments, the second expansion of TILs (including expansion referred to as REP) can be performed using previously described T-175 flasks and gas-permeable bags (Tran KQ, Zhou J, Durflinger KH, et al., 2008, J Immunother., 31:742-751 and Dudley ME, Wunderlich JR, Shelton TE, et al., 2003, J Immunother., 26:332-342) or gas-permeable G-Rex flasks. In some embodiments, the second expansion is performed using flasks. In some embodiments, the second expansion is performed using gas-permeable G-Rex flasks. In some embodiments, the second expansion is performed in T-175 flasks and approximately 1 x 10 6TILs are suspended in approximately 150 mL of medium and added to each T-175 flask. TILs were cultured at a 1:100 ratio using irradiated (50 Gy) allogeneic PBMCs as "feeder" cells, and the cells were 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% CO2. 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 reach approximately 0.5 to 2.0 x 10 cells. 6 Cell numbers between 100 and 150 cells / mL can be maintained.

[0428] In some embodiments, the second extension (including the extension 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) (Figure 1), 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 returned to the original G-Rex 100 flask. In an embodiment in which TILs are continuously expanded in G-Rex 100 flasks, on day 7, the TILs in each G-Rex 100 are suspended in 300 mL of medium present in each flask, and the cell suspension is divided into three 100 mL aliquots that are used to seed three G-Rex 100 flasks. 150 mL of AIM-V containing 5% human AB serum and 3000 IU / mL of IL-2 is then added to each flask. The G-Rex 100 flasks are incubated at 37°C in 5% CO2, and after 4 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.

[0429] The diverse antigen receptors of T and B lymphocytes are produced by somatic recombination of a limited but numerous 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 this method exhibit increased T cell repertoire diversity. In some embodiments, the 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 α, β, γ, and δ receptors. In some embodiments, there is increased expression of T cell receptor (TCR) α and / or β. In some embodiments, there is increased expression of T cell receptor (TCR) α. In some embodiments, there is increased expression of T cell receptor (TCR) β. In some embodiments, there is increased expression of T cell receptor (TCR) α. In some embodiments, there is increased expression of T cell receptor (TCR) β. In some embodiments, there is increased expression of TCRab (i.e., TCR α / β).

[0430] In some embodiments, the second expansion culture medium (e.g., sometimes 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.

[0431] In some embodiments, the second expansion, e.g., step D according to FIG. 1, is performed in a closed bioreactor. In some embodiments, a closed system is used for TIL expansion as described herein. In some embodiments, a single bioreactor is used. In some embodiments, the single bioreactor used is, for example, a G-REX-10 or G-REX-100. In some embodiments, the closed bioreactor is a single bioreactor.

[0432] 1. Feeder cells and antigen-presenting cells In 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. The PBMCs are obtained using standard methods, such as Ficoll-Paque gradient separation.

[0433] 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 capacity of irradiated allogeneic PBMCs.

[0434] In some embodiments, if the total number of viable cells on day 14 is less than the initial number of viable cells cultured on day 0 of REP and / or day 0 of second expansion (i.e., the start day of second expansion), the PBMCs are considered replication incompetent and are approved for use in the TIL expansion procedures described herein.

[0435] In some embodiments, if the total number of viable cells cultured in the presence of OKT3 and IL-2 on days 7 and 14 does not increase from the initial number of viable cells cultured on REP day 0 and / or second expansion day 0 (i.e., the start day of second 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.

[0436] In some embodiments, if the total number of viable cells cultured in the presence of OKT3 and IL-2 on days 7 and 14 does not increase from the initial number of viable cells cultured on day 0 of REP and / or day 0 of second expansion (i.e., the start day of second expansion), PBM C 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 25-35 ng / mL OKT3 antibody and 2500-3500 IU / mL IL-2.

[0437] 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 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 embodiments, the ratio of TILs to antigen-presenting feeder cells in the second expansion is between 1:50 and 1:300. In embodiments, the ratio of TILs to antigen-presenting feeder cells in the second expansion is between 1:100 and 1:200.

[0438] In embodiments, the second expansion procedure described herein involves approximately 2.5×10 9 Feeder cells per approximately 100 x 10 6 In another embodiment, the second expansion procedure described herein requires a ratio of about 2.5 x 10 TILs. 9 Feeder cells per approximately 50 x 10 6 In yet another embodiment, the second expansion procedure described herein requires a ratio of about 2.5 x 10 TILs. 9 Feeder cells per approximately 25 x 10 6 Requires TIL.

[0439] In 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 embodiments, artificial antigen-presenting (aAPC) cells are used in place of PBMCs.

[0440] 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.

[0441] In embodiments, artificial antigen-presenting cells are used in the second expansion as a replacement for or in combination with PBMCs.

[0442] 2. Cytokines The expansion methods described herein generally use culture media containing high doses of cytokines, particularly IL-2, as known in the art.

[0443] Alternatively, it is further possible to use combinations of cytokines for rapid and / or secondary expansion of TILS, such as in combinations of two or more of IL-2, IL-15, and IL-21, as generally outlined in International Publication Nos. WO2015 / 189356 and WO2015 / 189357 (expressly incorporated herein by reference in their entireties). 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 of which finds particular use in many embodiments. The use of combinations of cytokines is particularly useful for lymphocytes, particularly those described therein. This is particularly favorable for the generation of T cells, which are involved in the immune response.

[0444] E. Step E: Harvesting TILs After the second expansion step, the cells can be harvested. In some embodiments, the TILs are harvested after one, two, three, four, or more expansion steps, 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.

[0445] TILs can be collected in any suitable sterile manner, for example, by centrifugation. Methods for collecting TILs are well known in the art, and such known methods can be used in this process. In some embodiments, TILs are collected using an automated system.

[0446] Cell harvesters and / or cell processing systems are commercially available from a variety of sources, including, for example, Fresenius Kabi, Tomtec Life Science, Perkin Elmer, and Inotech Biosystems International, Inc. Any cell-based harvester can be used in the present methods. In some embodiments, the cell harvester and / or cell processing system is a membrane-based cell harvester. In some embodiments, cell harvesting is performed via a cell processing system, such as the LOVO system (manufactured by Fresenius Kabi). The term "LOVO cell processing system" also refers to any device or apparatus manufactured by any vendor that pumps a cell-containing solution through a membrane or filter, such as a spun membrane or spun filter, in a sterile and / or closed environment, allowing for continuous flow and cell processing to remove supernatant or cell culture medium without pelleting. In some embodiments, the cell harvester and / or cell processing system can perform cell separation, washing, fluid exchange, concentration, and / or other cell processing steps in a closed, sterile system.

[0447] In some embodiments, harvesting, e.g., step E according to FIG. 1, is performed from a closed bioreactor. In some embodiments, a closed system is used for TIL expansion as described herein. In some embodiments, a single bioreactor is used. In some embodiments, the single bioreactor used is, for example, a G-REX-10 or G-REX-100. In some embodiments, the closed bioreactor is a single bioreactor.

[0448] In some embodiments, step E according to Figure 1 is performed according to the process described in Example G. In some embodiments, the closed system is accessed via a syringe under sterile conditions to maintain the sterility and closure of the system. In some embodiments, a closed system such as that described in Example G is used.

[0449] In some embodiments, TILs are harvested according to the methods described in Example G. In some embodiments, TILs between days 1 and 11 are harvested using the methods described in Section 8.5 (referred to in Example G as Day 11 TIL harvest). In some embodiments, TILs between days 12 and 22 are harvested using the methods described in Section 8.12 (referred to in Example G as Day 22 TIL harvest).

[0450] F. Step F: Transfer to Final Formulation / Infusion Bag After completing steps A-E, provided in an exemplary order in FIG. 1 and outlined in detail above and herein, the cells are transferred to a container for use in administering to a patient. In some embodiments, once a therapeutically sufficient number of TILs are obtained using the expansion methods described above, they are is transferred to a container for administration to a patient.

[0451] In embodiments, TILs expanded using the APCs of the present disclosure are administered to a patient as a pharmaceutical composition. In embodiments, the pharmaceutical composition is a suspension of TILs 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 administration routes include intraperitoneal, intrathecal, and intralymphatic.

[0452] G. Optional Cell Culture Media Components 1. Anti-CD3 antibody In some embodiments, the culture medium (including what is referred to as REP; see, e.g., FIG. 1) used in the expansion methods described herein also contains anti-CD3 antibody. Anti-CD3 antibody in combination with IL-2 induces T cell activation and cell division of TIL populations. This effect is seen with full-length antibodies, Fab, and F(ab')2 fragments, the former being generally preferred. See, e.g., Tsoukas et al., J. Immunol. 1985, 135, 1719 (incorporated herein by reference in its entirety).

[0453] As will be appreciated by those skilled in the art, there are several suitable anti-human CD3 antibodies that find use in the present invention, including anti-human CD3 polyclonal and monoclonal antibodies from various mammals, including, but not limited to, mouse, human, primate, rat, and canine antibodies. In a specific embodiment, the OKT3 anti-CD3 antibody is used (commercially available from Ortho-McNeil, Raritan, NJ or Miltenyi Biotech, Auburn, CA). [Table 5]

[0454] 2.4-1BB (CD137) agonist In embodiments, the TNFRSF agonist is a 4-1BB (CD137) agonist. The 4-1BB agonist can be any 4-1BB binding molecule known in the art. The 4-1BB binding molecule can be a monoclonal antibody or fusion protein capable of binding to human or mammalian 4-1BB. The 4-1BB agonist or 4-1BB binding molecule can include an immunoglobulin heavy chain of any isotype (e.g., IgG, IgE, IgM, IgD, IgA, and IgY), class (e.g., IgG1, IgG2, IgG3, IgG4, IgA1, and IgA2), or subclass of immunoglobulin molecule. The 4-1BB agonist or 4-1BB binding molecule can have both a heavy chain and a light chain. As used herein, the term binding molecule also refers to an antibody (including a full-length antibody), a monoclonal antibody (including a full-length monoclonal antibody), a polyclonal antibody, a multispecific antibody (e.g., a bispecific antibody), a human, humanized or chimeric antibody, and antibody fragments, e.g., Fab fragments, F(ab') fragments, fragments produced by a Fab expression library, epitope-binding fragments of any of the above, as well as engineered forms of antibodies that bind to 4-1BB, e.g., scFv molecules. In embodiments, the 4-1BB agonist is an antigen-binding protein that is a fully human antibody. In embodiments, the 4-1BB agonist is an antigen-binding protein that is a humanized antibody. In some embodiments, 4-1BB agonists for use in the methods and compositions disclosed herein include anti-4-1BB antibodies, human anti-4-1BB antibodies, mouse anti-4-1BB antibodies, mammalian anti-4-1BB antibodies, monoclonal anti-4-1BB antibodies, polyclonal anti-4-1BB antibodies, chimeric anti-4-1BB antibodies, anti-4-1BB Adnectins, anti-4-1BB domain antibodies, single-chain anti-4-1BB fragments, heavy-chain anti-4-1BB fragments, light-chain anti-4-1BB fragments, anti-4-1BB fusion proteins, and fragments, derivatives, conjugates, variants, or biosimilars thereof. Agonistic anti-4-1BB antibodies are known to induce potent immune responses. Lee, et al., PLOS One 2013, 8, e69677. In a preferred embodiment, the 4-1BB agonist is an agonist, anti-4-1BB humanized, or fully human monoclonal antibody (i.e., an antibody derived from a single cell line). In an embodiment, the 4-1BB agonist is EU-101 (Eutilex Co. Ltd.), utomilumab, or urelumab, or a fragment, derivative, conjugate, variant, or biosimilar thereof. In a preferred embodiment, the 4-1BB agonist is utomilumab or urelumab, or a fragment, derivative, conjugate, variant, or biosimilar thereof.

[0455] In a preferred embodiment, the 4-1BB agonist or 4-1BB binding molecule can also be a fusion protein. In a preferred embodiment, a multimeric 4-1BB agonist, such as a trimeric or hexameric 4-1BB agonist (having three or six ligand binding domains), can induce superior receptor (4-1BBL) clustering and intracellular signaling complex formation compared to agonist monoclonal antibodies, which typically have two ligand binding domains. Trimeric (trivalent) or hexameric (or hexavalent) or higher fusion proteins containing three TNFRSF binding domains and IgG1-Fc, optionally further combining two or more of these fusion proteins, are described, for example, in Gieffers, et al., Mol. Cancer Therapeutics 2013, 12, 2735-47.

[0456] Agonistic 4-1BB antibodies and fusion proteins are known to induce strong immune responses. In preferred embodiments, the 4-1BB agonist is a monoclonal antibody or fusion protein that specifically binds to the 4-1BB antigen in a manner sufficient to reduce toxicity. In some embodiments, the 4-1BB agonist is an agonistic 4-1BB monoclonal antibody or fusion protein that abolishes antibody-dependent cellular cytotoxicity (ADCC), e.g., NK cell cytotoxicity. In some embodiments, the 4-1BB agonist is an agonistic 4-1BB monoclonal antibody or fusion protein that abolishes antibody-dependent cellular phagocytosis (ADCP). In some embodiments, the 4-1BB agonist is an agonistic 4-1BB monoclonal antibody or fusion protein that abolishes complement-dependent cytotoxicity (CDC). In some embodiments, the 4-1BB agonist is an agonistic 4-1BB monoclonal antibody or fusion protein that abolishes Fc region functionality.

[0457] In some embodiments, the 4-1BB agonist is characterized by binding to human 4-1BB (SEQ ID NO: 9) with high affinity and agonistic activity. In embodiments, the 4-1BB agonist is a binding molecule that binds to human 4-1BB (SEQ ID NO: 9). In embodiments, the 4-1BB agonist is a binding molecule that binds to mouse 4-1BB (SEQ ID NO: 10). The amino acid sequences of the 4-1BB antigens to which the 4-1BB agonists or binding molecules bind are summarized in Table 6. [Table 6]

[0458] In some embodiments, the compositions, processes, and methods described have a K of about 100 pM or less for human or mouse 4-1BB. D binds to human or mouse 4-1BB with a K of approximately 90 pM or less D binds to human or mouse 4-1BB with a K of approximately 80 pM or less D binds to human or mouse 4-1BB with a K of approximately 70 pM or less D binds to human or mouse 4-1BB with a K of approximately 60 pM or less D binds to human or mouse 4-1BB with a K of approximately 50 pM or less D binds to human or mouse 4-1BB with a K of approximately 40 pM or less D or binds to human or mouse 4-1BB with a K of about 30 pM or less D 4-1BB agonists that bind at

[0459] In some embodiments, the compositions, processes, and methods described provide for the delivery of approximately 7.5×10 human or mouse 4-1BB. 5 k greater than 1 / M s assoc Approximately 7.5 × 10 binds to human or mouse 4-1BB. 5 k greater than 1 / M s assoc Approximately 8 × 10 binds to human or mouse 4-1BB. 5 k greater than 1 / M s assoc Approximately 8.5 × 10 binds to human or mouse 4-1BB.5 k greater than 1 / M s assoc Approximately 9 × 10 binds to human or mouse 4-1BB. 5 k greater than 1 / M s assoc Approximately 9.5 × 10 binds to human or mouse 4-1BB. 5 k greater than 1 / M s assoc or binds to human or mouse 4-1BB at approximately 1 × 10 6 k greater than 1 / M s assoc 4-1BB agonists that bind at

[0460] In some embodiments, the compositions, processes, and methods described provide for the delivery of approximately 2×10 human or mouse 4-1BB. -5 k less than 1 / s dissoc Approximately 2.1 × 10 binds to human or mouse 4-1BB. -5 k less than 1 / s dissoc Approximately 2.2 × 10 binds to human or mouse 4-1BB. -5 k less than 1 / s dissoc Approximately 2.3 × 10 binds to human or mouse 4-1BB. -5 k less than 1 / s dissoc Approximately 2.4 × 10 binds to human or mouse 4-1BB. -5 k less than 1 / s dissoc Approximately 2.5 × 10 binds to human or mouse 4-1BB. -5 k less than 1 / s dissoc Approximately 2.6 × 10 binds to human or mouse 4-1BB. -5 k less than 1 / s dissoc or approximately 2.7 × 10 to human or mouse 4-1BB. -5 k less than 1 / s dissoc Approximately 2.8 × 10 binds to human or mouse 4-1BB. -5 k less than 1 / s dissoc It binds to human or mouse 4-1BB at approximately 2. 9×10 -5 k less than 1 / s dissoc or approximately 3 × 10 to human or mouse 4-1BB. -5 k less than 1 / s dissoc4-1BB agonists that bind at

[0461] In some embodiments, the compositions, processes, and methods described have an IC50 of about 10 nM or less for human or mouse 4-1BB. 50 Binds to human or mouse 4-1BB with an IC of approximately 9 nM or less 50 Binds to human or mouse 4-1BB with an IC of approximately 8 nM or less 50 Binds to human or mouse 4-1BB with an IC of approximately 7 nM or less 50 Binds to human or mouse 4-1BB with an IC of approximately 6 nM or less 50 Binds to human or mouse 4-1BB with an IC of approximately 5 nM or less 50 Binds to human or mouse 4-1BB with an IC of approximately 4 nM or less 50 Binds to human or mouse 4-1BB with an IC of approximately 3 nM or less 50 Binds to human or mouse 4-1BB with an IC of approximately 2 nM or less 50 Binds to human or mouse 4-1BB with an IC of approximately 1 nM or less 50 4-1BB agonists that bind at

[0462] In a preferred embodiment, the 4-1BB agonist is utomilumab, also known as PF-05082566 or MOR-7480, or a fragment, derivative, variant, or biosimilar thereof. Utomilumab is available from Pfizer, Inc. Utomilumab is an immunoglobulin G2-lambda, anti-[Homo sapiens TNFRSF9 (tumor necrosis factor receptor (TNFR) superfamily member 9, 4-1BB, T cell antigen ILA, CD137)], Homo sapiens (fully human) monoclonal antibody. The amino acid sequence of utomilumab is shown in Table EE. Utomilumab contains glycosylation sites at Asn59 and Asn292, and glycosylation sites at positions 22-96 (V H -V L ), 143-199 (C H 1-C L ), 256-316 (C H 2), and 362-420th place (C H3) heavy chain intrachain disulfide bridge; 22'-87' positions (V H -V L ) and positions 136′-195′ (C H 1-C L interchain heavy-heavy chain disulfide bridges at positions 218-218, 219-219, 222-222, and 225-225 for IgG2A isoforms, at positions 218-130, 219-219, 222-222, and 225-225 for IgG2A / B isoforms, and at positions 219-130(2), 222-222, and 225-225 for IgG2B isoforms; and interchain heavy-light chain disulfide bridges at positions 130-213′(2) for IgG2A isoforms, at positions 218-213′ and 130-213′ for IgG2A / B isoforms, and at position 218-213′(2) for IgG2B isoforms. The preparation and properties of utomilumab and its variants and fragments are described in U.S. Patent Nos. 8,821,867, 8,337,850, and 9,468,678, and International Patent Application Publication No. WO2012 / 032433A1, the disclosures of each of which are incorporated herein by reference. Preclinical characteristics of utomilumab are described in Fisher, et al., Cancer Immunolog. & Immunother. 2012, 61, 1721-33. Current clinical trials of utomilumab in various hematological and solid tumor indications include the National Institutes of Health clinicaltrials.gov identifiers NCT02444793, NCT01307267, NCT02315066, and NCT02554812.

[0463] In embodiments, the 4-1BB agonist comprises a heavy chain given by SEQ ID NO: 11 and a light chain given by SEQ ID NO: 12. In embodiments, the 4-1BB agonist comprises a heavy chain and a light chain having the sequences set forth in SEQ ID NO: 11 and SEQ ID NO: 12, respectively, or an antigen-binding fragment, Fab fragment, single-chain variable fragment (scFv), variant, or conjugate thereof. In embodiments, the 4-1BB agonist comprises a heavy chain and a light chain each at least 99% identical to the sequences set forth in SEQ ID NO: 11 and SEQ ID NO: 12, respectively. In embodiments, the 4-1BB agonist comprises a heavy chain and a light chain each having the sequences set forth in SEQ ID NO: 11 and SEQ ID NO: 12, respectively. In embodiments, the 4-1BB agonist comprises a heavy chain and a light chain that are at least 98% identical to the sequences set forth in SEQ ID NOs: 11 and 12. In embodiments, the 4-1BB agonist comprises a heavy chain and a light chain that are each at least 97% identical to the sequences set forth in SEQ ID NOs: 11 and 12, respectively. In embodiments, the 4-1BB agonist comprises a heavy chain and a light chain that are each at least 96% identical to the sequences set forth in SEQ ID NOs: 11 and 12, respectively. In embodiments, the 4-1BB agonist comprises a heavy chain and a light chain that are each at least 95% identical to the sequences set forth in SEQ ID NOs: 11 and 12, respectively.

[0464] In embodiments, the 4-1BB agonist comprises the heavy and light chain CDRs or variable regions (VRs) of utomilumab. In embodiments, the 4-1BB agonist heavy chain variable region (VR) H ) comprises the sequence set forth in SEQ ID NO: 13, and the 4-1BB agonist light chain variable region (V L ) comprises the sequence set forth in SEQ ID NO: 14, and conservative amino acid substitutions thereof. In embodiments, the 4-1BB agonist comprises a V that is at least 99% identical to the sequence set forth in SEQ ID NO: 13 and SEQ ID NO: 14, respectively. H and V L In embodiments, the 4-1BB agonist comprises a V region that is at least 98% identical to the sequences set forth in SEQ ID NO: 13 and SEQ ID NO: 14, respectively. H and V LIn embodiments, the 4-1BB agonist comprises a V region that is at least 97% identical to the sequences set forth in SEQ ID NO: 13 and SEQ ID NO: 14, respectively. H and V L In embodiments, the 4-1BB agonist comprises a V region that is at least 96% identical to the sequences set forth in SEQ ID NO: 13 and SEQ ID NO: 14, respectively. H and V L In embodiments, the 4-1BB agonist comprises a V region that is at least 95% identical to the sequences set forth in SEQ ID NO: 13 and SEQ ID NO: 14, respectively. H and V L In embodiments, the 4-1BB agonist comprises a V region that is at least 99% identical to the sequences set forth in SEQ ID NO:13 and SEQ ID NO:14, respectively. H and V L The present invention also includes scFv antibodies containing the IgG domain.

[0465] In embodiments, the 4-1BB agonist comprises heavy chain CDR1, CDR2, and CDR3 domains having the sequences shown in SEQ ID NO: 15, SEQ ID NO: 16, and SEQ ID NO: 17, respectively, and their conservative amino acid substitutions, and light chain CDR1, CDR2, and CDR3 domains having the sequences shown in SEQ ID NO: 18, SEQ ID NO: 19, and SEQ ID NO: 20, respectively, and their conservative amino acid substitutions.

[0466] In some embodiments, the 4-1BB agonist is a biosimilar monoclonal antibody of a 4-1BB agonist approved by a drug regulatory agency for utomilumab. In some embodiments, the biosimilar monoclonal antibody comprises a 4-1BB antibody that contains an amino acid sequence with at least 97% sequence identity, e.g., 97%, 98%, 99%, or 100% sequence identity, to the amino acid sequence of the reference drug or reference biological product, and that contains one or more post-translational modifications compared to the reference drug or reference biological product, where the reference drug or reference biological product is utomilumab. In some embodiments, the one or more post-translational modifications are selected from one or more of glycosylation, oxidation, deamidation, and cleavage. In some embodiments, the biosimilar is a 4-1BB agonist antibody that has been approved or submitted for approval, where the 4-1BB agonist antibody is provided in a formulation different from that of the reference drug or reference biological product, where the reference drug or reference biological product is utomilumab. The 4-1BB agonist antibody may be approved by a drug regulatory authority, such as the U.S. FDA and / or the European Union's EMA. In some embodiments, the biosimilar is provided as a composition further comprising one or more excipients, which are the same or different from the excipients contained in the reference drug or reference biological product, and the reference drug or reference biological product is utomilumab. In some embodiments, the biosimilar is provided as a composition further comprising one or more excipients, which are the same or different from the excipients contained in the reference drug or reference biological product, and the reference drug or reference biological product is utomilumab. [Table 7]

[0467] In a preferred embodiment, the 4-1BB agonist is the monoclonal antibody urelumab, also known as BMS-663513 and 20H4.9.h4a, or a fragment, derivative, variant, or biosimilar thereof. Urelumab is available from Bristol-Myers Squibb, Inc. and Creative Biolabs, Inc. Urelumab is an immunoglobulin G4-κ, anti-[Homo sapiens TNFRSF9 (tumor necrosis factor receptor superfamily member 9), 4-1BB, T-cell antigen ILA, CD137)], a homo sapiens (fully human) monoclonal antibody. The amino acid sequence of urelumab is shown in Table EE. Urelumab has an N-glycosylation site at positions 298 (and 298"); positions 22-95 (V H -V L ), 148-204 (C H 1-C L ), 262-322 (C H 2), and 368-426th place (C H 3) heavy chain intrachain disulfide bridges at positions 22''-95'', 148''-204'', 262''-322'', and 368''-426''; H -V L ) and positions 136′-196′ (C H 1-C L) (positions 23'"-88'" and 136'"-196'"); interchain heavy-heavy chain disulfide bridges at 227-227" and 230-230"; and interchain heavy-light chain disulfide bridges at 135-216' and 135"-216'". The preparation and properties of urelumab and its variants and fragments are described in U.S. Pat. Nos. 7,288,638 and 8,962,804, the disclosures of which are incorporated herein by reference. Preclinical and clinical characteristics of urelumab are described in Segal, et al., Clin. Cancer Res. 2016, available at http: / / dx.doi.org / 10.1158 / 1078-0432.CCR-16-1272. Current clinical trials of urelumab in various hematologic and solid tumor indications include the National Institutes of Health clinicaltrials.gov identifiers NCT01775631, NCT02110082, NCT02253992, and NCT01471210.

[0468] In embodiments, the 4-1BB agonist comprises a heavy chain given by SEQ ID NO:21 and a light chain given by SEQ ID NO:22. In embodiments, the 4-1BB agonist comprises a heavy chain and a light chain having the sequences set forth in SEQ ID NO:21 and SEQ ID NO:22, respectively, or an antigen-binding fragment, Fab fragment, single-chain variable fragment (scFv), variant, or conjugate thereof. In embodiments, the 4-1BB agonist comprises a heavy chain and a light chain, each of which is at least 99% identical to the sequences set forth in SEQ ID NO:21 and SEQ ID NO:22, respectively. In embodiments, the 4-1BB agonist comprises a heavy chain and a light chain, each of which is at least 98% identical to the sequences set forth in SEQ ID NO:21 and SEQ ID NO:22, respectively. In embodiments, the 4-1BB agonist comprises a heavy chain and a light chain, each of which is at least 97% identical to the sequences set forth in SEQ ID NO:21 and SEQ ID NO:22, respectively. In embodiments, the 4-1BB agonist comprises a heavy chain and a light chain that are each at least 96% identical to the sequences set forth in SEQ ID NO: 21 and SEQ ID NO: 22, respectively. In embodiments, the 4-1BB agonist comprises a heavy chain and a light chain that are each at least 95% identical to the sequences set forth in SEQ ID NO: 21 and SEQ ID NO: 22, respectively.

[0469] In embodiments, the 4-1BB agonist comprises the heavy and light chain CDRs or variable regions (VRs) of urelumab. In embodiments, the 4-1BB agonist heavy chain variable region (VR) H ) comprises the sequence set forth in SEQ ID NO: 23, and L ) comprises the sequence set forth in SEQ ID NO: 24, and conservative amino acid substitutions thereof. In embodiments, the 4-1BB agonist comprises a V that is at least 99% identical to the sequence set forth in SEQ ID NO: 23 and SEQ ID NO: 24, respectively. H and V L In embodiments, the 4-1BB agonist comprises a V region that is at least 98% identical to the sequences set forth in SEQ ID NO:23 and SEQ ID NO:24, respectively. H and V LIn embodiments, the 4-1BB agonist comprises a V region that is at least 97% identical to the sequences set forth in SEQ ID NO:23 and SEQ ID NO:24, respectively. H and V L In embodiments, the 4-1BB agonist comprises a V region that is at least 96% identical to the sequences set forth in SEQ ID NO:23 and SEQ ID NO:24, respectively. H and V L In embodiments, the 4-1BB agonist comprises a V region that is at least 95% identical to the sequences set forth in SEQ ID NO:23 and SEQ ID NO:24, respectively. H and V L In embodiments, the 4-1BB agonist comprises a V region that is at least 99% identical to the sequences set forth in SEQ ID NO:23 and SEQ ID NO:24, respectively. H and V L The present invention also includes scFv antibodies containing the IgG domain.

[0470] In embodiments, the 4-1BB agonist comprises heavy chain CDR1, CDR2, and CDR3 domains having the sequences shown in SEQ ID NO: 25, SEQ ID NO: 26, and SEQ ID NO: 27, respectively, and their conservative amino acid substitutions, and light chain CDR1, CDR2, and CDR3 domains having the sequences shown in SEQ ID NO: 28, SEQ ID NO: 29, and SEQ ID NO: 30, respectively, and their conservative amino acid substitutions.

[0471] In some embodiments, the 4-1BB agonist is a biosimilar monoclonal antibody of a 4-1BB agonist approved by a drug regulatory agency for urelumab. In some embodiments, the biosimilar monoclonal antibody comprises a 4-1BB antibody that contains an amino acid sequence with at least 97% sequence identity, e.g., 97%, 98%, 99%, or 100% sequence identity, to the amino acid sequence of the reference drug or reference biological product, and that contains one or more post-translational modifications compared to the reference drug or reference biological product, where the reference drug or reference biological product is urelumab. In some embodiments, the one or more post-translational modifications are selected from one or more of glycosylation, oxidation, deamidation, and cleavage. In some embodiments, the biosimilar is a 4-1BB agonist antibody that has been approved or submitted for approval, where the 4-1BB agonist antibody is provided in a formulation different from that of the reference drug or reference biological product, where the reference drug or reference biological product is urelumab. The 4-1BB agonist antibody may be approved by a drug regulatory authority, such as the U.S. FDA and / or the European Union's EMA. In some embodiments, the biosimilar is provided as a composition further comprising one or more excipients, which are the same or different from the excipients contained in the reference drug or reference biological product, and the reference drug or reference biological product is urelumab. In some embodiments, the biosimilar is provided as a composition further comprising one or more excipients, which are the same or different from the excipients contained in the reference drug or reference biological product, and the reference drug or reference biological product is urelumab. [Table 8]

[0472] In embodiments, the 4-1BB agonist is selected from the group consisting of 1D8, 3Elor, 4B4 (BioLegend 309809), H4-1BB-M127 (BD Pharmingen 552532), BBK2 (Thermo Fisher MS621PABX), 145501 (Leinco Technologies B591), the antibody produced by the cell line deposited under ATCC number HB-11248 and disclosed in U.S. Pat. No. 6,974,863, 5F4 (BioLegend 31 1503), C65-485 (BD Pharmingen 559446), antibodies disclosed in U.S. Patent Application Publication No. US2005 / 0095244, antibodies disclosed in U.S. Patent No. 7,288,638 (e.g., , 20H4.9-IgGl (BMS-663031)), antibodies disclosed in U.S. Pat. No. 6,887,673 (e.g., 4E9 or BMS-554271), antibodies disclosed in U.S. Pat. No. 7,214,493, antibodies disclosed in U.S. Pat. No. 6,303,121, antibodies disclosed in U.S. Pat. No. 6,569,997, antibodies disclosed in U.S. Pat. No. 6,905,685 (e.g., 4E9 or BMS-554271), antibodies disclosed in U.S. Pat. No. 6,362,325 (e.g., 1D8 or BMS-469492, 3H3 or BMS-469497, or 3El), antibodies disclosed in U.S. Pat. No. 6,974,863 No. 6,210,669 (e.g., 1D8, 3B8, or 3El), antibodies disclosed in U.S. Pat. No. 5,928,893, antibodies disclosed in U.S. Pat. No. 6,303,121, antibodies disclosed in U.S. Pat. No. 6,569,997, antibodies disclosed in International Patent Application Publication Nos. WO2012 / 177788, WO2015 / 119923, and WO2010 / 042433, and fragments, derivatives, conjugates, variants, or biosimilars thereof, the disclosures of each of the foregoing patents or patent application publications are incorporated herein by reference.

[0473] In embodiments, the 4-1BB agonist is a compound described in International Patent Application Publication Nos. WO2008 / 025516A1, WO2009 / 007120A1, WO2010 / 003766A1, WO2010 / 010051A1, and WO2010 / 078966A1; U.S. Patent Application Publication Nos. US2011 / 0027218A1, US2015 / 0126709A1, and 4-1BB agonist fusion proteins described in US2011 / 0111494A1, US2015 / 0110734A1, and US2015 / 0126710A1; and U.S. Patent Nos. 9,359,420, 9,340,599, 8,921,519, and 8,450,460, the disclosures of which are incorporated herein by reference.

[0474] In embodiments, the 4-1BB agonist is a 4-1BB agonist fusion protein as shown in Structure IA (C-terminal Fc antibody fragment fusion protein) or Structure IB (N-terminal Fc antibody fragment fusion protein) as provided in Figure 10, or a fragment, derivative, conjugate, variant, or biosimilar thereof.

[0475] In structures IA and IB, the cylinders refer to individual polypeptide binding domains. Structures IA and IB contain three linearly linked TNFRSF binding domains derived from antibodies that bind, for example, to 4-1BBL or 4-1BB, which fold to form a trivalent protein, which is then combined with IgG1-Fc (C H 3 and C HThe TNFRSF-binding domain is bound to a second trivalent protein by a V-shaped linker (comprising a V-shaped domain and a V-shaped domain), which is then used to link two of the trivalent proteins together by disulfide bonds (small oblong ellipses), stabilizing the structure and bringing together the intracellular signaling domains of the six receptors and signaling proteins to provide an agonist that can form a signaling complex. The TNFRSF-binding domain, shown as a cylinder, is connected by a V-shaped linker that may contain, for example, hydrophilic residues and Gly and Ser sequences for flexibility, and Glu and Lys for solubility. H and V L The scFv domain may be an scFv domain containing a chain. Any scFv domain design can be used, such as those described in de Marco, Microbial Cell Factories, 2011, 10, 44; Ahmad, et al., Clin. & Dev. Immunol. 2012, 980250; Monnier, et al., Antibodies, 2013, 2, 193-208, or other references incorporated herein. This type of fusion protein structure is described in U.S. Patent Nos. 9,359,420, 9,340,599, 8,921,519, and 8,450,460, the disclosures of which are incorporated herein by reference.

[0476] The amino acid sequences of other polypeptide domains of Structure IA are shown in Table GG. The Fc domain preferably comprises the complete constant domain (amino acids 17-230 of SEQ ID NO:31), the complete hinge domain (amino acids 1-16 of SEQ ID NO:31), or a portion of the hinge domain (e.g., amino acids 4-16 of SEQ ID NO:31). Preferred linkers for connecting the C-terminal Fc antibody may be selected from the embodiments shown in SEQ ID NOs:32-41, including linkers suitable for fusing additional polypeptides. [Table 9]

[0477] The amino acid sequences of the other polypeptide domains of Structure IB are set forth in Table HH. When an Fc antibody fragment is fused to the N-terminus of the TNRFSF fusion protein as in Structure IB, the sequence of the Fc module is preferably that set forth in SEQ ID NO:42, and the linker sequence is preferably selected from those embodiments set forth in SEQ ID NOs:43 to 45. [Table 10]

[0478] In embodiments, the 4-1BB agonist fusion protein according to structure IA or IB comprises one or more 4-1BB binding domains selected from the group consisting of the variable heavy and variable light chains of utomilumab, the variable heavy and variable light chains of urelumab, the variable heavy and variable light chains of utomilumab, a variable heavy and variable light chain selected from the variable heavy and variable light chains set forth in Table GG, any combination of the foregoing variable heavy and variable light chains, and fragments, derivatives, conjugates, variants, and biosimilars thereof.

[0479] In embodiments, a 4-1BB agonist fusion protein according to structure IA or IB comprises one or more 4-1BB binding domains comprising a 4-1BBL sequence. In embodiments, a 4-1BB agonist fusion protein according to structure IA or IB comprises one or more 4-1BB binding domains comprising a sequence according to SEQ ID NO: 46. In embodiments, a 4-1BB agonist fusion protein according to structure IA or IB comprises one or more 4-1BB binding domains comprising a soluble 4-1BBL sequence. In embodiments, a 4-1BB agonist fusion protein according to structure IA or IB comprises one or more 4-1BB binding domains comprising a sequence according to SEQ ID NO: 47.

[0480] In embodiments, the 4-1BB agonist fusion protein according to structure IA or IB each comprises a V sequence at least 95% identical to SEQ ID NO: 13 and SEQ ID NO: 14, respectively. H and V Lone or more 4-1BB binding domains, which are scFv domains containing the V H and V L The domains are connected by a linker. In embodiments, the 4-1BB agonist fusion proteins according to structure IA or IB each comprise a V domain that is at least 95% identical to SEQ ID NO:23 and SEQ ID NO:24, respectively. H and V L one or more 4-1BB binding domains, which are scFv domains containing the V H and V L The domains are connected by a linker. In embodiments, the 4-1BB agonist fusion protein according to structure IA or IB is a fusion protein having a V domain, each of which is shown in Table 11. H and V L V that is at least 95% identical to the sequence H and V L one or more 4-1BB binding domains, which are scFv domains containing the V H and V L The domains are connected by linkers. [Table 11]

[0481] In embodiments, the 4-1BB agonist is a 4-1BB agonist single-chain fusion polypeptide comprising (i) a first soluble 4-1BB binding domain, (ii) a first peptide linker, (iii) a second soluble 4-1BB binding domain, (iv) a second peptide linker, and (v) a third soluble 4-1BB binding domain, and further comprising an additional domain at the N-terminus and / or C-terminus, wherein the additional domain is a Fab or Fc fragment domain. In embodiments, the 4-1BB agonist is a 4-1BB agonist single-chain fusion polypeptide comprising (i) a first soluble 4-1BB binding domain, (ii) a first peptide linker, (iii) a second soluble 4-1BB binding domain, (iv) a second peptide linker, and (v) a third soluble 4-1BB binding domain, and further comprising additional domains at the N-terminus and / or C-terminus, wherein the additional domains are Fab or Fc fragment domains, each of the soluble 4-1BB domains lacks a stalk region (which contributes to trimerization and provides a certain distance to the cell membrane, but is not part of the 4-1BB binding domain), and the first and second peptide linkers independently have a length of 3 to 8 amino acids.

[0482] In embodiments, the 4-1BB agonist is a 4-1BB agonist single-chain fusion polypeptide comprising: (i) a first soluble tumor necrosis factor (TNF) superfamily cytokine domain, (ii) a first peptide linker, (iii) a second soluble TNF superfamily cytokine domain, (iv) a second peptide linker, and (v) a third soluble TNF superfamily cytokine domain, wherein each of the soluble TNF superfamily cytokine domains lacks a stalk region, and the first and second peptide linkers are independently 3 to 8 amino acids in length, and each TNF superfamily cytokine domain is a 4-1BB binding domain.

[0483] In embodiments, the 4-1BB agonist is a V L the aforementioned V bound to any of the domains H It is a 4-1BB agonist scFv antibody comprising any of the domains.

[0484] In embodiments, the 4-1BB agonist is BPS Bioscience 4-1BB agonist antibody catalog number 79097-2, commercially available from BPS Bioscience (San Diego, CA, USA). In embodiments, the 4-1BB agonist is Creative Biolabs 4-1BB agonist antibody catalog number MOM-18179, commercially available from Creative Biolabs (Shirley, NY, USA).

[0485] 3. OX40 (CD134) agonist In embodiments, the TNFRSF agonist is an OX40 (CD134) agonist. The OX40 agonist can be any OX40 binding molecule known in the art. The OX40 binding molecule can be a monoclonal antibody or fusion protein that can bind to human or mammalian OX40. The OX40 agonist or OX40 binding molecule can include an immunoglobulin heavy chain of any isotype (e.g., IgG, IgE, IgM, IgD, IgA, and IgY), class (e.g., IgG1, IgG2, IgG3, IgG4, IgA1, and IgA2), or subclass of immunoglobulin molecule. The OX40 agonist or OX40 binding molecule can have both a heavy chain and a light chain. As used herein, the term binding molecule also includes antibodies (including full-length antibodies), monoclonal antibodies (including full-length monoclonal antibodies), polyclonal antibodies, multispecific antibodies (e.g., bispecific antibodies), human, humanized, or chimeric antibodies, and antibody fragments, e.g., Fab fragments, F(ab') fragments, fragments produced by a Fab expression library, epitope-binding fragments of any of the above, as well as engineered forms of antibodies that bind to OX40, e.g., scFv molecules. In embodiments, the OX40 agonist is an antigen-binding protein that is a fully human antibody. In embodiments, the OX40 agonist is an antigen-binding protein that is a humanized antibody. In some embodiments, OX40 agonists for use in the presently disclosed methods and compositions include anti-OX40 antibodies, human anti-OX40 antibodies, murine anti-OX40 antibodies, mammalian anti-OX40 antibodies, monoclonal anti-OX40 antibodies, polyclonal anti-OX40 antibodies, chimeric anti-OX40 antibodies, anti-OX40 Adnectins, anti-OX40 domain antibodies, single-chain anti-OX40 fragments, heavy-chain anti-OX40 fragments, light-chain anti-OX40 fragments, anti-OX40 fusion proteins, and fragments, derivatives, conjugates, variants, or biosimilars thereof. In preferred embodiments, the OX40 agonist is an agonist, anti-OX40 humanized, or fully human monoclonal antibody (i.e., an antibody derived from a single cell line).

[0486] In a preferred embodiment, the OX40 agonist or OX40 binding molecule can also be a fusion protein. An OX40 fusion protein comprising an Fc domain fused to OX40L is described, for example, in Sadun, et al., J. Immunother. 2009, 182, 1481-89. In a preferred embodiment, trimeric or hexameric OX4 Multimeric OX40 agonists, such as OX40 agonists (having three or six ligand-binding domains), can induce superior receptor (OX40L) clustering and intracellular signaling complex formation compared to agonist monoclonal antibodies, which typically have two ligand-binding domains. Trimeric (trivalent) or hexameric (hexavalent) or higher fusion proteins containing three TNFRSF-binding domains and IgG1-Fc, optionally further combining two or more of these fusion proteins, are described, for example, in Gieffers, et al., Mol. Cancer Therapeutics 2013, 12, 2735-47.

[0487] Agonistic OX40 antibodies and fusion proteins are known to induce strong immune responses. Curti, et al., Cancer Res. 2013, 73, 7189-98. In a preferred embodiment, the OX40 agonist is a monoclonal antibody or fusion protein that specifically binds to the OX40 antigen in a manner sufficient to reduce toxicity. In some embodiments, the OX40 agonist is an agonistic OX40 monoclonal antibody or fusion protein that neutralizes antibody-dependent cellular cytotoxicity (ADCC), e.g., NK cell cytotoxicity. In some embodiments, the OX40 agonist is an agonistic OX40 monoclonal antibody or fusion protein that neutralizes antibody-dependent cellular phagocytosis (ADCP). In some embodiments, the OX40 agonist is an agonistic OX40 monoclonal antibody or fusion protein that neutralizes complement-dependent cytotoxicity (CDC). In some embodiments, the OX40 agonist is an agonistic OX40 monoclonal antibody or fusion protein that neutralizes Fc region functionality.

[0488] In some embodiments, the OX40 agonist is characterized by binding to human OX40 (SEQ ID NO: 54) with high affinity and agonistic activity. In embodiments, the OX40 agonist is a binding molecule that binds to human OX40 (SEQ ID NO: 54). In embodiments, the OX40 agonist is a binding molecule that binds to mouse OX40 (SEQ ID NO: 55). The amino acid sequences of the OX40 antigens to which the OX40 agonists or binding molecules bind are summarized in Table 12. [Table 12]

[0489] In some embodiments, the compositions, processes, and methods described have a K of about 100 pM or less for human or mouse OX40. D binds to human or mouse OX40 with a K of approximately 90 pM or less D binds to human or mouse OX40 with a K of approximately 80 pM or less D binds to human or mouse OX40 with a K of approximately 70 pM or less D binds to human or mouse OX40 with a K of approximately 60 pM or less D binds to human or mouse OX40 with a K of approximately 50 pM or less D binds to human or mouse OX40 with a K of approximately 40 pM or less D or binds to human or mouse OX40 with a K of about 30 pM or less D OX40 agonists that bind at

[0490] In some embodiments, the compositions, processes, and methods described provide for the production of approximately 7.5×10 human or mouse OX40. 5 k greater than 1 / M s assoc Approximately 7.5 × 10 binds to human or mouse OX40. 5 k greater than 1 / M s assoc Approximately 8 × 10 binds to human or mouse OX40. 5 k greater than 1 / M s assoc Approximately 8.5 × 10 binds to human or mouse OX40. 5k greater than 1 / M s assoc Approximately 9 × 10 binds to human or mouse OX40. 5 k greater than 1 / M s assoc Approximately 9.5 × 10 binds to human or mouse OX40. 5 k greater than 1 / M s assoc or binds to human or mouse OX40 at approximately 1 × 10 6 k greater than 1 / M s assoc OX40 agonists that bind at

[0491] In some embodiments, the compositions, processes, and methods described provide for the production of approximately 2×10 human or mouse OX40. -5 k less than 1 / s dissoc Approximately 2.1 × 10 binds to human or mouse OX40. -5 k less than 1 / s dissoc Approximately 2.2 × 10 binds to human or mouse OX40. -5 k less than 1 / s dissoc Approximately 2.3 × 10 binds to human or mouse OX40. -5 k less than 1 / s dissoc Approximately 2.4 × 10 binds to human or mouse OX40. -5 k less than 1 / s dissoc Approximately 2.5 × 10 binds to human or mouse OX40. -5 k less than 1 / s dissoc Approximately 2.6 × 10 binds to human or mouse OX40. -5 k less than 1 / s dissoc or binds to human or mouse OX40 at approximately 2.7 × 10 -5 k less than 1 / s dissoc Approximately 2.8 × 10 binds to human or mouse OX40. -5 k less than 1 / s dissoc Approximately 2.9 × 10 binds to human or mouse OX40. -5 k less than 1 / s dissoc or binds to human or mouse OX40 at approximately 3 × 10 -5 k less than 1 / s dissoc OX40 agonists that bind at

[0492] In some embodiments, the compositions, processes, and methods described have an IC of about 10 nM or less for human or mouse OX40. 50 Binds to human or mouse OX40 with an IC of approximately 9 nM or less 50 Binds to human or mouse OX40 with an IC of approximately 8 nM or less 50 Binds to human or mouse OX40 with an IC of approximately 7 nM or less 50 Binds to human or mouse OX40 with an IC of approximately 6 nM or less 50 Binds to human or mouse OX40 with an IC of approximately 5 nM or less 50 Binds to human or mouse OX40 with an IC of approximately 4 nM or less 50 Binds to human or mouse OX40 with an IC of approximately 3 nM or less 50 Binds to human or mouse OX40 with an IC of approximately 2 nM or less 50 Binds to human or mouse OX40 with an IC of approximately 1 nM or less 50 OX40 agonists that bind at

[0493] In some embodiments, the OX40 agonist is taborixizumab, also known as MEDI0562 or MEDI-0562. Taborixizumab is available from Medimmune, a subsidiary of AstraZeneca, Inc. Taborixizumab is an immunoglobulin G1-κ, anti-[Homo sapiens TNFRSF4 (tumor necrosis factor receptor (TNFR) superfamily member 4, OX40, CD134)], humanized and chimeric monoclonal antibody. The amino acid sequence of taborixizumab is shown in Table KK. Taborixizumab has N-glycosylation sites at positions 301 and 301" with fucosylated complex bisecting CHO-type glycans; N-glycosylation sites at positions 22-95 (V H -V L ), 148-204 (C H 1-C L ), 265-325 (C H 2), and 371-429th place (C H3) heavy chain intrachain disulfide bridges at positions 22''-95'', 148''-204'', 265''-325'', and 371''-429''; H -V L ) and positions 134′-194′ (C H 1-C L ) (positions 23''-88'''' and 134''-194''''); interchain heavy-heavy chain disulfide bridges at positions 230-230'' and 233-233''; and interchain heavy-light chain disulfide bridges at positions 224-214'' and 224''-214''''. Current status of tabolixizumab in various solid tumor indications The clinical trials include the National Institutes of Health clinicaltrials.gov identifiers NCT02318394 and NCT02705482.

[0494] In embodiments, the OX40 agonist comprises a heavy chain given by SEQ ID NO:56 and a light chain given by SEQ ID NO:57. In embodiments, the OX40 agonist comprises a heavy chain and a light chain having the sequences set forth in SEQ ID NO:56 and SEQ ID NO:57, respectively, or an antigen-binding fragment, Fab fragment, single-chain variable fragment (scFv), variant, or conjugate thereof. In embodiments, the OX40 agonist comprises a heavy chain and a light chain, each of which is at least 99% identical to the sequences set forth in SEQ ID NO:56 and SEQ ID NO:57, respectively. In embodiments, the OX40 agonist comprises a heavy chain and a light chain, each of which is at least 98% identical to the sequences set forth in SEQ ID NO:56 and SEQ ID NO:57, respectively. In embodiments, the OX40 agonist comprises a heavy chain and a light chain, each of which is at least 97% identical to the sequences set forth in SEQ ID NO:56 and SEQ ID NO:57, respectively. In embodiments, the OX40 agonist comprises a heavy chain and a light chain that are each at least 96% identical to the sequences set forth in SEQ ID NO: 56 and SEQ ID NO: 57, respectively. In embodiments, the OX40 agonist comprises a heavy chain and a light chain that are each at least 95% identical to the sequences set forth in SEQ ID NO: 56 and SEQ ID NO: 57, respectively.

[0495] In embodiments, the OX40 agonist comprises the heavy and light chain CDRs or variable regions (VRs) of taborixizumab. In embodiments, the OX40 agonist heavy chain variable region (VR) H ) comprises the sequence set forth in SEQ ID NO: 58 and is an OX40 agonist light chain variable region (V L ) comprises the sequence set forth in SEQ ID NO: 59, and conservative amino acid substitutions thereof. In embodiments, the OX40 agonist comprises a V that is at least 99% identical to the sequence set forth in SEQ ID NO: 58 and SEQ ID NO: 59, respectively. H and V L In embodiments, the OX40 agonist comprises a V region that is at least 98% identical to the sequences set forth in SEQ ID NO: 58 and SEQ ID NO: 59, respectively. H and V L In embodiments, the OX40 agonist comprises a V region that is at least 97% identical to the sequences set forth in SEQ ID NO: 58 and SEQ ID NO: 59, respectively. H and V L In embodiments, the OX40 agonist comprises a V region that is at least 96% identical to the sequences set forth in SEQ ID NO: 58 and SEQ ID NO: 59, respectively. H and V L In embodiments, the OX40 agonist comprises a V region that is at least 95% identical to the sequences set forth in SEQ ID NO: 58 and SEQ ID NO: 59, respectively. H and V L In embodiments, the OX40 agonist comprises a V region that is at least 99% identical to the sequences set forth in SEQ ID NO:58 and SEQ ID NO:59, respectively. H and V L The present invention also includes scFv antibodies containing the IgG domain.

[0496] In embodiments, the OX40 agonist comprises heavy chain CDR1, CDR2, and CDR3 domains having the sequences set forth in SEQ ID NO: 60, SEQ ID NO: 61, and SEQ ID NO: 62, respectively, and their conservative amino acid substitutions, and light chain CDR1, CDR2, and CDR3 domains having the sequences set forth in SEQ ID NO: 63, SEQ ID NO: 64, and SEQ ID NO: 65, respectively, and their conservative amino acid substitutions.

[0497] In some embodiments, the OX40 agonist is a biosimilar monoclonal antibody of an OX40 agonist approved by a drug regulatory agency for taborixizumab. In some embodiments, the biosimilar monoclonal antibody comprises an OX40 antibody that comprises an amino acid sequence having at least 97% sequence identity, e.g., 97%, 98%, 99%, or 100% sequence identity, to the amino acid sequence of the reference drug or reference biological product, and that comprises one or more post-translational modifications compared to the reference drug or reference biological product, wherein the reference drug or reference biological product is taborixizumab. In some embodiments, the one or more post-translational modifications are selected from one or more of glycosylation, oxidation, deamidation, and cleavage. In some embodiments, the biosimilar is an OX40 antibody that has been approved or submitted for approval. The biosimilar is an OX40 agonist antibody, wherein the OX40 agonist antibody is provided in a formulation that differs from the formulation of the reference drug or reference biological product, and the reference drug or reference biological product is taborixizumab. The OX40 agonist antibody may be approved by a drug regulatory authority, such as the U.S. FDA and / or the European Union's EMA. In some embodiments, the biosimilar is provided as a composition further comprising one or more excipients, wherein the one or more excipients are the same or different from the excipients contained in the reference drug or reference biological product, and the reference drug or reference biological product is taborixizumab. In some embodiments, the biosimilar is provided as a composition further comprising one or more excipients, wherein the one or more excipients are the same or different from the excipients contained in the reference drug or reference biological product, and the reference drug or reference biological product is taborixizumab. [Table 13]

[0498] In some embodiments, the OX40 agonist is 11D4, a fully human antibody available from Pfizer, Inc. The preparation and properties of 11D4 are described in U.S. Patent Nos. 7,960,515, 8,236,930, and 9,028,824, the disclosures of which are incorporated herein by reference. The amino acid sequence of 11D4 is: The columns are shown in Table LL.

[0499] In embodiments, the OX40 agonist comprises a heavy chain given by SEQ ID NO:66 and a light chain given by SEQ ID NO:67. In embodiments, the OX40 agonist comprises a heavy chain and a light chain having the sequences set forth in SEQ ID NO:66 and SEQ ID NO:67, respectively, or an antigen-binding fragment, Fab fragment, single-chain variable fragment (scFv), variant, or conjugate thereof. In embodiments, the OX40 agonist comprises a heavy chain and a light chain, each of which is at least 99% identical to the sequences set forth in SEQ ID NO:66 and SEQ ID NO:67, respectively. In embodiments, the OX40 agonist comprises a heavy chain and a light chain, each of which is at least 98% identical to the sequences set forth in SEQ ID NO:66 and SEQ ID NO:67, respectively. In embodiments, the OX40 agonist comprises a heavy chain and a light chain, each of which is at least 97% identical to the sequences set forth in SEQ ID NO:66 and SEQ ID NO:67, respectively. In embodiments, the OX40 agonist comprises a heavy chain and a light chain that are each at least 96% identical to the sequences set forth in SEQ ID NO: 66 and SEQ ID NO: 67, respectively. In embodiments, the OX40 agonist comprises a heavy chain and a light chain that are each at least 95% identical to the sequences set forth in SEQ ID NO: 66 and SEQ ID NO: 67, respectively.

[0500] In embodiments, the OX40 agonist comprises the heavy and light chain CDRs or variable regions (VRs) of 11D4. In embodiments, the OX40 agonist comprises the heavy chain variable region (VR) of 11D4. H) comprises the sequence set forth in SEQ ID NO: 68 and is an OX40 agonist light chain variable region (V L ) comprises the sequence set forth in SEQ ID NO: 69, and conservative amino acid substitutions thereof. In embodiments, the OX40 agonist comprises a V that is at least 99% identical to the sequence set forth in SEQ ID NO: 68 and SEQ ID NO: 69, respectively. H and V L In embodiments, the OX40 agonist comprises a V region that is at least 98% identical to the sequences set forth in SEQ ID NO: 68 and SEQ ID NO: 69, respectively. H and V L In embodiments, the OX40 agonist comprises a V region that is at least 97% identical to the sequences set forth in SEQ ID NO: 68 and SEQ ID NO: 69, respectively. H and V L In embodiments, the OX40 agonist comprises a V region that is at least 96% identical to the sequences set forth in SEQ ID NO: 68 and SEQ ID NO: 69, respectively. H and V L In embodiments, the OX40 agonist comprises a V region that is at least 95% identical to the sequences set forth in SEQ ID NO: 68 and SEQ ID NO: 69, respectively. H and V L Includes the area.

[0501] In embodiments, the OX40 agonist comprises heavy chain CDR1, CDR2, and CDR3 domains having the sequences set forth in SEQ ID NO: 70, SEQ ID NO: 71, and SEQ ID NO: 72, respectively, and their conservative amino acid substitutions, and light chain CDR1, CDR2, and CDR3 domains having the sequences set forth in SEQ ID NO: 73, SEQ ID NO: 74, and SEQ ID NO: 75, respectively, and their conservative amino acid substitutions.

[0502] In some embodiments, the OX40 agonist is a biosimilar monoclonal antibody of an OX40 agonist approved by a drug regulatory agency for 11D4. In some embodiments, the biosimilar monoclonal antibody comprises an OX40 antibody that comprises an amino acid sequence having at least 97% sequence identity, e.g., 97%, 98%, 99%, or 100% sequence identity, to the amino acid sequence of a reference drug or reference biological product, and that comprises one or more post-translational modifications compared to the reference drug or reference biological product, wherein the reference drug or reference biological product is 11D4. In some embodiments, the one or more post-translational modifications are selected from one or more of glycosylation, oxidation, deamidation, and cleavage. In some embodiments, the biosimilar is an OX40 agonist antibody that has been approved or submitted for approval, wherein the OX40 agonist antibody is provided in a formulation different from that of the reference drug or reference biological product, and wherein the reference drug or reference biological product is 11D4. OX40 agonist antibodies are being approved by drug regulatory authorities such as the US FDA and / or the European Union's EMA. In some embodiments, the biosimilar is provided as a composition further comprising one or more excipients, which are the same or different from the excipients contained in the reference pharmaceutical or reference biological product, and the reference pharmaceutical or reference biological product is 11D4. In some embodiments, the biosimilar is provided as a composition further comprising one or more excipients, which are the same or different from the excipients contained in the reference pharmaceutical or reference biological product, and the reference pharmaceutical or reference biological product is 11D4. [Table 14]

[0503] In some embodiments, the OX40 agonist is 18D8, a fully human antibody available from Pfizer, Inc. The preparation and properties of 18D8 are described in U.S. Patent Nos. 7,960,515, 8,236,930, and 9,028,824, the disclosures of which are incorporated herein by reference. The amino acid sequence of 18D8 is shown in Table MM.

[0504] In embodiments, the OX40 agonist comprises a heavy chain given by SEQ ID NO: 76 and a light chain given by SEQ ID NO: 77. In embodiments, the OX40 agonist comprises a heavy chain and a light chain having the sequences set forth in SEQ ID NO: 76 and SEQ ID NO: 77, respectively, or an antigen-binding fragment, Fab fragment, single-chain variable fragment (scFv), variant, or conjugate thereof. In embodiments, the OX40 agonist comprises a heavy chain and a light chain, each of which is at least 99% identical to the sequences set forth in SEQ ID NO: 76 and SEQ ID NO: 77, respectively. In embodiments, the OX40 agonist comprises a heavy chain and a light chain, each of which is at least 98% identical to the sequences set forth in SEQ ID NO: 76 and SEQ ID NO: 77, respectively. In embodiments, the OX40 agonist comprises a heavy chain and a light chain, each of which is at least 97% identical to the sequences set forth in SEQ ID NO: 76 and SEQ ID NO: 77, respectively. In embodiments, the OX40 agonist comprises a heavy chain and a light chain that are each at least 96% identical to the sequences set forth in SEQ ID NO: 76 and SEQ ID NO: 77, respectively. In embodiments, the OX40 agonist comprises a heavy chain and a light chain that are each at least 95% identical to the sequences set forth in SEQ ID NO: 76 and SEQ ID NO: 77, respectively.

[0505] In embodiments, the OX40 agonist comprises the heavy and light chain CDRs or variable regions (VRs) of 18D8. In embodiments, the OX40 agonist heavy chain variable region (VR) H ) comprises the sequence set forth in SEQ ID NO: 78 and is an OX40 agonist light chain variable region (V L) comprises the sequence set forth in SEQ ID NO: 79, and conservative amino acid substitutions thereof. In embodiments, the OX40 agonist comprises a V that is at least 99% identical to the sequence set forth in SEQ ID NO: 78 and SEQ ID NO: 79, respectively. H and V L In embodiments, the OX40 agonist comprises a V region that is at least 98% identical to the sequences set forth in SEQ ID NO: 78 and SEQ ID NO: 79, respectively. H and V L In embodiments, the OX40 agonist comprises a V region that is at least 97% identical to the sequences set forth in SEQ ID NO: 78 and SEQ ID NO: 79, respectively. H and V L In embodiments, the OX40 agonist comprises a V region that is at least 96% identical to the sequences set forth in SEQ ID NO: 78 and SEQ ID NO: 79, respectively. H and V L In embodiments, the OX40 agonist comprises a V region that is at least 95% identical to the sequences set forth in SEQ ID NO: 78 and SEQ ID NO: 79, respectively. H and V L Includes the area.

[0506] In embodiments, the OX40 agonist comprises heavy chain CDR1, CDR2, and CDR3 domains having the sequences set forth in SEQ ID NO: 80, SEQ ID NO: 81, and SEQ ID NO: 82, respectively, and their conservative amino acid substitutions, and light chain CDR1, CDR2, and CDR3 domains having the sequences set forth in SEQ ID NO: 83, SEQ ID NO: 84, and SEQ ID NO: 85, respectively, and their conservative amino acid substitutions.

[0507] In embodiments, the OX40 agonist is a biosimilar monoclonal antibody of an OX40 agonist approved by a drug regulatory agency for 18D8. In embodiments, the biosimilar monoclonal antibody comprises an OX40 antibody that comprises an amino acid sequence having at least 97% sequence identity, e.g., 97%, 98%, 99%, or 100% sequence identity, to the amino acid sequence of a reference pharmaceutical or reference biological product, and that comprises one or more post-translational modifications compared to the reference pharmaceutical or reference biological product, wherein the reference pharmaceutical or reference biological product is 18D8. In some embodiments, the one or more post-translational modifications are glycosyltransferases. The biosimilar may be an OX40 agonist antibody that has been approved or submitted for approval, wherein the OX40 agonist antibody is provided in a formulation that differs from that of the reference drug or reference biological product, and wherein the reference drug or reference biological product is 18D8. The OX40 agonist antibody may be approved by a drug regulatory authority, such as the U.S. FDA and / or the European Union's EMA. In some embodiments, the biosimilar is provided as a composition further comprising one or more excipients, wherein the one or more excipients are the same or different from the excipients contained in the reference drug or reference biological product, and wherein the reference drug or reference biological product is 18D8. In some embodiments, the biosimilar is provided as a composition further comprising one or more excipients, wherein the one or more excipients are the same or different from the excipients contained in the reference drug or reference biological product, and wherein the reference drug or reference biological product is 18D8. [Table 15]

[0508] In some embodiments, the OX40 agonist is Hu119-122, a humanized antibody available from GlaxoSmithKline plc. The preparation and properties of Hu119-122 are described in U.S. Patent Nos. 9,006,399 and 9,163,085, and International Patent Publication No. WO2012 / 027328, the disclosures of which are incorporated herein by reference. The amino acid sequence of Hu119-122 is shown in Table NN.

[0509] In embodiments, the OX40 agonist comprises the heavy and light chain CDRs or variable regions (VRs) of Hu119-122. In embodiments, the OX40 agonist heavy chain variable region (VR) H ) comprises the sequence set forth in SEQ ID NO: 86 and is an OX40 agonist light chain variable region (V L ) comprises the sequence set forth in SEQ ID NO: 87, and conservative amino acid substitutions thereof. In embodiments, the OX40 agonist comprises a V that is at least 99% identical to the sequence set forth in SEQ ID NO: 86 and SEQ ID NO: 87, respectively. H and V L In embodiments, the OX40 agonist comprises a V region that is at least 98% identical to the sequences set forth in SEQ ID NO: 86 and SEQ ID NO: 87, respectively. H and V L In embodiments, the OX40 agonist comprises a V region that is at least 97% identical to the sequences set forth in SEQ ID NO: 86 and SEQ ID NO: 87, respectively. H and V L In embodiments, the OX40 agonist comprises a V region that is at least 96% identical to the sequences set forth in SEQ ID NO: 86 and SEQ ID NO: 87, respectively. H and V L In embodiments, the OX40 agonist comprises a V region that is at least 95% identical to the sequences set forth in SEQ ID NO: 86 and SEQ ID NO: 87, respectively. H and V L Includes the area.

[0510] In embodiments, the OX40 agonist comprises heavy chain CDR1, CDR2, and CDR3 domains having the sequences set forth in SEQ ID NO: 88, SEQ ID NO: 89, and SEQ ID NO: 90, respectively, and their conservative amino acid substitutions, and light chain CDR1, CDR2, and CDR3 domains having the sequences set forth in SEQ ID NO: 91, SEQ ID NO: 92, and SEQ ID NO: 93, respectively, and their conservative amino acid substitutions.

[0511] In some embodiments, the OX40 agonist is a biosimilar monoclonal antibody of an OX40 agonist approved by a drug regulatory agency for Hu119-122. In some embodiments, the biosimilar monoclonal antibody comprises an OX40 antibody that comprises an amino acid sequence having at least 97% sequence identity, e.g., 97%, 98%, 99%, or 100% sequence identity, to the amino acid sequence of a reference drug or reference biological product, and that comprises one or more post-translational modifications compared to the reference drug or reference biological product, where the reference drug or reference biological product is Hu119-122. In some embodiments, the one or more post-translational modifications are selected from one or more of glycosylation, oxidation, deamidation, and cleavage. In some embodiments, the biosimilar is an OX40 agonist antibody that has been approved or submitted for approval, where the OX40 agonist antibody is provided in a formulation that is different from the formulation of the reference drug or reference biological product, where the reference drug or reference biological product is Hu119-122. The OX40 agonist antibody may be approved by a drug regulatory authority, such as the U.S. FDA and / or the European Union's EMA. In some embodiments, the biosimilar is provided as a composition further comprising one or more excipients, which are the same or different from the excipients contained in the reference drug or reference biological product, and the reference drug or reference biological product is Hu119-122. In some embodiments, the biosimilar is provided as a composition further comprising one or more excipients, which are the same or different from the excipients contained in the reference drug or reference biological product, and the reference drug or reference biological product is Hu119-122. [Table 16]

[0512] In some embodiments, the OX40 agonist is Hu106-222, a humanized antibody available from GlaxoSmithKline plc. The preparation and properties of Hu106-222 are described in U.S. Patent Nos. 9,006,399 and 9,163,085, and International Patent Publication No. WO2012 / 027328, the disclosures of which are incorporated herein by reference. The amino acid sequence of Hu106-222 is shown in Table 00.

[0513] In embodiments, the OX40 agonist comprises the heavy and light chain CDRs or variable regions (VRs) of Hu106-222. In embodiments, the OX40 agonist heavy chain variable region (VR) H ) comprises the sequence set forth in SEQ ID NO: 94 and is an OX40 agonist light chain variable region (V L ) comprises the sequence set forth in SEQ ID NO: 95, and conservative amino acid substitutions thereof. In embodiments, the OX40 agonist comprises a V that is at least 99% identical to the sequence set forth in SEQ ID NO: 94 and SEQ ID NO: 95, respectively. H and V L In embodiments, the OX40 agonists each comprise at least one region having the sequence shown in SEQ ID NO: 94 and SEQ ID NO: 95, respectively. V that is at least 98% identical H and V L In embodiments, the OX40 agonist comprises a V region that is at least 97% identical to the sequences set forth in SEQ ID NO: 94 and SEQ ID NO: 95, respectively. H and V L In embodiments, the OX40 agonist comprises a V region that is at least 96% identical to the sequences set forth in SEQ ID NO: 94 and SEQ ID NO: 95, respectively. H and V L In embodiments, the OX40 agonist comprises a V region that is at least 95% identical to the sequences set forth in SEQ ID NO: 94 and SEQ ID NO: 95, respectively. Hand V L Includes the area.

[0514] In embodiments, the OX40 agonist comprises heavy chain CDR1, CDR2, and CDR3 domains having the sequences set forth in SEQ ID NO: 96, SEQ ID NO: 97, and SEQ ID NO: 98, respectively, and their conservative amino acid substitutions, and light chain CDR1, CDR2, and CDR3 domains having the sequences set forth in SEQ ID NO: 99, SEQ ID NO: 100, and SEQ ID NO: 101, respectively, and their conservative amino acid substitutions.

[0515] In some embodiments, the OX40 agonist is a biosimilar monoclonal antibody of an OX40 agonist approved by a drug regulatory agency for Hu106-222. In some embodiments, the biosimilar monoclonal antibody comprises an OX40 antibody that comprises an amino acid sequence having at least 97% sequence identity, e.g., 97%, 98%, 99%, or 100% sequence identity, to the amino acid sequence of a reference drug or reference biological product, and that comprises one or more post-translational modifications compared to the reference drug or reference biological product, where the reference drug or reference biological product is Hu106-222. In some embodiments, the one or more post-translational modifications are selected from one or more of glycosylation, oxidation, deamidation, and cleavage. In some embodiments, the biosimilar is an OX40 agonist antibody that has been approved or submitted for approval, where the OX40 agonist antibody is provided in a formulation that is different from the formulation of the reference drug or reference biological product, where the reference drug or reference biological product is Hu106-222. The OX40 agonist antibody may be approved by a drug regulatory authority, such as the U.S. FDA and / or the European Union's EMA. In some embodiments, the biosimilar is provided as a composition further comprising one or more excipients, which are the same or different from the excipients contained in the reference drug or reference biological product, and the reference drug or reference biological product is Hu106-222. In some embodiments, the biosimilar is provided as a composition further comprising one or more excipients, which are the same or different from the excipients contained in the reference drug or reference biological product, and the reference drug or reference biological product is Hu106-222. [Table 17]

[0516] In some embodiments, the OX40 agonist antibody is MEDI6469 (also referred to as 9B12). MEDI6469 is a mouse monoclonal antibody. Weinberg, et al., J. Immunother. 2006, 29, 575-585. In some embodiments, the OX40 agonist is an antibody produced by the 9B12 hybridoma deposited with Biovest Inc. (Malvern, MA, USA), as described in Weinberg, et al., J. Immunother. 2006, 29, 575-585, the disclosure of which is incorporated herein by reference in its entirety. In some embodiments, the antibody comprises the CDR sequence of MEDI6469. In some embodiments, the antibody comprises the heavy chain variable region sequence and / or the light chain variable region sequence of MEDI6469.

[0517] In embodiments, the OX40 agonist is L106 BD (Pharmingen Product No. 340420). In some embodiments, the OX40 agonist comprises the CDRs of antibody L106 (BD Pharmingen Product No. 340420). In some embodiments, the OX40 agonist comprises the heavy chain variable region sequence and / or the light chain variable region sequence of antibody L106 (BD Pharmingen Product No. 340420). In some embodiments, the OX40 agonist is ACT35 (Santa Cruz Biotechnology, catalog number 20073). In some embodiments, the OX40 agonist comprises the CDRs of antibody ACT35 (Santa Cruz Biotechnology, catalog number 20073). In some embodiments, the OX40 agonist comprises the heavy chain variable region sequence and / or the light chain variable region sequence of antibody ACT35 (Santa Cruz Biotechnology, catalog number 20073). In embodiments, the OX40 agonist is InVivoMAb, a murine monoclonal antibody anti-mCD134 / mOX40 (clone OX86) commercially available from BioXcell Inc (West Lebanon, NH).

[0518] In embodiments, the OX40 agonist is any of the compounds described in International Patent Application Publication Nos. WO95 / 12673, WO95 / 21925, WO2006 / 121810, WO2012 / 027328, WO2013 / 028231, WO2013 / 038191, and WO2014 / 148895; European Patent Application No. EP0672141; U.S. Patent Application Publication Nos. US2010 / 136030, US2014 / 377284, US2015 / 190506, and US2015 / 132288 (including clones 20E5 and 12H3); and the OX40 agonists described in U.S. Patent Nos. 7,504,101, 7,550,140, ​​7,622,444, 7,696,175, 7,960,515, 7,961,515, 8,133,983, 9,006,399, and 9,163,085, the disclosures of each of which are incorporated herein by reference in their entirety.

[0519] In embodiments, the OX40 agonist is an OX40 agonist fusion protein, such as those shown in Structure IA (C-terminal Fc antibody fragment fusion protein) or Structure IB (N-terminal Fc antibody fragment fusion protein), or a fragment, derivative, conjugate, variant, or biosimilar thereof. The properties of Structures IA and IB are described above and in U.S. Patent Nos. 9,359,420, 9,340,599, 8,921,519, and 8,450,460, the disclosures of which are incorporated herein by reference. The amino acid sequence of the polypeptide domain of Structure IA is set forth in Table GG. The Fc domain preferably includes the complete constant domain (amino acids 17-230 of SEQ ID NO:31), the complete hinge domain (amino acids 1-16 of SEQ ID NO:31), or a portion of the hinge domain (e.g., amino acids 4-16 of SEQ ID NO:31). Preferred linkers for connecting the C-terminal Fc antibody may be selected from the embodiments shown in SEQ ID NO: 32 to SEQ ID NO: 41, including linkers suitable for fusing additional polypeptides. Similarly, the amino acid sequences of the polypeptide domains of structure IB are shown in Table HH. When an Fc antibody fragment is fused to the N-terminus of a TNRFSF fusion protein as in structure IB, the sequence of the Fc module is preferably that shown in SEQ ID NO: 42, and the linker sequence is preferably selected from those embodiments shown in SEQ ID NO: 43 to SEQ ID NO: 45.

[0520] In embodiments, the OX40 agonist fusion protein according to structure IA or IB comprises one or more OX40 binding domains selected from the group consisting of the variable heavy and variable light chains of taborixizumab, the variable heavy and variable light chains of 11D4, the variable heavy and variable light chains of 18D8, the variable heavy and variable light chains of Hu119-122, the variable heavy and variable light chains of Hu106-222, a variable heavy and variable light chain selected from the variable heavy and variable light chains set forth in Table 00, any combination of the foregoing variable heavy and variable light chains, and fragments, derivatives, conjugates, variants, and biosimilars thereof.

[0521] In embodiments, the OX40 agonist fusion protein according to structure IA or IB comprises one or more OX40 binding domains comprising an OX40L sequence. In embodiments, the OX40 agonist fusion protein according to structure IA or IB comprises one or more OX40 binding domains comprising an OX40L sequence according to SEQ ID NO: 102. In embodiments, an OX40 agonist fusion protein according to structure IA or IB comprises one or more OX40 binding domains comprising a soluble OX40L sequence. In embodiments, an OX40 agonist fusion protein according to structure IA or IB comprises one or more OX40 binding domains comprising a sequence according to SEQ ID NO: 103. In embodiments, an OX40 agonist fusion protein according to structure IA or IB comprises one or more OX40 binding domains comprising a sequence according to SEQ ID NO: 104.

[0522] In embodiments, the OX40 agonist fusion protein according to structure IA or IB each comprises a V sequence at least 95% identical to SEQ ID NO:58 and SEQ ID NO:59, respectively. H and V L one or more OX40-binding domains, which are scFv domains containing the V H and V L The domains are connected by a linker. In embodiments, the OX40 agonist fusion proteins according to structure IA or IB each comprise a V domain that is at least 95% identical to SEQ ID NO:68 and SEQ ID NO:69, respectively. H and V L one or more OX40-binding domains, which are scFv domains containing the V H and V L The domains are connected by a linker. In embodiments, the OX40 agonist fusion proteins according to structure IA or IB each comprise a V domain that is at least 95% identical to SEQ ID NO:78 and SEQ ID NO:79, respectively. H and V L one or more OX40-binding domains, which are scFv domains containing the V H and V LThe domains are connected by a linker. In embodiments, the OX40 agonist fusion proteins according to structure IA or IB each comprise a V domain that is at least 95% identical to SEQ ID NO:86 and SEQ ID NO:87, respectively. H and V L one or more OX40-binding domains, which are scFv domains containing the V H and V L The domains are connected by a linker. In embodiments, the OX40 agonist fusion proteins according to structure IA or IB each comprise a V domain that is at least 95% identical to SEQ ID NO:94 and SEQ ID NO:95, respectively. H and V L one or more OX40-binding domains, which are scFv domains containing the V H and V L The domains are connected by a linker. In embodiments, the OX40 agonist fusion protein according to structure IA or IB comprises a V domain, each of which is shown in Table 18. H and V L V that is at least 95% identical to the sequence H and V L one or more OX40-binding domains, which are scFv domains containing the V H and V L The domains are connected by linkers. [Table 18] [Table 19]

[0523] In embodiments, the OX40 agonist is an OX40 agonist single-chain fusion polypeptide comprising (i) a first soluble OX40 binding domain, (ii) a first peptide linker, (iii) a second soluble OX40 binding domain, (iv) a second peptide linker, and (v) a third soluble OX40 binding domain, and further comprising an additional domain at the N-terminus and / or C-terminus, wherein the additional domain is a Fab or Fc fragment domain. In embodiments, the OX40 agonist is an OX40 agonist single-chain fusion polypeptide comprising (i) a first soluble OX40 binding domain, (ii) a first peptide linker, (iii) a second soluble OX40 binding domain, (iv) a second peptide linker, and (v) a third soluble OX40 binding domain, and further comprising additional domains at the N-terminus and / or C-terminus, wherein the additional domains are Fab or Fc fragment domains, each of the soluble OX40 domains lacks a stalk region (which contributes to trimerization and provides a distance to the cell membrane, but is not part of the OX40 binding domain), and the first and second peptide linkers independently have a length of 3 to 8 amino acids.

[0524] In embodiments, the OX40 agonist comprises (i) a first soluble tumor necrosis factor (TNF) superfamily cytokine domain, (ii) a first peptide linker, (iii) a second soluble TNF superfamily cytokine domain, (iv) a second peptide a linker; and (v) a third soluble TNF superfamily cytokine domain, each of the soluble TNF superfamily cytokine domains lacking a stalk region, the first and second peptide linkers independently being 3 to 8 amino acids in length, and the TNF superfamily cytokine domain is an OX40-binding domain.

[0525] In some embodiments, the OX40 agonist is MEDI6383. MEDI6383 is an OX40 agonist fusion protein and can be prepared as described in U.S. Patent No. 6,312,700, the disclosure of which is incorporated herein by reference.

[0526] In embodiments, the OX40 agonist is selected from the group consisting of the aforementioned V L the aforementioned V bound to any of the domains H It is an OX40 agonist scFv antibody comprising any of the domains.

[0527] In embodiments, the OX40 agonist is Creative Biolabs OX40 agonist monoclonal antibody MOM-18455, commercially available from Creative Biolabs, Inc. (Shirley, NY, USA).

[0528] In embodiments, the OX40 agonist is the OX40 agonist antibody clone Ber-ACT35, commercially available from BioLegend, Inc. (San Diego, CA, USA).

[0529] H. Optional Cell Viability Analysis Optionally, cell viability assays can be performed after the first expansion (sometimes referred to as initial bulk expansion) using standard assays known in the art. For example, trypan blue exclusion assays can be performed on samples of bulk TILs, which selectively label dead cells and allow for assessment of viability. Other assays used to test viability include, but are not limited to, Alamar Blue assays and MTT assays.

[0530] 1. Cell Count, Viability, and Flow Cytometry In some embodiments, cell number and / or viability are measured. Expression of markers such as, but not limited to, CD3, CD4, CD8, and CD56, as well as any other markers disclosed or described herein, can be measured by flow cytometry using antibodies, for example, but not limited to, those commercially available from BD Biosciences (BD Biosciences, San Jose, CA), using a FACSCanto™ flow cytometer (BD Biosciences). Cells can be counted manually using a disposable c-chip hemocytometer (VWR, Batavia, IL), and viability can be assessed using any method known in the art, including, but not limited to, trypan blue staining. Cell viability can also be assayed according to U.S. Pat. No. 15 / 863,634, the entire contents of which are incorporated herein by reference.

[0531] In some cases, the bulk TIL population can be immediately cryopreserved using the protocol discussed below. Alternatively, the bulk TIL population can be subjected to REP and cryopreserved as discussed below. Similarly, when genetically modified TILs are used for treatment, the bulk or REP TIL population can be subjected to genetic modification for the appropriate treatment.

[0532] According to the present disclosure, T Methods for Assaying ILs. In some embodiments, a method for assaying tumor infiltrating lymphocytes (TILs) comprises: (i) obtaining a first population of TILs; (ii) performing a first expansion by culturing the first TIL population in a cell culture medium containing IL-2 and optionally OKT-3 to produce a second TIL population; (iii) 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 produce a third TIL population, wherein the third TIL population is at least 50-fold more numerous than the second TIL population; (iv) harvesting, washing, and cryopreserving the third population of TILs; (v) storing the cryopreserved TILs at cryogenic temperatures; and (vi) thawing the third TIL population to provide a thawed third TIL population; (vii) performing an additional second expansion of a portion of the thawed third TIL population by supplementing the cell culture medium of the third population with IL-2, OKT-3, and APC for an additional expansion period of at least 3 days (sometimes referred to as the reREP period), wherein the third expansion results in a fourth TIL population, and the number of TILs in the fourth TIL population is compared with the number of TILs in the third TIL population to determine a ratio; (viii) determining whether the population of thawed TILs is suitable for administration to the patient based on the ratio of step (vii); (ix) if the ratio of the number of TILs in the fourth TIL population to the number of TILs in the third TIL population is determined to be greater than 5:1 in step (viii), administering a therapeutically effective dose of the thawed third TIL population to the patient.

[0533] In some embodiments, the TILs are assayed for viability after step (vii).

[0534] The present disclosure also provides additional methods for assaying TILs. In some embodiments, the present disclosure provides a method for assaying TILs, comprising: (i) obtaining a portion of the cryopreserved first TIL population; (ii) thawing a portion of the cryopreserved first population of TILs; (iii) performing a first expansion by culturing a portion of the first TIL population in a cell culture medium comprising IL-2, OKT-3, and antigen-presenting cells (APCs) for an additional expansion period (sometimes referred to as the reREP period) of at least 3 days to produce a second TIL population, wherein the portion from the first TIL population is compared with the second TIL population to determine a ratio of the number of TILs, and performing the first expansion such that the ratio of the number of TILs in the second TIL population to the number of TILs in the portion of the first TIL population is greater than 5:1; (iv) determining whether the first population of TILs is suitable for use in therapeutic administration to the patient based on the ratio in step (iii); (v) determining that the first TIL population is suitable for use in therapeutic administration if the ratio of the number of TILs in the second TIL population to the number of TILs in the first TIL population is determined to be greater than 5:1 in step (iv).

[0535] In some embodiments, the ratio of the number of TILs in the second TIL population to the number of TILs in the first TIL population is greater than 50:1.

[0536] In some embodiments, the method further comprises expanding the entire cryopreserved first TIL population from step (i) according to a method described in any of the embodiments provided herein.

[0537] In some embodiments, the method further comprises administering the entire cryopreserved first population of TILs from step (i) to the patient.

[0538] 2.Cell culture In embodiments, methods for expanding TILs, including those discussed above and illustrated in FIG. 1, may include using about 5,000 mL to about 25,000 mL of cell culture medium, about 5,000 mL to about 10,000 mL of cell culture medium, or about 5,800 mL to about 8,700 mL of cell culture medium. In some embodiments, the medium is serum-free medium. In some embodiments, the medium in the first expansion is serum-free. In some embodiments, the medium in the second expansion is serum-free. In some embodiments, the medium in both the first and second expansions is serum-free. In embodiments, expanding the number of TILs uses only one type of cell culture medium. Any suitable cell culture medium, for example, AIM-V cell culture medium (L-glutamine, 50 μM streptomycin sulfate, and 10 μM gentamicin sulfate) cell culture medium (Invitrogen, Carlsbad, CA) may be used. CA) can be used. In this regard, the methods of the present invention advantageously reduce the amount of medium and the number of medium types required to expand the number of TILs. In embodiments, expanding the number of TILs can include feeding the cells no more frequently than once every three or four days. Expanding the number of cells in a gas-permeable container simplifies the procedures required to expand the number of cells by reducing the feeding frequency required to expand the cells.

[0539] In some embodiments, the cell culture medium in the first and / or second gas-permeable containers is unfiltered. The use of unfiltered cell culture medium can simplify the steps required to expand the number of cells. In some embodiments, the cell culture medium in the first and / or second gas-permeable containers lacks beta-mercaptoethanol (BME).

[0540] In embodiments, the duration of a method comprising obtaining a tumor tissue sample from a mammal, culturing the tumor tissue sample in a first gas-permeable container containing cell culture medium therein, obtaining TILs from the tumor tissue sample, and expanding the number of TILs in a second gas-permeable container containing cell culture medium is about 7-14 days, e.g., about 11 days. In some embodiments, the pre-REP is about 7-14 days, e.g., about 11 days. In some embodiments, the REP is about 7-14 days, e.g., about 11 days.

[0541] In embodiments, TILs are expanded in a gas-permeable container. Gas-permeable containers have been used to expand TILs using PBMCs using methods, compositions, and devices known in the art, including those described in U.S. Patent Application Publication No. 2005 / 0106717A1 (the disclosure of which is incorporated herein by reference). In embodiments, TILs are expanded in a gas-permeable bag. In embodiments, TILs are expanded using a cell expansion system that expands TILs in a gas-permeable bag, such as the Xuri Cell Expansion System W25 (GE Healthcare). In embodiments, TILs are expanded using a cell expansion system that expands TILs in a gas-permeable bag, such as the WAVE Bioreactor System, also known as the Xuri Cell Expansion System W5 (GE Healthcare). In embodiments, the cell expansion system comprises a gas permeable cell bag having a volume selected from the group consisting of about 100 mL, about 200 mL, about 300 mL, about 400 mL, about 500 mL, about 600 mL, about 700 mL, about 800 mL, about 900 mL, about 1 L, about 2 L, about 3 L, about 4 L, about 5 L, about 6 L, about 7 L, about 8 L, about 9 L, and about 10 L.

[0542] In an embodiment, TILs are cultured in G-Rex flasks (Wilson Wolf Manuf Such an embodiment may be used to expand cell populations up to about 5×10 5 cells / cm 2 From 10 x 10 6 ~30×106 cells / cm 2 In embodiments, this is without feeding. In embodiments, this is without feeding, as long as the medium is present to a height of about 10 cm in the G-Rex flask. In embodiments, this is without feeding, but with the addition of one or more cytokines. In embodiments, the cytokines can be added as a bolus without having to be mixed with the medium. Such vessels, devices, and methods are known in the art and have been used to expand TILs, and are described in U.S. Patent Application Publication No. US2014 / 0377739A1, International Publication No. WO2014 / 210036A1, U.S. Patent Application Publication No. US2013 / 0115617A1, International Publication No. WO2013 / 188427A1, U.S. Patent Application Publication No. US2011 / 0136228A1, U.S. Patent No. US8,809,050B2, International Publication No. WO2011 / 072088A1, and the like. 2, U.S. Patent Application Publication No. US2016 / 0208216A1, U.S. Patent Application Publication No. US2012 / 0244133A1, International Publication No. WO2012 / 129201A1, U.S. Patent Application Publication No. US2013 / 0102075A1, U.S. Patent No. US8,956,860B2, International Publication No. WO2013 / 173835A1, U.S. Patent Application Publication No. US2015 / 0175966A1 (the disclosures of which are incorporated herein by reference). Such processes are also described in Jin et al., J. Immunotherapy, 2012, 35:283-292.

[0543] I. Optional genetic manipulation of TILs In some embodiments, TILs are optionally genetically engineered to contain additional functionality, including, but not limited to, a high-affinity T cell receptor (TCR), e.g., a TCR targeted with a tumor-associated antigen such as MAGE-1, HER2, or NY-ESO-1, or a chimeric antigen receptor (CAR) that binds to a tumor-associated cell surface molecule (e.g., mesothelin) or a lineage-restricted cell surface molecule (e.g., CD19).

[0544] J.Optional cryopreservation of TILs As discussed above, cryopreservation can occur at various points throughout the TIL expansion process, as exemplified by steps A-E shown in FIG. 1 . In some embodiments, the expanded TIL population after the second expansion (e.g., provided according to step D of FIG. 1 ) can be cryopreserved. Cryopreservation can generally be achieved by placing the TIL population in a freezing solution, such as 85% complement-inactivated AB serum and 15% dimethyl sulfoxide (DMSO). The cells in the solution are placed in a cryogenic vial, stored at -80°C for 24 hours, and optionally transferred to a gaseous nitrogen freezer for cryopreservation. See Sadeghi, et al., Acta Oncologica 2013, 52, 978-986. In some embodiments, TILs are cryopreserved in 5% DMSO. In some embodiments, TILs are cryopreserved in cell culture medium supplemented with 5% DMSO. In some embodiments, TILs are cryopreserved according to the methods provided in Examples F and G.

[0545] If necessary, remove the cells from the freezer and thaw in a 37°C water bath until approximately 4 / 5 of the solution has thawed. The cells are generally resuspended in complete medium and optionally washed one or more times. In some embodiments, the thawed TILs can be counted and assessed for viability as known in the art.

[0546] Closed system for K.TIL production The present invention provides the use of a closed system during the TIL culture process. Such a closed system can prevent and / or reduce microbial contamination, allow the use of fewer flasks, and reduce costs. In some embodiments, the closed system uses two vessels.

[0547] Such closed systems are well known in the art and can be found, for example, at http: / / www.fda.gov / cber / guidelines.htm and https: / / www.fda.gov / BiologicsBloodVaccines / GuidanceComplianceRegulatoryInformation / Guidances / Blood / ucm076779.htm.

[0548] A sterile connection device (STCD) performs a sterile weld between two compatible tubes. This procedure allows for a variety of container and tube diameters to be sterilely connected. In some embodiments, the closed system includes a luer lock system and a heat seal system, for example, as described in Example G. In some embodiments, the closed system is accessed via a syringe under sterile conditions to maintain the sterility and closure of the system. In some embodiments, a closed system such as that described in Example G is used. In some embodiments, the TILs are compounded into the final product formulation container according to the method described in the section "Final Formulation and Filling" of Example G.

[0549] In some embodiments, a closed system uses one container from the time the tumor fragments are obtained until the TILs are ready for administration to the patient or cryopreservation. In some embodiments, when two containers are used, the first container is a closed G container, and the TIL population is centrifuged and transferred to an infusion bag without opening the first closed G container. In some embodiments, when two containers are used, the infusion bag is an infusion bag containing HypoThermosol. A closed system or closed TIL cell culture system is characterized in that once the tumor sample and / or tumor fragments are added, the system is tightly sealed from the outside, forming a closed environment that is impervious to bacterial, fungal, and / or any other microbial contamination.

[0550] In some embodiments, the reduction in microbial contamination is between about 5% and about 100%. In some embodiments, the reduction in microbial contamination is between about 5% and about 95%. In some embodiments, the reduction in microbial contamination is between about 5% and about 90%. In some embodiments, the reduction in microbial contamination is between about 10% and about 90%. In some embodiments, the reduction in microbial contamination is between about 15% and about 85%. In some embodiments, the reduction in microbial contamination is about 5%, about 10%, about 15%, about 20%, about 25%, about 30%, about 35%, about 40%, about 45%, about 50%, about 55%, about 60%, about 65%, about 70%, about 75%, about 80%, about 85%, about 90%, about 95%, about 97%, about 98%, about 99%, or about 100%.

[0551] The closed system allows for the growth of TILs in the absence of and / or with greatly reduced microbial contamination.

[0552] Furthermore, the pH, carbon dioxide partial pressure, and oxygen partial pressure of the TIL cell culture environment each change as the cells are cultured. Therefore, even if a medium suitable for cell culture is circulated, it is necessary to maintain a constant closed environment as an optimal environment for TIL growth. To this end, it is desirable to monitor the physical factors of pH, carbon dioxide partial pressure, and oxygen partial pressure in the culture medium of the closed environment using sensors, use the signals to control a gas exchanger installed at the inlet of the culture environment, and adjust the gas partial pressure of the closed environment in real time in response to changes in the culture medium to optimize the cell culture environment. In some embodiments, the present invention provides a closed cell culture system incorporating a gas exchanger at the inlet to the closed environment equipped with a monitoring device that measures the pH, carbon dioxide partial pressure, and oxygen partial pressure of the closed environment, and automatically adjusts the gas concentrations based on signals from the monitoring device to optimize the cell culture environment.

[0553] In some embodiments, the pressure in the closed environment is controlled continuously or intermittently. That is, the pressure in the closed environment can be varied, for example, by a pressure maintenance device, thereby ensuring that the space is suitable for TIL growth under positive pressure conditions, or promoting fluid exudation under negative pressure conditions, thereby promoting cell proliferation. Furthermore, by applying negative pressure intermittently, temporary contraction of the volume of the closed environment can uniformly and efficiently replace the circulating liquid in the closed environment.

[0554] In some embodiments, optimal culture components for the expansion of TILs can be substituted or added, and factors such as IL-2 and / or OKT3, as well as combinations, can be added.

[0555] Optional cryopreservation of L.TILs Either the bulk TIL population or the expanded population of TILs can be optionally cryopreserved. In some embodiments, cryopreservation occurs on a therapeutic TIL population. In some embodiments, cryopreservation occurs on TILs harvested after the second expansion. In some embodiments, cryopreservation occurs on TILs at exemplary step F in FIG. 1 . In some embodiments, TILs are cryopreserved in an infusion bag. In some embodiments, TILs are cryopreserved before being placed in an infusion bag. In some embodiments, TILs are cryopreserved and not in an infusion bag. In some embodiments, cryopreservation is performed using a cryopreservation medium. In some embodiments, the cryopreservation medium contains dimethyl sulfoxide (DMSO). This is generally accomplished by placing the TIL population in a freezing solution, such as 85% complement-inactivated AB serum and 15% dimethyl sulfoxide (DMSO). The cells in solution are placed in a cryovial, stored at -80°C for 24 hours, and optionally transferred to a gaseous nitrogen freezer for cryopreservation. See Sadeghi, et al., Acta Oncologica 2013, 52, 978-986.

[0556] If necessary, remove the cells from the freezer and thaw in a 37°C water bath until approximately 4 / 5 of the solution has thawed. The cells are generally resuspended in complete medium and optionally washed one or more times. In some embodiments, the thawed TILs can be counted and assessed for viability as known in the art.

[0557] In a preferred embodiment, the population of TILs is cryopreserved using CS10 cryopreservation medium (CryoStor 10, BioLife Solutions). In a preferred embodiment, the population of TILs is cryopreserved using a cryopreservation medium containing dimethyl sulfoxide (DMSO). In a preferred embodiment, the population of TILs is cryopreserved using a 1:1 (volume:volume) ratio of CS10 and cell culture medium. In a preferred embodiment, the population of TILs is cryopreserved using an approximately 1:1 (volume:volume) ratio of CS10 and cell culture medium, further comprising additional IL-2.

[0558] As discussed in steps A-E above, cryopreservation can occur at various points throughout the TIL expansion process. In some embodiments, the bulk TIL population after the first expansion according to step B or the expanded TIL population after one or more second expansions according to step D can be cryopreserved. Cryopreservation can generally be achieved by placing the TIL population in a freezing solution, such as 85% complement-inactivated AB serum and 15% dimethyl sulfoxide (DMSO). The cells in the solution are placed in a cryogenic vial and stored at -80°C for 24 hours, and optionally transferred to a gaseous nitrogen freezer for cryopreservation. See Sadeghi, et al., Acta Oncologica 2013, 52, 978-986.

[0559] If necessary, remove the cells from the freezer and thaw for 3 minutes until approximately 4 / 5 of the solution has thawed. Thaw in a water bath at 7° C. Cells are generally resuspended in complete medium and optionally washed one or more times. In some embodiments, thawed TILs can be counted and assessed for viability as known in the art.

[0560] In some cases, the Step B TIL population can be immediately cryopreserved using the protocols discussed below. Alternatively, the bulk TIL population can be subjected to Steps C and D and cryopreserved after Step D. Similarly, if genetically engineered TILs are to be used therapeutically, the Step B or Step D TIL population can be subjected to genetic modification for the appropriate therapy.

[0561] IV. Pharmaceutical Compositions, Dosages, and Dosing Regimen In embodiments, TILs expanded using the methods of the present disclosure are administered to a patient as a pharmaceutical composition. In embodiments, the pharmaceutical composition is a suspension of TILs in a sterile buffer. TILs expanded using 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 administration routes include intraperitoneal, intrathecal, and intralymphatic administration.

[0562] Any suitable dose of TILs can be administered. In some embodiments, particularly when the cancer is NSCLC, about 2.3 x 10 10 ~Approx. 13.7×10 10 TILs were administered, with an average of approximately 7.8 × 10 10 In an embodiment, the TILs are about 1.2 x 10 10 ~Approx. 4.3×10 10 In some embodiments, about 3 x 10 TILs are administered. 10 ~Approx. 12×10 10 In some embodiments, about 4 x 10 TILs are administered. 10 ~About 10×10 10 In some embodiments, about 5 x 10 TILs are administered.10 ~Approx. 8×10 10 In some embodiments, about 6 x 10 TILs are administered. 10 ~Approx. 8×10 10 In some embodiments, about 7 x 10 TILs are administered. 10 ~Approx. 8×10 10 In some embodiments, the therapeutically effective dose is about 2.3 x 10 10 ~Approx. 13.7×10 10 In some embodiments, particularly when the cancer is NSCLC, the therapeutically effective dose is about 7.8 x 10 10 In some embodiments, the therapeutically effective dose is about 1.2 x 10 10 ~Approx. 4.3×10 10 In some embodiments, the therapeutically effective dose is about 3 x 10 TILs. 10 ~Approx. 12×10 10 In some embodiments, the therapeutically effective dose is about 4 x 10 10 ~About 10×10 10 In some embodiments, the therapeutically effective dose is about 5×10 10 ~Approx. 8×10 10 In some embodiments, the therapeutically effective dose is about 6×10 10 ~Approx. 8×10 10 In some embodiments, the therapeutically effective dose is about 7 x 10 10 ~Approx. 8×10 10 It's TIL.

[0563] In some embodiments, particularly when the cancer is NSCLC, about 1 x 10 9 ~Approx. 150×10 9 In some embodiments, the therapeutically effective dose is about 1×10 9 ~Approx. 150×10 9 In some embodiments, the therapeutically effective dose is about 1 x 10 9 ~Approx. 140×10 9 In some embodiments, the therapeutically effective dose is about 1 x 10 9~Approx. 130×10 9 In some embodiments, the therapeutically effective dose is about 1 x 10 9 ~Approx. 120×10 9 In some embodiments, the therapeutically effective dose is about 1 x 10 9 ~Approx. 110×10 9 In some embodiments, the therapeutically effective dose is about 1 x 10 9 ~About 100×10 9 In some embodiments, the therapeutically effective dose is about 1 x 10 9 ~About 90×10 9 In some embodiments, the therapeutically effective dose is about 1 x 10 9 ~About 80×10 9 In some embodiments, the therapeutically effective dose is about 1 x 10 9 ~Approx. 70×10 9 In some embodiments, the therapeutically effective dose is about 1 x 10 9 ~Approx. 60×10 9 In some embodiments, the therapeutically effective dose is is approximately 1 x 10 9 ~About 50×10 9 In some embodiments, the therapeutically effective dose is about 1 x 10 9 ~About 40×10 9 In some embodiments, the therapeutically effective dose is about 1 x 10 9 ~Approx. 30×10 9 In some embodiments, the therapeutically effective dose is about 1 x 10 9 ~About 20×10 9 In some embodiments, the therapeutically effective dose is about 1 x 10 9 ~About 10×10 9 In some embodiments, the therapeutically effective dose is about 1 x 10 9 ~Approx. 5×10 9 It's TIL.

[0564] In some embodiments, the number of TILs provided in the pharmaceutical composition of the present invention is about 1 x 10 6 , 2 × 10 6 , 3×10 6 , 4×10 6 , 5×10 6 , 6×10 6 , 7×10 6 , 8×10 6 , 9×10 6 , 1×10 7 , 2 × 10 7 , 3×10 7 , 4×10 7 , 5×10 7 , 6×10 7 , 7×10 7 , 8×10 7 , 9×10 7 , 1×10 8 , 2 × 10 8 , 3×10 8 , 4×10 8 , 5×10 8 , 6×10 8 , 7×10 8 , 8×10 8 , 9×10 8 , 1×10 9 , 2 × 10 9 , 3×10 9 , 4×10 9 , 5×10 9 , 6×10 9 , 7×10 9 , 8×10 9 , 9×10 9 , 1×10 10 , 2 × 10 10 , 3×10 10 , 4×10 10 , 5×10 10 , 6×10 10 , 7×10 10 , 8×10 10 , 9×10 10 , 1×10 11 , 2 × 10 11 , 3×10 11 , 4×10 11 , 5×10 11 , 6×10 11 , 7×10 11 , 8×10 11 , 9×1011 , 1×10 12 , 2 × 10 12 , 3×10 12 , 4×10 12 , 5×10 12 , 6×10 12 , 7×10 12 , 8×10 12 , 9×10 12 , 1×10 13 , 2 × 10 13 , 3×10 13 , 4×10 13 , 5×10 13 , 6×10 13 , 7×10 13 , 8×10 13 , and 9×10 13 In an embodiment, the number of TILs provided in the pharmaceutical composition of the present invention is 1 x 10 6 ~5×10 6 , 5×10 6 ~1×10 7 , 1×10 7 ~5×10 7 , 5×10 7 ~1×10 8 , 1×10 8 ~5×10 8 , 5×10 8 ~1×10 9 , 1×10 9 ~5×10 9 , 5×10 9 ~1×10 10 , 1×10 10 ~5×10 10 , 5×10 10 ~1×10 11 , 5×10 11 ~1×10 12 , 1×10 12 ~5×10 12 , and 5 × 10 12 ~1×10 13 is within the range.

[0565] In some embodiments, the concentration of TILs provided in the pharmaceutical compositions of the present invention is, for example, 100%, 90%, 80%, 70%, 60%, 50%, 40%, 30%, 20%, 19%, 18%, 17%, 16%, 15%, 14%, 13%, 12%, 11%, 10%, 9%, 8%, 7%, 6%, 5%, 4%, 3%, 2%, 1%, 0.5%, 0.4%, 0.3%, 0.2%, 0.1%, 0.09%, 0.08%, 0.07%, 0.06% , 0.05%, 0.04%, 0.03%, 0.02%, 0.01%, 0.009%, 0.008%, 0.007%, 0.006%, 0.005%, 0.004%, 0.003%, 0.002%, 0.001%, 0.0009%, 0.0008%, 0.0007%, 0.0006%, 0.0005%, 0.0004%, 0.0003%, 0.0002%, or 0.0001% w / w, w / v, or v / v of the pharmaceutical composition.

[0566] In some embodiments, the concentration of TILs provided in the pharmaceutical compositions of the present invention is 90%, 80%, 70%, 60%, 50%, 40%, 30%, 20%, 19.75%, 19.50%, 19.25%, 19%, 18.75%, 18.50%, 18.25%, 18%, 17.75%, 17.50%, 17.25%, 17%, 16.75%, 16.50%, 16.25%, 16%, 15.75%, 15.50%, 15.25%, 15%, 14.75%, 14.50%, 14.25%, 14. %,13.75%,13.50%,13.25%,13%,12.75%,12.50%,12.25%,12%,11.75%,11.50%,11.25%,11%,10.75%,10.50%,10.25%,10%,9.75%,9.50%,9.25%,9%,8.75%,8.50%,8.25%,8%,7.75%,7.50%,7.25%,7%,6.75%,6.50%,6.25%,6%,5.75%,5.50%,5.25%,5%,4.75%,4.50%,4 .25%,4%,3.75%,3.50%,3.25%,3%,2.75%,2.50%,2.25%,2%,1.75%,1.50%,125%,1%,0.5%,0.4%,0.3%,0.2%,0.1%,0.09%,0.08%,0.07%,0.06%,0.05%,0.04%,0.03%,0.02%,0.01%,0.009%, The pharmaceutical composition is greater than 0.008%, 0.007%, 0.006%, 0.005%, 0.004%, 0.003%, 0.002%, 0.001%, 0.0009%, 0.0008%, 0.0007%, 0.0006%, 0.0005%, 0.0004%, 0.0003%, 0.0002%, or 0.0001% w / w, w / v, or v / v.

[0567] In some embodiments, the concentration of TILs provided in the pharmaceutical compositions of the present invention is from about 0.0001% to about 50%, from about 0.001% to about 40%, from about 0.01% to about 30%, from about 0.02% to about 29%, from about 0.03% to about 28%, from about 0.04% to about 27%, from about 0.05% to about 26%, from about 0.06% to about 25%, from about 0.07% to about 24%, from about 0.08% to about 10%, from about 0.09% to about 12%, from about 0.10% to about 13%, from about 0.11% to about 14%, from about 0.12% to about 15%, from about 0.13% to about 16%, from about 0.14% to about 17%, from about 0.15% to about 18%, from about 0.16% to about 19%, from about 0.17% to about 20%, from about 0.18% to about 22%, from about 0.19% to about 23%, from about 0.19% to about 24%, from about 0.19% to about 25%, from about 0.19% to about 26%, from about 0.19% to about 25%, from about 0.19% to about 26%, from about 0.19% to about 27%, from about 0.19% to about 28%, from about 0.19% to about 29%, from about 0.19% to about 29%, from about 0.19% to about 25%, from about 0.19% to about 24%, from about 0.19% to about 25%, from about 0.19% to about 26%, from about 0.19% to about 2 The range is about 23%, about 0.09% to about 22%, about 0.1% to about 21%, about 0.2% to about 20%, about 0.3% to about 19%, about 0.4% to about 18%, about 0.5% to about 17%, about 0.6% to about 16%, about 0.7% to about 15%, about 0.8% to about 14%, about 0.9% to about 12%, or about 1% to about 10% w / w, w / v, or v / v of the pharmaceutical composition.

[0568] In some embodiments, the concentration of TILs provided in the pharmaceutical compositions of the present invention is within the range of about 0.001% to about 10%, about 0.01% to about 5%, about 0.02% to about 4.5%, about 0.03% to about 4%, about 0.04% to about 3.5%, about 0.05% to about 3%, about 0.06% to about 2.5%, about 0.07% to about 2%, about 0.08% to about 1.5%, about 0.09% to about 1%, or about 0.1% to about 0.9% w / w, w / v, or v / v of the pharmaceutical composition.

[0569] In some embodiments, the amount of TILs provided in the pharmaceutical compositions of the present invention is 10 g, 9.5 g, 9.0 g, 8.5 g, 8.0 g, 7.5 g, 7.0 g, 6.5 g, 6.0 g, 5.5 g, 5.0 g, 4.5 g, 4.0 g, 3.5 g, 3.0 g, 2.5 g, 2.0 g, 1.5 g, 1.0 g, 0.95 g, 0.9 g, 0.85 g, 0.8 g, 0.75 g, 0.7 g, 0.65 g, 0.6 g, 0.55 g, 0.5 g, 0.45 g, 0.4 g, 0.35 g, 0.3 g, 0.25 g , 0.2g, 0.15g, 0.1g, 0.09g, 0.08g, 0.07g, 0.06g, 0.05g, 0.04g, 0.03g, 0.02g, 0.01g, 0.009g, 0.008g, 0.007g, 0.006g, 0.005g, 0.004g, 0.003g, 0.002g, 0.001g, 0.0009g, 0.0008g, 0.0007g, 0.0006g, 0.0005g, 0.0004g, 0.0003g, 0.0002g, or 0.0001g or less.

[0570] In some embodiments, the amount of TILs provided in the pharmaceutical compositions of the present invention is 0.0001g, 0.0002g, 0.0003g, 0.0004g, 0.0005g, 0.0006g, 0.0007g, 0.0008g, 0.0009g, 0.001g, 0.0015g, 0.002g, 0.0025g, 0.003g, 0.0035g, 0.004g, 0.0045g, 0.005g, 0.0055g, 0.006g, 0.0065g, 0.007g, 0.0075g, 0.008g, 0.0085g, 0.009g, 0.0095g, 0.01g, 0.015 ... .02g, 0.025g, 0.03g, 0.035g, 0.04g, 0.045g, 0.05g, 0.055g, 0.06g, 0.065g, 0 .07g, 0.075g, 0.08g, 0.085g, 0.09g, 0.095g, 0.1g, 0.15g, 0.2g, 0.25g, 0.3g, 0.35g, 0.4g, 0.45g, 0.5g, 0.55g, 0.6g, 0.65g, 0.7g, 0.75g, 0.8g, 0.85g, 0.9g , 0.95g, 1g, 1.5g, 2g, 2.5, 3g, 3.5, 4g, 4.5g, 5g, 5.5g, 6g, 6.5g, 7g, 7.5g, 8g, 8 More than .5g, 9g, 9.5g, or 10g.

[0571] The TILs provided in the pharmaceutical compositions of the present invention are effective over a wide dosage range. The exact dosage will depend on the route of administration, the form in which the compound is administered, the gender and age of the subject being treated, the weight of the subject being treated, and the preferences and experience of the attending physician. If necessary, clinically established dosages of TILs can also be used. The amount of the pharmaceutical composition administered using the methods herein, such as the dosage of TILs, will depend on the human or mammal being treated, the severity of the disorder or condition, the rate of administration, the nature of the active pharmaceutical ingredient, and the discretion of the prescribing physician.

[0572] In some embodiments, TILs can be administered in a single dose. Such administration can be by injection, for example, intravenous injection. In some embodiments, TILs can be administered in multiple doses. Administration can be once, twice, three times, four times, five times, six times, or more than six times per year. Administration can be once a month, once every two weeks, once a week, or once every other day. Administration of TILs can be continued as long as necessary.

[0573] In some embodiments, an effective dose of TILs is about 1×10 6 , 2 × 10 6 , 3×10 6 , 4×10 6 , 5×10 6 , 6×10 6 , 7×10 6 , 8×10 6 , 9×10 6 , 1×10 7 , 2 × 10 7 , 3×10 7 , 4×10 7 , 5×10 7 , 6×10 7 , 7×10 7 , 8×10 7 , 9×10 7 , 1×10 8 , 2 × 10 8 , 3×10 8 , 4×10 8, 5×10 8 , 6×10 8 , 7×10 8 , 8×10 8 , 9×10 8 , 1×10 9 , 2 × 10 9 , 3×10 9 , 4×10 9 , 5×10 9 , 6×10 9 , 7×10 9 , 8×10 9 , 9×10 9 , 1×10 10 , 2 × 10 10 , 3×10 10 , 4×10 10 , 5×10 10 , 6×10 10 , 7×10 10 , 8×10 10 , 9×10 10 , 1×10 11 , 2 × 10 11 , 3×10 11 , 4×10 11 , 5×10 11 , 6×10 11 , 7×10 11 , 8×10 11 , 9×10 11 , 1×10 12 , 2 × 10 12 , 3×10 12 , 4×10 12 , 5×10 12 , 6×10 12 , 7×10 12 , 8×10 12 , 9×10 12 , 1×10 13 , 2 × 10 13 , 3×10 13 , 4×10 13 , 5×10 13 , 6×10 13 , 7×10 13 , 8×10 13 , and 9×10 13 In some embodiments, the effective dose of TILs is 1 x 10 6 ~5×10 6 , 5×10 6 ~1×10 7, 1×10 7 ~5×10 7 , 5×10 7 ~1×10 8 , 1×10 8 ~5×10 8 , 5×10 8 ~1×10 9 , 1×10 9 ~5×10 9 , 5×10 9 ~1×10 10 , 1×10 10 ~5×10 10 , 5×10 10 ~1×10 11 , 5×10 11 ~1×10 12 , 1×10 12 ~5×10 12 , and 5 × 10 12 ~1×10 13 is within the range.

[0574] In some embodiments, an effective dosage of TILs is about 0.01 mg / kg to about 4.3 mg / kg, about 0.15 mg / kg to about 3.6 mg / kg, about 0.3 mg / kg to about 3.2 mg / kg, about 0.35 mg / kg to about 2.85 mg / kg, about 0.15 mg / kg to about 2.85 mg / kg, about 0.3 mg to about 2.15 mg / kg, about 0.45 mg / kg to about 1.7 mg / kg, about 0.15 mg / kg to about 1.3 mg / kg, about 0.3 mg / kg to about 1.15 mg / kg, about 0.45 mg / kg to about 1 mg / kg, about 0.55 mg / kg to about 0.85 mg / kg, about 0.65 mg / kg to about 0.8 mg / kg, or about 0.7 mg / kg about 0.75 mg / kg, about 0.7 mg / kg to about 2.15 mg / kg, about 0.85 mg / kg to about 2 mg / kg, about 1 mg / kg to about 1.85 mg / kg, about 1.15 mg / kg to about 1.7 mg / kg, about 1.3 mg / kg to about 1.6 mg / kg, about 1.35 mg / kg to about 1.5 mg / kg, about 2.15 mg / kg to about 3.6 mg / kg, about 2.3 mg / kg to about 3.4 mg / kg, about 2.4 mg / kg to about 3.3 mg / kg, about 2.6 mg / kg to about 3.15 mg / kg, about 2.7 mg / kg to about 3 mg / kg, about 2.8 mg / kg to about 3 mg / kg, or about 2.85 mg / kg to about 2.95 mg / kg.

[0575] In some embodiments, an effective dose of TILs is about 1 mg to about 500 mg, about 10 mg to about 300 mg, about 20 mg to about 250 mg, about 25 mg to about 200 mg, about 1 mg to about 50 mg, about 5 mg to about 45 mg, about 10 mg to about 40 mg, about 15 mg to about 35 mg, about 20 mg to about 30 mg, about 23 mg to about 28 mg, about 50 mg to about 150 mg, about 60 mg to about 140 mg, or about The range is from 70 mg to about 130 mg, from about 80 mg to about 120 mg, from about 90 mg to about 110 mg, or from about 95 mg to about 105 mg, from about 98 mg to about 102 mg, from about 150 mg to about 250 mg, from about 160 mg to about 240 mg, from about 170 mg to about 230 mg, from about 180 mg to about 220 mg, from about 190 mg to about 210 mg, from about 195 mg to about 205 mg, or from about 198 mg to about 207 mg.

[0576] An effective amount of TILs can be administered by any of the accepted modes of administration for agents with similar utility, including intranasal and transdermal routes, by intra-arterial injection, intravenously, intraperitoneally, parenterally, intramuscularly, subcutaneously, topically, by implantation, or by inhalation, either in a single dose or in multiple doses.

[0577] V. How to Treat a Patient Therapeutic methods begin with initial TIL collection and TIL culture. Both of these methods are described in the art, for example, by Jin et al., J. Immunotherapy, 2012, 35(3):283-292, which is incorporated herein by reference in its entirety. Embodiments of therapeutic methods are described throughout the following sections, including the Examples.

[0578] Expanded TILs produced according to the methods described herein, e.g., as described in steps A-F above, or according to steps A-F above (and e.g., as shown in Figure 1), find particular use in treating patients with cancer (e.g., as described in Goff, et al., J. Clinical Oncology, 2016, 34(20):2389-239, and supplementary content, which are incorporated herein by reference in their entirety). In some embodiments, TILs were grown from excised deposits of metastatic melanoma, as previously described (see Dudley, et al., J. Immunother., 2003, 26:332-342, which are incorporated herein by reference in their entirety). Fresh tumors can be excised under sterile conditions. Representative samples can be collected for formal pathological analysis. 2 mm 3 ~3mm 3Single fragments of 1000 μg of TILs can be used. In some embodiments, 5, 10, 15, 20, 25, or 30 samples are obtained per patient. In some embodiments, 20, 25, or 30 samples are obtained per patient. In some embodiments, 20, 22, 24, 26, or 28 samples are obtained per patient. In some embodiments, 24 samples are obtained per patient. Samples can be placed in individual wells of a 24-well plate and maintained in growth medium containing high-dose IL-2 (6,000 IU / mL) to monitor tumor destruction and / or TIL proliferation. Tumors with viable cells remaining after treatment can be enzymatically digested into single-cell suspensions and cryopreserved as described herein.

[0579] In some embodiments, successfully grown TILs can be sampled for phenotypic analysis (CD3, CD4, CD8, and CD56) and tested against autologous tumor cells, if available. TILs can be considered reactive if interferon-gamma (IFN-γ) levels exceed 200 pg / mL, twice background, in overnight coculture. (Goff, et al., J Immunother., 2010, 33:840-847, incorporated herein by reference in its entirety.) In some embodiments, cultures with evidence of autoreactivity or sufficient growth patterns are further expanded (e.g., as shown in Figure 1), including a second expansion, sometimes referred to as rapid expansion (REP). In some embodiments, expanded TILs with high autoreactivity (e.g., high proliferation during the second expansion) are selected for an additional second expansion, as provided in step D of FIG. 1 . In some embodiments, expanded TILs with high autoreactivity (e.g., high proliferation during the second expansion) are selected for an additional second expansion, as provided in step D of FIG. 1 .

[0580] The cell phenotype of cryopreserved samples of infusion bag TILs can be analyzed by flow cytometry (e.g., FlowJo) for surface markers CD3, CD4, CD8, CCR7, and CD45RA (BD BioSciences) and any of the methods described herein. Serum cytokines were measured using standard enzyme-linked immunosorbent assays. Elevated serum IFN-g was defined as >100 pg / mL and above baseline levels.

[0581] In some embodiments, TILs produced by the methods provided herein, e.g., the method illustrated in FIG. 1, provide a surprising improvement in the clinical efficacy of TILs. In some embodiments, TILs produced by the methods provided herein, e.g., the method illustrated in FIG. 1, exhibit increased clinical efficacy compared to TILs produced by methods other than those described herein, including, e.g., methods other than those illustrated in FIG. 1. In some embodiments, methods other than those described herein include a method referred to as Process 1C and / or first generation (Gen1). In some embodiments, the increased efficacy is measured by DCR, ORR, and / or other clinical response. In some embodiments, TILs produced by the methods provided herein, e.g., the method illustrated in FIG. 1, exhibit a similar response time and safety profile compared to TILs produced by methods other than those described herein, including, e.g., the Gen1 process, other than those illustrated in FIG. 1.

[0582] In some embodiments, IFN-gamma (IFN-γ) indicates a therapeutic effect and / or an increased clinical effect. In some embodiments, IFN-γ in the blood of a subject treated with TILs indicates active TILs. In some embodiments, a potency assay for IFN-γ production is used. IFN-γ production is another measure of cytotoxicity. IFN-γ production can be measured by determining the level of the cytokine IFN-γ in the ex vivo blood, serum, or TILs of a subject treated with TILs prepared by the methods of the invention, including, for example, those described in FIG. 1. In some embodiments, an increase in IFN-γ indicates a therapeutic effect in patients treated with TILs produced by the methods of the invention. In some embodiments, IFN-γ is increased by 1-fold, 2-fold, 3-fold, 4-fold, or 5-fold or more compared to untreated patients and / or compared to patients treated with TILs prepared using methods other than those provided herein, including, for example, methods other than those embodied in FIG. 1. In some embodiments, IFN-γ secretion is increased 1-fold compared to untreated patients and / or compared to patients treated with TILs prepared using a method other than those provided herein, including, for example, a method other than those embodied in FIG. 1. In some embodiments, IFN-γ secretion is increased 2-fold compared to untreated patients and / or compared to patients treated with TILs prepared using a method other than those provided herein, including, for example, a method other than those embodied in FIG. 1. In some embodiments, IFN-γ secretion is increased 3-fold compared to untreated patients and / or compared to patients treated with TILs prepared using a method other than those provided herein, including, for example, a method other than those embodied in FIG. 1. In some embodiments, IFN-γ secretion is increased 4-fold compared to untreated patients and / or compared to patients treated with TILs prepared using a method other than those provided herein, including, for example, a method other than those embodied in FIG. 1. In some embodiments, IFN-γ secretion is increased 4-fold compared to untreated patients and / or compared to patients treated with TILs prepared using a method other than those provided herein, including, for example, a method other than those embodied in FIG. 1. For example, a 5-fold increase compared to patients treated with TILs prepared using methods other than those provided herein, including methods other than those embodied in FIG. 1. In some embodiments, IFN-γ is measured using a Quantikine ELISA kit. In some embodiments, IFN-γ is measured in ex vivo TILs of subjects treated with TILs prepared by the methods of the invention, including, for example, those described in FIG. 1. In some embodiments, IFN-γ is measured in the blood of subjects treated with TILs prepared by the methods of the invention, including, for example, those described in FIG. 1. In some embodiments, IFN-γ is measured in TIL serum of subjects treated with TILs prepared by the methods of the invention, including, for example, those described in FIG. 1.

[0583] In some embodiments, TILs prepared by the methods of the invention, including those described in FIG. 1, exhibit increased polyclonality compared to TILs produced by other methods, including those not illustrated in FIG. 1, such as the method designated Process 1C. In some embodiments, significantly improved and / or increased polyclonality indicates therapeutic and / or increased clinical efficacy. In some embodiments, polyclonality refers to the diversity of the T cell repertoire. In some embodiments, increased polyclonality can indicate a therapeutic effect for administration of TILs produced by the methods of the invention. In some embodiments, polyclonality is increased by 1-fold, 2-fold, 10-fold, 100-fold, 500-fold, or 1000-fold compared to TILs prepared using methods other than those provided herein, including, for example, methods other than those embodied in FIG. 1. In some embodiments, polyclonality is increased by 1-fold compared to untreated patients and / or compared to patients treated with TILs prepared using methods other than those provided herein, including, for example, methods other than those embodied in FIG. 1. In some embodiments, polyclonality is increased 2-fold compared to untreated patients and / or compared to patients treated with TILs prepared using methods other than those provided herein, including, for example, methods other than those embodied in FIG. 1. In some embodiments, polyclonality is increased 10-fold compared to untreated patients and / or compared to patients treated with TILs prepared using methods other than those provided herein, including, for example, methods other than those embodied in FIG. 1. In some embodiments, polyclonality is increased 100-fold compared to untreated patients and / or compared to patients treated with TILs prepared using methods other than those provided herein, including, for example, methods other than those embodied in FIG. 1. In some embodiments, polyclonality is increased 500-fold compared to untreated patients and / or compared to patients treated with TILs prepared using methods other than those provided herein, including, for example, methods other than those embodied in FIG. 1.In some embodiments, polyclonality is increased 1000-fold compared to untreated patients and / or compared to patients treated with TILs prepared using methods other than those provided herein, including, for example, methods other than those embodied in FIG. 1.

[0584] Measures of efficacy can include disease control rate (DCR) and overall response rate (ORR), as known in the art and as described herein.

[0585] 1. How to treat NSCLC The compositions and methods described herein can be used in methods for treating non-small cell lung cancer (NSCLC), where the NSCLC is refractory to treatment with an anti-PD-1 antibody. In some embodiments, the anti-PD-1 antibody includes, for example, nivolumab (BMS-936558, Bristol-Myers Squibb; Opdivo®), pembrolizumab (lambrolizumab, MK03475 or MK-3475, Merck; Keytruda®), ipilimumab (Yervoy®), humanized anti-PD-1 antibody JS001 (ShangHai JunShi), monoclonal antibody PD-1 (PD-1), ... Examples of suitable PD-1 antibodies include, but are not limited to, monoclonal anti-PD-1 antibody TSR-042 (Tesaro, Inc.), pidilizumab (anti-PD-1 mAb CT-011, Medivation), anti-PD-1 monoclonal antibody BGB-A317 (BeiGene), and / or anti-PD-1 antibody SHR-1210 (ShangHai HengRui), human monoclonal antibody REGN2810 (Regeneron), human monoclonal antibody MDX-1106 (Bristol-Myers Squibb), and / or humanized anti-PD-1 IgG4 antibody PDR001 (Novartis). In some embodiments, the PD-1 antibody is derived from clone: ​​RMP1-14 (rat IgG) - BioXcell catalog number BP0146. Other suitable antibodies for use in the co-administration method with TILs produced according to steps A-F described herein are the anti-PD-1 antibodies disclosed in U.S. Patent No. 8,008,449, incorporated herein by reference. In some embodiments, the antibody or antigen-binding portion thereof specifically binds to PD-L1 and inhibits its interaction with PD-1, thereby increasing immune activity. Included are any antibodies known in the art that bind to PD-L1, disrupt the interaction between PD-1 and PD-L1, and stimulate an anti-tumor immune response. For example, antibodies targeting PD-L1 and currently in clinical trials include BMS-936559 (Bristol-Myers Squibb) and MPDL3280A (Genentech). Other suitable antibodies targeting PD-L1 are disclosed in U.S. Patent No. 7,943,743, incorporated herein by reference. Those skilled in the art will recognize that the term "antibody" includes any antibody that binds to PD-1 or PD-L1, disrupts the PD-1 / PD-L1 interaction, and stimulates an anti-tumor immune response.

[0586] In some embodiments, the NSCLC has been treated with an anti-PD-1 antibody. In some embodiments, the NSCLC has been treated with an anti-PD-L1 antibody. In some embodiments, the NSCLC subject is treatment-naive. In some embodiments, the NSCLC has not been treated with an anti-PD-1 antibody. In some embodiments, the NSCLC has not been treated with an anti-PD-L1 antibody. In some embodiments, the NSCLC has been previously treated with a chemotherapeutic agent. In some embodiments, the NSCLC has been previously treated with a chemotherapeutic agent but is no longer being treated with the chemotherapeutic agent. In some embodiments, the NSCLC patient is anti-PD-1 / PD-L1 naive. In some embodiments, the NSCLC subject has low PD-L1 expression. In some embodiments, the NSCLC subject has treatment-naive NSCLC or is post-chemotherapeutic treatment but is anti-PD-1 / PD-L1 naive. In some embodiments, the NSCLC subject has treatment-naive NSCLC or post-chemotherapy treatment, but is anti-PD-1 / PD-L1 naive and has low PD-L1 expression. In some embodiments, the NSCLC subject has bulky mass disease at baseline. In some embodiments, the subject has bulky mass disease at baseline and low PD-L1 expression. In some embodiments, the NSCLC subject has treatment-naive NSCLC or post-chemotherapy treatment, but is anti-PD-1 / PD-L1 naive with low PD-L1 expression and / or bulky mass disease at baseline. In some embodiments, bulky mass disease is indicated when the maximum tumor diameter is greater than 7 cm measured in either the transverse or coronal plane. In some embodiments, bulky mass disease is indicated when there are enlarged lymph nodes with a short-axis diameter of 20 mm or greater. In some embodiments, the chemotherapeutic agent comprises a standard of care treatment for NSCLC.

[0587] In some embodiments, TILs prepared by the methods of the present invention, including, for example, those described in FIG. 1, exhibit increased polyclonality compared to TILs produced by other methods, including those not illustrated in FIG. 1, such as, for example, the method referred to as the Process 1C method. In some embodiments, significantly improved and / or increased polyclonality indicates a therapeutic effect and / or increased clinical efficacy for cancer treatment. In some embodiments, polyclonality refers to the diversity of the T cell repertoire. In some embodiments, an increase in polyclonality can indicate a therapeutic effect for administration of TILs produced by the methods of the invention. In some embodiments, polyclonality is increased 1-fold, 2-fold, 10-fold, 100-fold, 500-fold, or 1000-fold compared to TILs prepared using methods other than those provided herein, including, for example, methods other than those embodied in FIG. 1. In some embodiments, polyclonality is increased 1-fold compared to untreated patients and / or compared to patients treated with TILs prepared using methods other than those provided herein, including, for example, methods other than those embodied in FIG. 1. In some embodiments, polyclonality is increased 2-fold compared to untreated patients and / or compared to patients treated with TILs prepared using methods ot...

Claims

1. 1. A method of treating non-small cell lung cancer (NSCLC) with a population of tumor infiltrating lymphocytes (TILs), comprising: (a) obtaining and / or receiving a first TIL population from a surgical resection, needle biopsy, core biopsy, mini-biopsy, or other means for obtaining a sample containing a mixture of tumor and TIL cells from a patient's NSCLC tumor, including from multiple tumor fragments or biopsies; (c) contacting the tumor fragment with a first cell culture medium; (d) performing an initial expansion of the first TIL population in the first cell culture medium to obtain a second TIL population, the second TIL population being at least 5-fold more numerous than the first TIL population, and the first cell culture medium comprising IL-2; (e) performing rapid expansion of the second TIL population in a second cell culture medium to obtain a third TIL population, the third TIL population being at least 50 times more numerous than the second TIL population 7 days after the start of the 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), and the rapid expansion is performed for a period of 14 days or less; (f) harvesting the third population of TILs; (g) administering a therapeutically effective portion of the third population of TILs to the patient with NSCLC; The method, wherein the NSCLC is refractory to treatment with an anti-PD-1 antibody.

2. The method of claim 1 , wherein obtaining the first population of TILs comprises a multi-lesion sampling method.

3. The method of claim 1, wherein the refractory NSCLC has been previously treated with an anti-PD-1 and / or anti-PD-L1 and / or anti-PD-L2 antibody.

4. The method of claim 1, wherein the refractory NSCLC has not been previously treated with an anti-PD-1 and / or anti-PD-L1 antibody.

5. 10. The method of claim 1, wherein the refractory NSCLC is being treated with a chemotherapeutic agent.

6. The method of claim 1, wherein the refractory NSCLC has been previously treated with an anti-PD-1 and / or anti-PD-L1 antibody and has been previously treated with a chemotherapeutic agent.

7. The method of claim 1, wherein the refractory NSCLC has not been previously treated with an anti-PD-1 and / or anti-PD-L1 antibody and has been previously treated with a chemotherapeutic agent.

8. The method of claims 5-7, wherein the refractory NSCLC has been treated with a chemotherapeutic agent but is not currently being treated with a chemotherapeutic agent.

9. The method of claim 1, wherein the refractory NSCLC has low expression of PD-L1.

10. The method of claim 1, wherein the refractory NSCLC has been previously treated with an anti-PD-1 and / or anti-PD-L1 antibody and has low PD-L1 expression.

11. The refractory NSCLC has been previously treated with an anti-PD-1 and / or anti-PD-L1 antibody. The method of claim 1, wherein the patient is not afflicted by PD-L1 and has low PD-L1 expression.

12. The method of claim 1, wherein the refractory NSCLC has been treated with a chemotherapeutic agent and has low PD-L1 expression.

13. 2. The method of claim 1, wherein the refractory NSCLC has been treated with a chemotherapeutic agent, but is not currently being treated with a chemotherapeutic agent, and has low PD-L1 expression.

14. The method of claim 1, wherein the refractory NSCLC has not been previously treated with an anti-PD-1 and / or anti-PD-L1 antibody and has bulky disease at baseline.

15. The method of claim 1, wherein the refractory NSCLC has been previously treated with an anti-PD-1 and / or anti-PD-L1 antibody and has bulky disease at baseline.

16. 2. The method of claim 1, wherein the refractory NSCLC has been treated with a chemotherapy agent and has bulky disease at baseline.

17. 2. The method of claim 1, wherein the refractory NSCLC has been treated with a chemotherapeutic agent, but is not currently being treated with a chemotherapeutic agent, and has bulky disease at baseline.

18. The method of claims 14 to 17, wherein a bulky mass lesion is indicated when the maximum tumor diameter is greater than 7 cm measured in either the transverse or coronal plane or when an engorged lymph node has a short axis diameter of 20 mm or greater.

19. 10. The method of claim 1, wherein the refractory NSCLC is refractory to at least two prior courses of systemic therapy, not including neoadjuvant or adjuvant therapy.

20. 2. The method of claim 1, wherein the refractory NSCLC is refractory to an anti-PD-1 antibody selected from the group consisting of nivolumab, pembrolizumab, ipilimumab, JS001, TSR-042, pidilizumab, (BGB-A317, SHR-1210, REGN2810, MDX-1106, PDR001, clonally derived anti-PD-1: RMP1-14, and the anti-PD-1 antibodies disclosed in U.S. Pat. No. 8,008,449, durvalumab, atezolizumab, avelumab, and fragments, derivatives, variants, and biosimilars thereof.

21. 2. The method of claim 1, wherein the refractory NSCLC is refractory to pembrolizumab or a biosimilar thereof.

22. 2. The method of claim 1, wherein the refractory NSCLC is refractory to nivolumab or a biosimilar thereof.

23. 2. The method of claim 1, wherein the refractory NSCLC is refractory to ipilimumab or a biosimilar thereof.

24. 2. The method of claim 1, wherein the refractory NSCLC is refractory to ipilimumab or a biosimilar thereof and pembrolizumab or a biosimilar thereof.

25. 2. The method of claim 1, wherein the refractory NSCLC is refractory to ipilimumab or a biosimilar thereof and nivolumab or a biosimilar thereof.

26. 2. The method of claim 1, wherein the refractory NSCLC is refractory to durvalumab or a biosimilar thereof.

27. 2. The method of claim 1, wherein the refractory NSCLC is refractory to atezolizumab or a biosimilar thereof.

28. 2. The method of claim 1, wherein the refractory NSCLC is refractory to avelumab or a biosimilar thereof.

29. 29. The method of any one of claims 1 to 28, wherein the initial expansion is performed over a period of no more than 21 days.

30. 30. The method of any one of claims 1 to 29, wherein the initial expansion is performed over a period of no more than 14 days.

31. 31. The method of any one of claims 1 to 30, wherein the initial expansion is performed over a period of about 11 days and the rapid expansion is performed over a period of about 11 days.

32. 32. The method of any one of claims 1 to 31, wherein the IL-2 is present in the first cell culture medium at an initial concentration of between 1000 IU / mL and 6000 IU / mL.

33. 33. The method of any one of claims 1 to 32, wherein the IL-2 is present at an initial concentration of between 1000 IU / mL and 6000 IU / mL and the OKT-3 antibody is present in the second cell culture medium at an initial concentration of about 30 ng / mL.

34. The method of any one of claims 1 to 33, wherein the initial expansion is performed using a gas permeable container.

35. The method of any one of claims 1 to 34, wherein the rapid expansion is performed using a gas permeable container.

36. 36. The method of any one of claims 1 to 35, wherein 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.

37. 37. The method of any one of claims 1 to 36, wherein 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.

38. 38. The method of any one of claims 1-37, further comprising treating the patient with a non-myeloablative lymphodepleting regimen prior to administering the third population of TILs to the patient.

39. The non-myeloablative lymphodepleting regimen comprises 60 mg / m ２ / day for 2 days followed by administration of cyclophosphamide at a dose of 25 mg / m ２ 39. The method of claim 38, comprising administering fludarabine at a dose of 100 mg / kg / day for 5 days.

40. 40. The method of any one of claims 1-39, further comprising treating said patient with an IL-2 regimen beginning the day after administration of said third population of TILs to said patient.

41. 41. The method of claim 40, wherein 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.

42. 1. A method of treating non-small cell lung cancer (NSCLC) with a population of tumor infiltrating lymphocytes (TILs), comprising: (a) resecting one or more tumors from a patient, said one or more tumors comprising a first population of TILs; (b) fragmenting the one or more tumors into tumor fragments; (c) contacting the tumor fragment with a first cell culture medium; (d) performing an initial expansion of the first TIL population in the first cell culture medium to obtain a second TIL population, the second TIL population being at least 5-fold more numerous than the first TIL population, and the first cell culture medium comprising IL-2; (e) performing rapid expansion of the second TIL population in a second cell culture medium to obtain a third TIL population, the third TIL population being at least 50 times more numerous than the second TIL population 7 days after the start of the 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), and the rapid expansion is performed for a period of 14 days or less; (f) harvesting the third population of TILs; (g) administering a therapeutically effective portion of the third population of TILs to the patient with NSCLC; The method, wherein the NSCLC is refractory to treatment with an anti-PD-1 antibody.

43. 43. The method of claim 42, wherein the tumor is resected from one or more tumor foci.

44. The method of claim 42, wherein the refractory NSCLC has been previously treated with an anti-PD-1 and / or anti-PD-L1 and / or anti-PD-L2 antibody.

45. The method of claim 42, wherein the refractory NSCLC has not been previously treated with an anti-PD-1 and / or anti-PD-L1 antibody.

46. 43. The method of claim 42, wherein the refractory NSCLC is being treated with a chemotherapeutic agent.

47. The method of claim 42, wherein the refractory NSCLC has been previously treated with an anti-PD-1 and / or anti-PD-L1 antibody and has been previously treated with a chemotherapeutic agent.

48. The method of claim 42, wherein the refractory NSCLC has not been previously treated with an anti-PD-1 and / or anti-PD-L1 antibody and has been previously treated with a chemotherapeutic agent.

49. The method of claims 46-48, wherein the refractory NSCLC has been treated with a chemotherapeutic agent but is not currently being treated with a chemotherapeutic agent.

50. The method of claim 42, wherein the refractory NSCLC has low expression of PD-L1.

51. The method of claim 42, wherein the refractory NSCLC has been previously treated with an anti-PD-1 and / or anti-PD-L1 antibody and has low PD-L1 expression.

52. The method of claim 42, wherein the refractory NSCLC has not been previously treated with an anti-PD-1 and / or anti-PD-L1 antibody and has low PD-L1 expression.

53. 43. The method of claim 42, wherein the refractory NSCLC has been treated with a chemotherapeutic agent and has low PD-L1 expression.

54. 43. The method of claim 42, wherein the refractory NSCLC has been treated with a chemotherapeutic agent, but is not currently being treated with a chemotherapeutic agent, and has low PD-L1 expression.

55. The method of claim 42, wherein the refractory NSCLC has not been previously treated with an anti-PD-1 and / or anti-PD-L1 antibody and has bulky disease at baseline.

56. The method of claim 42, wherein the refractory NSCLC has been previously treated with an anti-PD-1 and / or anti-PD-L1 antibody and has bulky disease at baseline.

57. 43. The method of claim 42, wherein the refractory NSCLC has been treated with a chemotherapy agent and has bulky disease at baseline.

58. 43. The method of claim 42, wherein the refractory NSCLC has been treated with a chemotherapeutic agent, but is not currently being treated with a chemotherapeutic agent, and has bulky disease at baseline.

59. 59. The method of claims 55-58, wherein said bulky mass lesion is indicated when the maximum tumor diameter is greater than 7 cm measured in either the transverse or coronal plane or when an enlarged lymph node has a short axis diameter of 20 mm or greater.

60. 43. The method of claim 42, wherein the refractory NSCLC is refractory to at least two prior courses of systemic therapy, not including neoadjuvant or adjuvant therapy.

61. 43. The method of claim 42, wherein the refractory NSCLC is refractory to an anti-PD-1 antibody selected from the group consisting of nivolumab, pembrolizumab, ipilimumab, JS001, TSR-042, pidilizumab, (BGB-A317, SHR-1210, REGN2810, MDX-1106, PDR001, clonally derived anti-PD-1: RMP1-14, and the anti-PD-1 antibodies disclosed in U.S. Pat. No. 8,008,449, durvalumab, atezolizumab, avelumab, and fragments, derivatives, variants, and biosimilars thereof.

62. 43. The method of claim 42, wherein the refractory NSCLC is refractory to pembrolizumab or a biosimilar thereof.

63. 43. The method of claim 42, wherein the refractory NSCLC is refractory to nivolumab or a biosimilar thereof.

64. 43. The method of claim 42, wherein the refractory NSCLC is refractory to ipilimumab or a biosimilar thereof.

65. 43. The method of claim 42, wherein the refractory NSCLC is refractory to ipilimumab or a biosimilar thereof and pembrolizumab or a biosimilar thereof.

66. 43. The method of claim 42, wherein the refractory NSCLC is refractory to ipilimumab or a biosimilar thereof and nivolumab or a biosimilar thereof.

67. 43. The method of claim 42, wherein the refractory NSCLC is refractory to durvalumab or a biosimilar thereof.

68. 43. The method of claim 42, wherein the refractory NSCLC is refractory to atezolizumab or a biosimilar thereof.

69. 43. The method of claim 42, wherein the refractory NSCLC is refractory to avelumab or a biosimilar thereof.

70. 70. The method of any one of claims 42 to 69, wherein the initial expansion is performed for a period of no more than 21 days.

71. 71. The method of any one of claims 42 to 70, wherein the initial expansion is performed for a period of no more than 14 days.

72. 72. The method of any one of claims 42-71, wherein the initial expansion is performed over a period of about 11 days and the rapid expansion is performed over a period of about 11 days.

73. 73. The method of any one of claims 42 to 72, wherein the IL-2 is present in the first cell culture medium at an initial concentration of between 1000 IU / mL and 6000 IU / mL.

74. 74. The method of any one of claims 42-73, wherein the IL-2 is present at an initial concentration of between 1000 IU / mL and 6000 IU / mL and the OKT-3 antibody is present in the second cell culture medium at an initial concentration of about 30 ng / mL.

75. 75. The method of any one of claims 42 to 74, wherein the initial expansion is performed using a gas permeable container.

76. 76. The method of any one of claims 42 to 75, wherein the rapid expansion is performed using a gas permeable container.

77. 77. The method of any one of claims 42-76, wherein 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.

78. 78. The method of any one of claims 42-77, wherein 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.

79. 79. The method of any one of claims 42-78, further comprising treating the patient with a non-myeloablative lymphodepleting regimen prior to administering the third population of TILs to the patient.

80. The non-myeloablative lymphodepleting regimen comprises 60 mg / m ２ / day for 2 days followed by administration of cyclophosphamide at a dose of 25 mg / m ２ 80. The method of claim 79, comprising administering fludarabine at a dose of 100 mg / kg / day for 5 days.

81. 81. The method of any one of claims 42-80, further comprising treating the patient with an IL-2 regimen beginning the day after administration of the third population of TILs to the patient.

82. 82. The method of claim 81, wherein 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.

83. 1. A method for treating a subject having non-small cell lung cancer (NSCLC), the cancer being refractory to treatment with an anti-PD-1 antibody, comprising administering expanded tumor infiltrating lymphocytes (TILs), (a) obtaining and / or receiving a first population of TILs from one or more tumors excised from a subject by processing one or more tumors obtained from the subject into a plurality of tumor fragments; (b) adding the tumor fragment to a closed system; (c) performing a first expansion by culturing the first TIL population in a cell culture medium comprising IL-2 to produce a second TIL population, wherein the first expansion is performed in a closed vessel providing a first gas permeable surface area, and wherein the first expansion is performed for about 3-14 days to obtain the 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 produce a third TIL population, wherein the second expansion is performed for about 7-14 days to obtain the third TIL population, the third TIL population being a therapeutic TIL population that comprises an increased subpopulation of effector T cells and / or central memory T cells compared to the second TIL population, wherein the second expansion is performed in a sealed 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; and (f) transferring the harvested TIL population from step (e) to an infusion bag, wherein the transition 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 to the subject from the infusion bag of step (g).

84. 84. The method of claim 83, wherein the tumor sample is derived from a multiple lesion sampling method.

85. The method of claim 83, wherein the refractory NSCLC has been previously treated with an anti-PD-1 and / or anti-PD-L1 antibody.

86. The method of claim 83, wherein the refractory NSCLC has not been previously treated with an anti-PD-1 and / or anti-PD-L1 antibody.

87. 84. The method of claim 83, wherein the refractory NSCLC is being treated with a chemotherapeutic agent.

88. The method of claim 83, wherein the refractory NSCLC has been previously treated with an anti-PD-1 and / or anti-PD-L1 antibody and has been previously treated with a chemotherapeutic agent.

89. The method of claim 83, wherein the refractory NSCLC has not been previously treated with an anti-PD-1 and / or anti-PD-L1 antibody and has been previously treated with a chemotherapeutic agent.

90. The method of claims 87-89, wherein the refractory NSCLC has been treated with a chemotherapeutic agent but is not currently being treated with a chemotherapeutic agent.

91. 84. The method of claim 83, wherein the refractory NSCLC has low expression of PD-L1.

92. 84. The method of claim 83, wherein the refractory NSCLC has been previously treated with an anti-PD-1 and / or anti-PD-L1 antibody and has low PD-L1 expression.

93. 84. The method of claim 83, wherein the refractory NSCLC has not been previously treated with an anti-PD-1 and / or anti-PD-L1 antibody and has low PD-L1 expression.

94. 84. The method of claim 83, wherein the refractory NSCLC has been treated with a chemotherapeutic agent and has low PD-L1 expression.

95. 84. The method of claim 83, wherein the refractory NSCLC has been treated with a chemotherapeutic agent, but is not currently being treated with a chemotherapeutic agent, and has low PD-L1 expression.

96. The method of claim 83, wherein the refractory NSCLC has not been previously treated with an anti-PD-1 and / or anti-PD-L1 antibody and has bulky disease at baseline.

97. The method of claim 83, wherein the refractory NSCLC has been previously treated with an anti-PD-1 and / or anti-PD-L1 antibody and has bulky disease at baseline.

98. 84. The method of claim 83, wherein the refractory NSCLC has been treated with a chemotherapy agent and has bulky disease at baseline.

99. 84. The method of claim 83, wherein the refractory NSCLC has been treated with a chemotherapeutic agent, but is not currently being treated with a chemotherapeutic agent, and has bulky disease at baseline.

100. 100. The method of claims 96-99, wherein a bulky mass lesion is indicated when the maximum tumor diameter is greater than 7 cm measured in either the transverse or coronal plane or when an enlarged lymph node has a short axis diameter of 20 mm or greater.

101. 84. The method of claim 83, wherein the refractory NSCLC is refractory to at least two prior courses of systemic therapy, not including neoadjuvant or adjuvant therapy.

102. 84. The method of claim 83, wherein the refractory NSCLC is refractory to an anti-PD-1 antibody selected from the group consisting of nivolumab, pembrolizumab, ipilimumab, JS001, TSR-042, pidilizumab, (BGB-A317, SHR-1210, REGN2810, MDX-1106, PDR001, clonally derived anti-PD-1: RMP1-14, and the anti-PD-1 antibodies disclosed in U.S. Pat. No. 8,008,449, durvalumab, atezolizumab, avelumab, and fragments, derivatives, variants, and biosimilars thereof.

103. 84. The method of claim 83, wherein the refractory NSCLC is refractory to pembrolizumab or a biosimilar thereof.

104. 84. The method of claim 83, wherein the refractory NSCLC is refractory to nivolumab or a biosimilar thereof.

105. 84. The method of claim 83, wherein the refractory NSCLC is refractory to ipilimumab or a biosimilar thereof.

106. 84. The method of claim 83, wherein the refractory NSCLC is refractory to ipilimumab or a biosimilar thereof and pembrolizumab or a biosimilar thereof.

107. 84. The method of claim 83, wherein the refractory NSCLC is refractory to ipilimumab or a biosimilar thereof and nivolumab or a biosimilar thereof.

108. 84. The method of claim 83, wherein the refractory NSCLC is refractory to durvalumab or a biosimilar thereof.

109. 84. The method of claim 83, wherein the refractory NSCLC is refractory to atezolizumab or a biosimilar thereof.

110. 84. The method of claim 83, wherein the refractory NSCLC is refractory to avelumab or a biosimilar thereof.

111. 111. The method of any one of claims 83 to 110, wherein the initial expansion is performed for a period of no more than 21 days.

112. 112. The method of any one of claims 83 to 111, wherein the initial expansion is performed for a period of no more than 14 days.

113. 113. The method of any one of claims 83 to 112, wherein the initial expansion is performed over a period of about 3 to 11 days and the second expansion is performed over a period of about 7 to 11 days.

114. 114. The method of any one of claims 83-113, wherein the initial expansion is performed over a period of about 11 days and the rapid expansion is performed over a period of about 11 days.

115. 115. The method of any one of claims 83 to 114, wherein the IL-2 is present in the first cell culture medium at an initial concentration of between 1000 IU / mL and 6000 IU / mL.

116. 116. The method of any one of claims 83-115, wherein the IL-2 is present at an initial concentration of between 1000 IU / mL and 6000 IU / mL and the OKT-3 antibody is present in the second cell culture medium at an initial concentration of about 30 ng / mL.

117. 117. The method of any one of claims 83 to 116, wherein the initial expansion is performed using a gas permeable container.

118. 118. The method of any one of claims 83 to 117, wherein the rapid expansion is performed using a gas permeable container.

119. 119. The method of any one of claims 83-118, wherein 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.

120. 120. The method of any one of claims 83-119, wherein 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.

121. 121. The method of any one of claims 83-120, further comprising treating the patient with a non-myeloablative lymphodepleting regimen prior to administering the third population of TILs to the patient.

122. The non-myeloablative lymphodepleting regimen comprises 60 mg / m ２ / day for 2 days followed by administration of cyclophosphamide at a dose of 25 mg / m ２ 122. The method of claim 121, comprising administering fludarabine at a dose of 100 mg / kg / day for 5 days.

123. 123. The method of any one of claims 83-122, further comprising treating said patient with an IL-2 regimen beginning the day after administration of said third population of TILs to said patient.

124. 124. The method of claim 123, wherein 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.

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