Chimeric costimulatory receptors, chemokine receptors, and their uses in cellular immunotherapy - Patents.com

Abstract

The present invention provides compositions comprising chimeric receptors, including chimeric costimulatory receptors (CCRs) and / or chemokine receptors, methods for preparing CCRs and / or chemokine receptors, and therapeutic populations of tumor-infiltrating lymphocytes, bone marrow-infiltrating lymphocytes, and peripheral blood lymphocytes expressing CCRs and / or chemokine receptors, which have increased therapeutic performance and other advantages for the treatment of cancer, including solid tumor cancers.

Description

[Technical Field]

[0001] CROSS-REFERENCE TO RELATED APPLICATIONS This application claims priority to U.S. Provisional Patent No. 63 / 176,675, filed April 19, 2021, U.S. Provisional Patent No. 63 / 223,925, filed July 20, 2021, U.S. Provisional Patent No. 63 / 254,297, filed October 11, 2021, and U.S. Provisional Patent No. 63 / 284,177, filed November 30, 2021, the entire disclosures of each of which are incorporated herein by reference in their entirety. [Background technology]

[0002] Treatment of solid tumor cancers remains challenging, especially for patients who do not respond to commonly used initial therapies, including chemotherapy, targeted therapies, and checkpoint inhibitors such as nivolumab, pembrolizumab, ipilimumab, atezolizumab, avelumab, and durvalumab, as well as combinations of these immunotherapies (e.g., nivolumab and ipilimumab) or combinations of these immunotherapies with chemotherapy. Treating bulky, refractory cancers using adoptive transfer of tumor-infiltrating lymphocytes (TILs) offers a powerful approach to treating patients with poor prognosis solid tumor cancers, including those who have failed initial therapy. (Gattinoni, et al., Nat. Rev. Immunol. 2006, 6, 383-393) Successful immunotherapy requires large numbers of active 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)) as feeder cells, often from multiple donors, 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 a 14-day period. (Dudley, et al., J. Immunother. 2003, 26, 332-42) TILs that have undergone the REP procedure have been successfully used in adoptive cell therapy following host immunosuppression in patients with melanoma, head and neck cancer, non-small cell lung cancer, and cervical cancer in both frontline and post-treatment settings.Jimeno,et al.,Poster 353,SITC Annual Meeting,Nov.9-14,2020, Sarnaik,et al.,Oral Presentation at ASCO Annual Meeting,May 29-30,2020,Jazaeri,et al.,Poster 182,ASCO Annual Meeting,May 31-June 4,2019.

[0003] Chimeric costimulatory receptors (CCRs) are genetically engineered chimeric receptors designed to provide costimulatory signals to effector cells, such as T cells, to promote activation. Sadelain, et al., Cancer Discovery, 2013, 3, 388-398; Liao, et al., Biomarker Res. 2020, 8, 57. CCRs are most commonly associated with T cell therapies based on chimeric antigen receptor (CAR)-modified T cell (CAR-T) products and can be used in combination with CARs to enhance activation. However, CCRs have not been extensively explored alongside other emerging cell therapies, including polyclonal TIL therapy in solid tumor cancers, which use tumor-associated antigens or other antigens for activation. While current TIL therapies have demonstrated safety and efficacy, there remains a significant unmet need for TIL therapies with improved efficacy, duration of response, and safety, particularly for patients with locally advanced or metastatic solid tumor cancers. Summary of the Invention

[0004] The present invention provides methods for generating and using TILs with enhanced properties using CCRs and compositions thereof for use in treating patients with solid tumors.

[0005] In some embodiments, the present invention includes methods of treating cancer by administering to a patient in need of cancer treatment a population of tumor-infiltrating lymphocytes (TILs), bone marrow-infiltrating lymphocytes (MILs), or peripheral blood lymphocytes (PBLs), wherein the TILs, MILs, or PBLs are genetically modified to express a chimeric costimulatory receptor (CCR), wherein the CCR comprises: i. an extracellular domain; ii. optionally, a hinge domain; and iii. optionally a transmembrane domain; and iv. at least one intracellular domain.

[0006] In some embodiments, the present invention provides a method of treating cancer by administering to a patient in need thereof a population of tumor-infiltrating lymphocytes (TILs), bone marrow-infiltrating lymphocytes (MILs), or peripheral blood lymphocytes (PBLs), wherein the TILs, MILs, or PBLs are genetically modified to express a chimeric costimulatory receptor (CCR), wherein the CCR comprises: i. an extracellular domain; ii. optionally, a hinge domain; and iii. optionally a transmembrane domain; and iv. at least one intracellular domain; The cancer is treated by administering a population of TILs, and the method comprises: (a) obtaining and / or receiving a first population of TILs from a tumor excised from a patient by processing a tumor sample obtained from the patient into a plurality of tumor fragments or tumor digests; (b) adding the first TIL population to the closed system; (c) performing a first expansion by culturing the first TIL population in a first cell culture medium comprising IL-2 and optionally an OKT-3 antibody, and antigen-presenting cells (APCs) to produce a second TIL population, wherein the first expansion is performed in a sealed container that provides a first gas-permeable surface area, and the first expansion is performed for about 3 to 14 days to obtain the second TIL population, and the transition from step (b) to step (c) occurs without opening the system. (d) genetically modifying the second TIL population to express a CCR; (e) performing a second expansion of the second TIL population in a second cell culture medium comprising IL-2, OKT-3 antibody, and APC to produce a third TIL population, wherein the second expansion is performed for about 3 to 14 days to obtain the third TIL population, the third TIL population being a therapeutic TIL population, and wherein the second expansion is performed in a sealed container providing a second gas permeable surface area; (f) harvesting the therapeutic TIL population obtained from step (e); (g) transferring the harvested TIL population from step (f) into an infusion bag, wherein the transition from step (e) to (f) occurs without opening the system; (h) cryopreserving the infusion bag containing the harvested TIL population from step (f) using a cryopreservation process; (i) administering a therapeutically effective dose of the third population of TILs to the patient from the infusion bag of step (g).

[0007] In some embodiments, the present invention provides a method of treating cancer by administering to a patient in need thereof a population of tumor-infiltrating lymphocytes (TILs), bone marrow-infiltrating lymphocytes (MILs), or peripheral blood lymphocytes (PBLs), wherein the TILs, MILs, or PBLs are genetically modified to express a chimeric costimulatory receptor (CCR), wherein the CCR comprises: i. an extracellular domain; ii. optionally, a hinge domain; and iii. optionally a transmembrane domain; and iv. at least one intracellular domain.

[0008] In some embodiments, the present invention provides a method of treating cancer by administering to a patient in need thereof a population of tumor-infiltrating lymphocytes (TILs), bone marrow-infiltrating lymphocytes (MILs), or peripheral blood lymphocytes (PBLs), wherein the TILs, MILs, or PBLs are genetically modified to express a chimeric costimulatory receptor (CCR), wherein the CCR comprises: i. an extracellular domain; ii. optionally, a hinge domain; and iii. optionally a transmembrane domain; and iv. at least one intracellular domain; The cancer is treated by administering a population of TILs, and the method comprises: (a) obtaining and / or receiving a first population of TILs from a tumor excised from a patient by processing a tumor sample obtained from the patient into a plurality of tumor fragments or tumor digests; (b) adding the first TIL population to the closed system; (c) performing a first expansion by culturing the first TIL population in a first cell culture medium comprising IL-2 and optionally an OKT-3 antibody, and antigen-presenting cells (APCs) to produce a second TIL population, wherein the first expansion is performed in a sealed container that provides a first gas-permeable surface area, and the first expansion is performed for about 3 to 14 days to obtain the second TIL population, and the transition from step (b) to step (c) occurs without opening the system. (d) genetically modifying the second TIL population to express a CCR; (e) performing a second expansion of the second TIL population in a second cell culture medium comprising IL-2, OKT-3 antibody, and APC to produce a third TIL population, wherein the second expansion is performed for about 3 to 14 days to obtain the third TIL population, the third TIL population being a therapeutic TIL population, and wherein the second expansion is performed in a sealed container providing a second gas permeable surface area; (f) harvesting the therapeutic TIL population obtained from step (e); (g) transferring the harvested TIL population from step (f) into an infusion bag, wherein the transition from step (e) to (f) occurs without opening the system; (h) cryopreserving the infusion bag containing the harvested TIL population from step (f) using a cryopreservation process; (i) administering a therapeutically effective dose of the third population of TILs to the patient from the infusion bag of step (g); The extracellular domain comprises an scFv binding domain.

[0009] In some embodiments, the present invention provides a method of treating cancer by administering to a patient in need thereof a population of tumor-infiltrating lymphocytes (TILs), bone marrow-infiltrating lymphocytes (MILs), or peripheral blood lymphocytes (PBLs), wherein the TILs, MILs, or PBLs are genetically modified to express a chimeric costimulatory receptor (CCR), wherein the CCR comprises: i. an extracellular domain; ii. optionally, a hinge domain; and iii. optionally a transmembrane domain; and iv. at least one intracellular domain; The cancer is treated by administering a population of TILs, and the method comprises: (a) obtaining and / or receiving a first population of TILs from a tumor excised from a patient by processing a tumor sample obtained from the patient into a plurality of tumor fragments or tumor digests; (b) adding the first TIL population to the closed system; (c) performing a first expansion by culturing the first TIL population in a first cell culture medium comprising IL-2 and optionally an OKT-3 antibody, and antigen-presenting cells (APCs) to produce a second TIL population, wherein the first expansion is performed in a sealed container that provides a first gas-permeable surface area, and the first expansion is performed for about 3 to 14 days to obtain the second TIL population, and the transition from step (b) to step (c) occurs without opening the system. (d) genetically modifying the second TIL population to express a CCR; (e) performing a second expansion of the second TIL population in a second cell culture medium comprising IL-2, OKT-3 antibody, and APC to produce a third TIL population, wherein the second expansion is performed for about 3 to 14 days to obtain the third TIL population, the third TIL population being a therapeutic TIL population, and wherein the second expansion is performed in a sealed container providing a second gas permeable surface area; (f) harvesting the therapeutic TIL population obtained from step (e); (g) transferring the harvested TIL population from step (f) into an infusion bag, wherein the transition from step (e) to (f) occurs without opening the system; (h) cryopreserving the infusion bag containing the harvested TIL population from step (f) using a cryopreservation process; (i) administering a therapeutically effective dose of the third population of TILs to the patient from the infusion bag of step (g); The extracellular domain comprises an scFv binding domain, which binds to a protein selected from the group consisting of CD19, CD20, CD22, CD24, CD33, CD38, CD39, CD73, CD123, CD138, CD228, LRRC15, CEA, FRα, EPCAM, PD-L1, PSMA, gp100, MUC1, MCSP, EGFR, GD2, TROP-2, GPC3, MICA, MICB, VISTA, ULBP, HER2, MCM5, FAP, 5T4, LFA-1, B7-H3, IL-13Rα2, FAS, TGFβRII, and MUC16.

[0010] In some embodiments, the present invention provides a method of treating cancer by administering to a patient in need thereof a population of tumor-infiltrating lymphocytes (TILs), bone marrow-infiltrating lymphocytes (MILs), or peripheral blood lymphocytes (PBLs), wherein the TILs, MILs, or PBLs are genetically modified to express a chimeric costimulatory receptor (CCR), wherein the CCR comprises: i. an extracellular domain; ii. optionally, a hinge domain; and iii. optionally a transmembrane domain; and iv. at least one intracellular domain; The cancer is treated by administering a population of TILs, and the method comprises: (a) obtaining and / or receiving a first population of TILs from a tumor excised from a patient by processing a tumor sample obtained from the patient into a plurality of tumor fragments or tumor digests; (b) adding the first TIL population to the closed system; (c) performing a first expansion by culturing the first TIL population in a first cell culture medium comprising IL-2 and optionally an OKT-3 antibody, and antigen-presenting cells (APCs) to produce a second TIL population, wherein the first expansion is performed in a sealed container that provides a first gas-permeable surface area, and the first expansion is performed for about 3 to 14 days to obtain the second TIL population, and the transition from step (b) to step (c) occurs without opening the system. (d) genetically modifying the second TIL population to express a CCR; (e) performing a second expansion of the second TIL population in a second cell culture medium comprising IL-2, OKT-3 antibody, and APC to produce a third TIL population, wherein the second expansion is performed for about 3 to 14 days to obtain the third TIL population, the third TIL population being a therapeutic TIL population, and wherein the second expansion is performed in a sealed container providing a second gas permeable surface area; (f) harvesting the therapeutic TIL population obtained from step (e); (g) transferring the harvested TIL population from step (f) into an infusion bag, wherein the transition from step (e) to (f) occurs without opening the system; (h) cryopreserving the infusion bag containing the harvested TIL population from step (f) using a cryopreservation process; (i) administering a therapeutically effective dose of the third population of TILs to the patient from the infusion bag of step (g); The extracellular domain is the PD-1 domain.

[0011] In some embodiments, the present invention provides a method of treating cancer by administering to a patient in need thereof a population of tumor-infiltrating lymphocytes (TILs), bone marrow-infiltrating lymphocytes (MILs), or peripheral blood lymphocytes (PBLs), wherein the TILs, MILs, or PBLs are genetically modified to express a chimeric costimulatory receptor (CCR), wherein the CCR comprises: i. an extracellular domain; ii. optionally, a hinge domain; and iii. optionally a transmembrane domain; and iv. at least one intracellular domain; The cancer is treated by administering a population of TILs, and the method comprises: (a) obtaining and / or receiving a first population of TILs from a tumor excised from a patient by processing a tumor sample obtained from the patient into a plurality of tumor fragments or tumor digests; (b) adding the first TIL population to the closed system; (c) performing a first expansion by culturing the first TIL population in a first cell culture medium comprising IL-2 and optionally an OKT-3 antibody, and antigen-presenting cells (APCs) to produce a second TIL population, wherein the first expansion is performed in a sealed container that provides a first gas-permeable surface area, and the first expansion is performed for about 3 to 14 days to obtain the second TIL population, and the transition from step (b) to step (c) occurs without opening the system. (d) genetically modifying the second TIL population to express a CCR; (e) performing a second expansion of the second TIL population in a second cell culture medium comprising IL-2, OKT-3 antibody, and APC to produce a third TIL population, wherein the second expansion is performed for about 3 to 14 days to obtain the third TIL population, the third TIL population being a therapeutic TIL population, and wherein the second expansion is performed in a sealed container providing a second gas permeable surface area; (f) harvesting the therapeutic TIL population obtained from step (e); (g) transferring the harvested TIL population from step (f) into an infusion bag, wherein the transition from step (e) to (f) occurs without opening the system; (h) cryopreserving the infusion bag containing the harvested TIL population from step (f) using a cryopreservation process; (i) administering a therapeutically effective dose of the third population of TILs to the patient from the infusion bag of step (g); The intracellular domain is selected from the group consisting of CD28, CD134 (OX40), CD278 (ICOS), CD137 (4-1BB), CD27, IL-2Rβ, IL-2Rγ, IL-18R1, IL-7Rα, IL-12R1, IL-12R2, IL-15Rα, IL-21R, and combinations thereof.

[0012] In some embodiments, the present invention provides a method of treating cancer by administering to a patient in need thereof a population of tumor-infiltrating lymphocytes (TILs), bone marrow-infiltrating lymphocytes (MILs), or peripheral blood lymphocytes (PBLs), wherein the TILs, MILs, or PBLs are genetically modified to express a chimeric costimulatory receptor (CCR), wherein the CCR comprises: i. an extracellular domain; ii. optionally, a hinge domain; and iii. optionally a transmembrane domain; and iv. at least one intracellular domain; The cancer is treated by administering a population of TILs, and the method comprises: (a) obtaining and / or receiving a first population of TILs from a tumor excised from a patient by processing a tumor sample obtained from the patient into a plurality of tumor fragments or tumor digests; (b) adding the first TIL population to the closed system; (c) performing a first expansion by culturing the first TIL population in a first cell culture medium comprising IL-2 and optionally an OKT-3 antibody, and antigen-presenting cells (APCs) to produce a second TIL population, wherein the first expansion is performed in a sealed container that provides a first gas-permeable surface area, and the first expansion is performed for about 3 to 14 days to obtain the second TIL population, and the transition from step (b) to step (c) occurs without opening the system. (d) genetically modifying the second TIL population to express a CCR; (e) performing a second expansion of the second TIL population in a second cell culture medium comprising IL-2, OKT-3 antibody, and APC to produce a third TIL population, wherein the second expansion is performed for about 3 to 14 days to obtain the third TIL population, the third TIL population being a therapeutic TIL population, and wherein the second expansion is performed in a sealed container providing a second gas permeable surface area; (f) harvesting the therapeutic TIL population obtained from step (e); (g) transferring the harvested TIL population from step (f) into an infusion bag, wherein the transition from step (e) to (f) occurs without opening the system; (h) cryopreserving the infusion bag containing the harvested TIL population from step (f) using a cryopreservation process; (i) administering a therapeutically effective dose of the third population of TILs to the patient from the infusion bag of step (g); The intracellular domain is selected from the group consisting of CD28, CD134 (OX40), CD278 (ICOS), CD137 (4-1BB), CD27, IL-2Rβ, IL-2Rγ, IL-18R1, IL-7Rα, IL-12R1, IL-12R2, IL-15Rα, IL-21R, and combinations thereof, and the transmembrane domain is selected from the group consisting of the transmembrane regions of CD3α, CD3β, CDζ, CD3ε, CD4, CD5, CD8α, CD9, CD16, CD22, CD27, CD28, CD33, CD37, CD45, CD64, CD80, CD86, CD134, CD137, CD154, IgG1, IgG4, IgD, IL-2Rα, IL-2Rβ, and IL-2Rγ.

[0013] In some embodiments, the present invention provides a method of treating cancer by administering to a patient in need thereof a population of tumor-infiltrating lymphocytes (TILs), bone marrow-infiltrating lymphocytes (MILs), or peripheral blood lymphocytes (PBLs), wherein the TILs, MILs, or PBLs are genetically modified to express a chimeric costimulatory receptor (CCR), wherein the CCR comprises: i. an extracellular domain; ii. optionally, a hinge domain; and iii. optionally a transmembrane domain; and iv. at least one intracellular domain; The cancer is treated by administering a population of TILs, and the method comprises: (a) obtaining and / or receiving a first population of TILs from a tumor excised from a patient by processing a tumor sample obtained from the patient into a plurality of tumor fragments or tumor digests; (b) adding the first TIL population to the closed system; (c) performing a first expansion by culturing the first TIL population in a first cell culture medium comprising IL-2 and optionally an OKT-3 antibody, and antigen-presenting cells (APCs) to produce a second TIL population, wherein the first expansion is performed in a sealed container that provides a first gas-permeable surface area, and the first expansion is performed for about 3 to 14 days to obtain the second TIL population, and the transition from step (b) to step (c) occurs without opening the system. (d) genetically modifying the second TIL population to express a CCR; (e) performing a second expansion of the second TIL population in a second cell culture medium comprising IL-2, OKT-3 antibody, and APC to produce a third TIL population, wherein the second expansion is performed for about 3 to 14 days to obtain the third TIL population, the third TIL population being a therapeutic TIL population, and wherein the second expansion is performed in a sealed container providing a second gas permeable surface area; (f) harvesting the therapeutic TIL population obtained from step (e); (g) transferring the harvested TIL population from step (f) into an infusion bag, wherein the transition from step (e) to (f) occurs without opening the system; (h) cryopreserving the infusion bag containing the harvested TIL population from step (f) using a cryopreservation process; (i) administering a therapeutically effective dose of the third population of TILs to the patient from the infusion bag of step (g); The intracellular domain is selected from the group consisting of CD28, CD134 (OX40), CD278 (ICOS), CD137 (4-1BB), CD27, IL-2Rβ, IL-2Rγ, IL-18R1, IL-7Rα, IL-12R1, IL-12R2, IL-15Rα, IL-21R, and combinations thereof, and the transmembrane domain is selected from the group consisting of CD3α, CD3β, CDζ, CD3ε, CD4, CD5, and CD8α. , CD9, CD16, CD22, CD27, CD28, CD33, CD37, CD45, CD64, CD80, CD86, CD134, CD137, CD154, IgG1, IgG4, IgD, IL-2Rα, IL-2Rβ, and the transmembrane domain of IL-2Rγ, and step (d) further comprises genetically modifying the TIL using a lentivirus to express the CCR.

[0014] In some embodiments, the present invention provides a method of treating cancer by administering to a patient in need thereof a population of tumor-infiltrating lymphocytes (TILs), bone marrow-infiltrating lymphocytes (MILs), or peripheral blood lymphocytes (PBLs), wherein the TILs, MILs, or PBLs are genetically modified to express a chimeric costimulatory receptor (CCR), wherein the CCR comprises: i. an extracellular domain; ii. optionally, a hinge domain; and iii. optionally a transmembrane domain; and iv. at least one intracellular domain; The cancer is treated by administering a population of TILs, and the method comprises: (a) obtaining and / or receiving a first population of TILs from a tumor excised from a patient by processing a tumor sample obtained from the patient into a plurality of tumor fragments or tumor digests; (b) adding the first TIL population to the closed system; (c) performing a first expansion by culturing the first TIL population in a first cell culture medium comprising IL-2 and optionally an OKT-3 antibody, and antigen-presenting cells (APCs) to produce a second TIL population, wherein the first expansion is performed in a sealed container that provides a first gas-permeable surface area, and the first expansion is performed for about 3 to 14 days to obtain the second TIL population, and the transition from step (b) to step (c) occurs without opening the system. (d) genetically modifying the second TIL population to express a CCR; (e) performing a second expansion of the second TIL population in a second cell culture medium comprising IL-2, OKT-3 antibody, and APC to produce a third TIL population, wherein the second expansion is performed for about 3 to 14 days to obtain the third TIL population, the third TIL population being a therapeutic TIL population, and wherein the second expansion is performed in a sealed container providing a second gas permeable surface area; (f) harvesting the therapeutic TIL population obtained from step (e); (g) transferring the harvested TIL population from step (f) into an infusion bag, wherein the transition from step (e) to (f) occurs without opening the system; (h) cryopreserving the infusion bag containing the harvested TIL population from step (f) using a cryopreservation process; (i) administering a therapeutically effective dose of the third population of TILs to the patient from the infusion bag of step (g); The intracellular domain is selected from the group consisting of CD28, CD134 (OX40), CD278 (ICOS), CD137 (4-1BB), CD27, IL-2Rβ, IL-2Rγ, IL-18R1, IL-7Rα, IL-12R1, IL-12R2, IL-15Rα, IL-21R, and combinations thereof; the transmembrane domain is selected from the group consisting of CD3α, CD3β, CDζ, CD3ε, CD4, CD5, CD8α, CD9, CD16, CD22, CD27, CD28, CD33, CD37, CD45, CD64, CD80, CD86, CD134, CD137, CD154, IgG1, The TIL, MIL, or PBL is further genetically modified to stably or transiently reduce expression of a gene selected from the group consisting of PD-1, LAG-3, TIM-3, CTLA-4, TIGIT, CISH, TGFβR2, PKA, CBL-B, BAFF(BR3), SOCS1, ANKRD11, BCOR, and a combination thereof.

[0015] In some embodiments, the present invention provides a method of treating cancer by administering to a patient in need thereof a population of tumor-infiltrating lymphocytes (TILs), bone marrow-infiltrating lymphocytes (MILs), or peripheral blood lymphocytes (PBLs), wherein the TILs, MILs, or PBLs are genetically modified to express a chimeric costimulatory receptor (CCR), wherein the CCR comprises: i. an extracellular domain; ii. optionally, a hinge domain; and iii. optionally a transmembrane domain; and iv. at least one intracellular domain; The cancer is treated by administering a population of TILs, and the method comprises: (a) obtaining and / or receiving a first population of TILs from a tumor excised from a patient by processing a tumor sample obtained from the patient into a plurality of tumor fragments or tumor digests; (b) adding the first TIL population to the closed system; (c) performing a first expansion by culturing the first TIL population in a first cell culture medium comprising IL-2 and optionally an OKT-3 antibody, and antigen-presenting cells (APCs) to produce a second TIL population, wherein the first expansion is performed in a sealed container that provides a first gas-permeable surface area, and the first expansion is performed for about 3 to 14 days to obtain the second TIL population, and the transition from step (b) to step (c) occurs without opening the system. (d) genetically modifying the second TIL population to express a CCR; (e) performing a second expansion of the second TIL population in a second cell culture medium comprising IL-2, OKT-3 antibody, and APC to produce a third TIL population, wherein the second expansion is performed for about 3 to 14 days to obtain the third TIL population, the third TIL population being a therapeutic TIL population, and wherein the second expansion is performed in a sealed container providing a second gas permeable surface area; (f) harvesting the therapeutic TIL population obtained from step (e); (g) transferring the harvested TIL population from step (f) into an infusion bag, wherein the transition from step (e) to (f) occurs without opening the system; (h) cryopreserving the infusion bag containing the harvested TIL population from step (f) using a cryopreservation process; (i) administering a therapeutically effective dose of the third population of TILs to the patient from the infusion bag of step (g); The intracellular domain is selected from the group consisting of CD28, CD134 (OX40), CD278 (ICOS), CD137 (4-1BB), CD27, IL-2Rβ, IL-2Rγ, IL-18R1, IL-7Rα, IL-12R1, IL-12R2, IL-15Rα, IL-21R, and combinations thereof, and the transmembrane domain is selected from the group consisting of CD3α, CD3β, CDζ, CD3ε, CD4, CD5, CD8α, CD9, CD16, CD22, CD27, CD28, CD33, CD37, CD45, CD64, CD80, CD86, CD134, CD137, CD154, IgG1, IgG4, IgD, and IL-2R. the transmembrane regions of IL-2Rα, IL-2Rβ, and IL-2Rγ are selected from the group consisting of IL-2Rβ, IL-2Rγ, and IL-2Rγ; step (d) further comprises genetically modifying the TILs using a lentivirus to express a CCR; the TILs, MILs, or PBLs are further genetically modified to stably or transiently reduce expression of a gene selected from the group consisting of PD-1, LAG-3, TIM-3, CTLA-4, TIGIT, CISH, TGFβR2, PKA, CBL-B, BAFF (BR3), SOCS1, ANKRD11, BCOR, and combinations thereof; and the cancer is a solid tumor cancer treated by administering the TILs.

[0016] In any of the foregoing embodiments, the cancer is selected from the group consisting of sarcoma, pancreatic cancer, liver cancer, glioblastoma, gastrointestinal cancer, melanoma, ovarian cancer, endometrial cancer, thyroid cancer, colorectal cancer, cervical cancer, lung cancer, non-small cell lung cancer, small cell lung cancer, mesothelioma, bladder cancer, breast cancer, cancer caused by human papillomavirus, head and neck cancer, renal cancer, and renal cell carcinoma, and the patient is human.

[0017] In any of the foregoing embodiments, the cancer is further treated with a PD-1 or PD-L1 inhibitor in combination with TILs, wherein the PD-1 or PD-L1 inhibitor is selected from the group consisting of nivolumab, pembrolizumab, cemiplimab, tislelizumab, sintilimab, toripalimab, dostarimab, durvalumab, avelumab, atezolizumab, retifanlimab, and fragments, variants, and biosimilars thereof.

[0018] In any of the preceding embodiments, the cancer is non-small cell lung cancer and the patient is 1) a predefined tumor proportion score (TPS) of PD-L1 of <1%; 2) PD-L1 TPS score of 1% to 49%, or 3) have at least one of the predetermined absences of one or more driver mutations.

[0019] In any of the foregoing embodiments, the cancer is non-small cell lung cancer and the patient has a TPS of PD-L1 of <1%.

[0020] In any of the foregoing embodiments, the patient has a cancer that is not indicated for treatment with an EGFR inhibitor, a BRAF inhibitor, an ALK inhibitor, a c-Ros inhibitor, a RET inhibitor, an ERBB2 inhibitor, a BRCA inhibitor, a MAP2K1 inhibitor, a PIK3CA inhibitor, a CDKN2A inhibitor, a PTEN inhibitor, a UMD inhibitor, an NRAS inhibitor, a KRAS inhibitor, an NF1 inhibitor, a MET inhibitor, a TP53 inhibitor, a CREBBP inhibitor, a KMT2C inhibitor, a KMT2D mutation, an ARID1A mutation, an RB1 inhibitor, an ATM inhibitor, a SETD2 inhibitor, a FLT3 inhibitor, a PTPN11 inhibitor, an FGFR1 inhibitor, an EP300 inhibitor, a MYC inhibitor, an EZH2 inhibitor, a JAK2 inhibitor, an FBXW7 inhibitor, a CCND3 inhibitor, and a GNA11 inhibitor.

[0021] In any of the foregoing embodiments, the patient has the absence of one or more driver mutations, wherein the one or more driver mutations are an EGFR mutation, an EGFR insertion, an EGFR exon 20, a KRAS mutation, a BRAF mutation, a BRAF V600 mutation, ALK mutation, c-ROS mutation (ROS1 mutation), ROS1 fusion, RET mutation, RET fusion, ERBB2 mutation, ERBB2 amplification, BRCA mutation, MAP2K1 mutation, PIK3CA, CDKN2A, PTEN mutation, UMD mutation, NRAS mutation, KRAS mutation, NF1 mutation, MET mutation, MET splice and / or altered MET signaling, TP53 mutation, CREBBP mutation, KMT2C mutation, KMT2D mutation, ARID1A mutation, RB1 mutation, ATM mutation, SETD2 mutation, FLT3 mutation, PTPN11 mutation, FGFR1 mutation, EP300 mutation, MYC mutation, EZH2 mutation, JAK2 mutation, FBXW7 mutation, CCND3 mutation, and GNA11 mutation.

[0022] In any of the foregoing embodiments, the cancer is refractory or resistant to treatment with a chemotherapeutic agent or chemotherapy regimen.

[0023] In any of the foregoing embodiments, the cancer is refractory or resistant to treatment with a VEGF-A inhibitor, and the VEGF-A inhibitor is selected from the group consisting of bevacizumab, ranibizumab, icrucumab, and fragments, variants, and biosimilars thereof.

[0024] In any of the foregoing embodiments, the cancer is refractory or resistant to treatment with a PD-1 or PD-L1 inhibitor, and the PD-1 or PD-L1 inhibitor is selected from the group consisting of nivolumab, pembrolizumab, cemiplimab, tislelizumab, sintilimab, toripalimab, dostarimab, durvalumab, avelumab, atezolizumab, retifanlimab, and fragments, variants, and biosimilars thereof.

[0025] In any of the foregoing embodiments, the cancer is refractory or resistant to treatment with a CTLA-4 inhibitor, and the CTLA-4 inhibitor is selected from the group consisting of ipilimumab, tremelimumab, zalifrelimab, and fragments, variants, and biosimilars thereof.

[0026] In any of the foregoing embodiments, IL-2 is initially present in the first cell culture medium and the second cell culture medium at an initial concentration of 1000 IU / mL to 6000 IU / mL.

[0027] In any of the foregoing embodiments, the OKT-3 antibody is initially present in the second cell culture medium at an initial concentration of about 30 ng / mL.

[0028] In any of the preceding 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.

[0029] In any of the preceding 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.

[0030] In any of the foregoing embodiments, the method may further comprise treating the patient with a non-myeloablative lymphodepletion regimen prior to administering the third population of TILs to the patient.

[0031] In any of the foregoing embodiments, the method may further include treating the patient with a non-myeloablative lymphodepletion regimen prior to administering the third population of TILs to the patient, the non-myeloablative lymphodepletion regimen including cyclophosphamide at 60 mg / m 2 / day for 2 days, followed by fludarabine at 25 mg / m 2 / day for 5 days.

[0032] In any of the foregoing embodiments, the method may further include treating the patient with a non-myeloablative lymphodepletion regimen prior to administering the third population of TILs to the patient, the non-myeloablative lymphodepletion regimen including cyclophosphamide at 60 mg / m 2 / day and fludarabine at a dose of 25 mg / m 2 / day for 2 days, followed by fludarabine at 25 mg / m 2 / day for 3 days.

[0033] In any of the foregoing embodiments, the method may further include treating the patient with an IL-2 regimen starting the day after the third population of TILs is administered to the patient.

[0034] In any of the foregoing embodiments, the method may further comprise treating the patient with an IL-2 regimen beginning on the same day that the third population of TILs is administered to the patient.

[0035] In any of the foregoing embodiments, the method may further include treating the patient with an IL-2 regimen, wherein the IL-2 regimen is a high-dose IL-2 regimen comprising 600,000 or 720,000 IU / kg aldesleukin, or a fragment, variant, or biosimilar thereof, administered as a 15-minute bolus intravenous infusion every 8 hours until tolerated.

[0036] In any of the foregoing embodiments, the method may further include treating the patient with an IL-2 regimen, wherein the IL-2 regimen includes administration of bempegaldesleukin, or a fragment, variant, or biosimilar thereof.

[0037] In any of the foregoing embodiments, the method may further include treating the patient with an IL-2 regimen, wherein the IL-2 regimen includes administration of THOR-707, or a fragment, variant, or biosimilar thereof.

[0038] In any of the foregoing embodiments, the method may further include treating the patient with an IL-2 regimen, wherein the IL-2 regimen includes administration of nembareukin alfa, or a fragment, variant, or biosimilar thereof.

[0039] In any of the foregoing embodiments, the method may further include treating the patient with an IL-2 regimen, wherein the IL-2 regimen comprises administering an antibody, or a fragment, variant, or biosimilar thereof, comprising a heavy chain selected from the group consisting of SEQ ID NO:29 and SEQ ID NO:38, and a light chain selected from the group consisting of SEQ ID NO:37 and SEQ ID NO:39.

[0040] In any of the foregoing embodiments, a therapeutically effective population of TILs is administered, and is about 2×10 9 ~Approx. 15×10 10 Includes TILs.

[0041] In any of the foregoing embodiments, the first expansion is performed for a period of 11 days or less.

[0042] In any of the foregoing embodiments, the second expansion is performed over a period of no more than 11 days.

[0043] In some embodiments, the invention includes compositions comprising tumor-infiltrating lymphocytes (TILs), bone marrow-infiltrating lymphocytes (MILs), or peripheral blood lymphocytes (PBLs) that have been genetically modified to express a chimeric costimulatory receptor (CCR), wherein the CCR is i. an extracellular domain; ii. optionally, a hinge domain; and iii. optionally a transmembrane domain; and iv. at least one intracellular domain.

[0044] In some embodiments, the invention includes compositions comprising tumor-infiltrating lymphocytes (TILs), bone marrow-infiltrating lymphocytes (MILs), or peripheral blood lymphocytes (PBLs) that have been genetically modified to express a chimeric costimulatory receptor (CCR), wherein the CCR is i. an extracellular domain; ii. optionally, a hinge domain; and iii. optionally a transmembrane domain; and iv. at least one intracellular domain; The extracellular domain comprises an scFv binding domain.

[0045] In some embodiments, the invention includes compositions comprising tumor-infiltrating lymphocytes (TILs), bone marrow-infiltrating lymphocytes (MILs), or peripheral blood lymphocytes (PBLs) that have been genetically modified to express a chimeric costimulatory receptor (CCR), wherein the CCR is i. an extracellular domain; ii. optionally, a hinge domain; and iii. optionally a transmembrane domain; and iv. at least one intracellular domain; The extracellular domain comprises an scFv binding domain, and the scFv binding domain is selected from the group consisting of an anti-CD19 domain, an anti-CD20 domain, an anti-CD22 domain, an anti-CD24 domain, an anti-CD33 domain, an anti-CD38 domain, an anti-CD39 domain, an anti-CD73 domain, an anti-CD123 domain, an anti-CD138 domain, an anti-CD228 domain, an anti-LRRC15 domain, an anti-CEA domain, an anti-FRα domain, an anti-EPCAM domain, an anti-PD-L1 domain, an anti-PSMA domain, an anti-gp100 domain, an anti-MUC1 domain, an anti-MCSP domain, an anti-EGFR domain, an anti-GD2 domain, an anti-TROP-2 domain, an anti-GPC3 domain, an anti-MICA domain, an anti-MICB domain, an anti-VISTA domain, an anti-ULBP domain, an anti-HER2 domain, an anti-MCM5 domain, an anti-FAP domain, an anti-5T4 domain, an anti-LFA-1 domain, an anti-B7-H3 domain, and an anti-MUC16 domain.

[0046] In some embodiments, the invention includes compositions comprising tumor-infiltrating lymphocytes (TILs), bone marrow-infiltrating lymphocytes (MILs), or peripheral blood lymphocytes (PBLs) that have been genetically modified to express a chimeric costimulatory receptor (CCR), wherein the CCR is i. an extracellular domain; ii. optionally, a hinge domain; and iii. optionally a transmembrane domain; and iv. at least one intracellular domain; The extracellular domain is the PD-1 domain.

[0047] In some embodiments, the invention includes compositions comprising tumor-infiltrating lymphocytes (TILs), bone marrow-infiltrating lymphocytes (MILs), or peripheral blood lymphocytes (PBLs) that have been genetically modified to express a chimeric costimulatory receptor (CCR), wherein the CCR is i. an extracellular domain; ii. optionally, a hinge domain; and iii. optionally a transmembrane domain; and iv. at least one intracellular domain; The intracellular domain is selected from the group consisting of a CD28 domain, a CD134 (OX40) domain, a CD278 (ICOS) domain, a CD137 (4-1BB) domain, a CD27 domain, an IL-2Rβ domain, an IL-2Rγ domain, an IL-18R1 domain, an IL-7Rα domain, an IL-12R1 domain, an IL-12R2 domain, an IL-15Rα domain, an IL-21R domain, and combinations thereof.

[0048] In some embodiments, the invention includes compositions comprising tumor-infiltrating lymphocytes (TILs), bone marrow-infiltrating lymphocytes (MILs), or peripheral blood lymphocytes (PBLs) that have been genetically modified to express a chimeric costimulatory receptor (CCR), wherein the CCR is i. an extracellular domain; ii. optionally, a hinge domain; and iii. optionally a transmembrane domain; and iv. at least one intracellular domain; The intracellular domain is selected from the group consisting of a CD28 domain, a CD134 (OX40) domain, a CD278 (ICOS) domain, a CD137 (4-1BB) domain, a CD27 domain, an IL-2Rβ domain, an IL-2Rγ domain, an IL-18R1 domain, an IL-7Rα domain, an IL-12R1 domain, an IL-12R2 domain, an IL-15Rα domain, an IL-21R domain, and combinations thereof; and the transmembrane domain is selected from the group consisting of a CD3α domain, a CD3β domain, a CDζ domain, a CD3ε domain, a CD3ζ ... domain, a CD4 domain, a CD5 domain, a CD8α domain, a CD9 domain, a CD16 domain, a CD22 domain, a CD27 domain, a CD28 domain, a CD33 domain, a CD37 domain, a CD45 domain, a CD64 domain, a CD80 domain, a CD86 domain, a CD134 domain, a CD137 domain, a CD154 domain, an IgG1 domain, an IgG4 domain, an IgD domain, an IL-2Rα domain, an IL-2Rβ domain, and an IL-2Rγ domain.

[0049] In some embodiments, the invention includes compositions comprising tumor-infiltrating lymphocytes (TILs), bone marrow-infiltrating lymphocytes (MILs), or peripheral blood lymphocytes (PBLs) that have been genetically modified to express a chimeric costimulatory receptor (CCR), wherein the CCR is i. an extracellular domain; ii. optionally, a hinge domain; and iii. optionally a transmembrane domain; and iv. at least one intracellular domain; The intracellular domain is selected from the group consisting of a CD28 domain, a CD134 (OX40) domain, a CD278 (ICOS) domain, a CD137 (4-1BB) domain, a CD27 domain, an IL-2Rβ domain, an IL-2Rγ domain, an IL-18R1 domain, an IL-7Rα domain, an IL-12R1 domain, an IL-12R2 domain, an IL-15Rα domain, an IL-21R domain, and combinations thereof; and the transmembrane domain is selected from the group consisting of a CD3α domain, a CD3β domain, a CDζ domain, a CD3ε domain, a CD4 domain, a CD5 domain, a CD8α domain, a CD9 domain, a CD16 domain, a CD22 domain, a CD27 domain, a CD2 8 domain, CD33 domain, CD37 domain, CD45 domain, CD64 domain, CD80 domain, CD86 domain, CD134 domain, CD137 domain, CD154 domain, IgG1 domain, IgG4 domain, IgD domain, IL-2Rα domain, IL-2Rβ domain, and IL-2Rγ domain, and the TIL, MIL, or PBL is further genetically modified to stably or transiently reduce expression of a gene selected from the group consisting of PD-1, LAG-3, TIM-3, CTLA-4, TIGIT, CISH, TGFβR2, PKA, CBL-B, BAFF(BR3), and combinations thereof.

[0050] In some embodiments, the present invention provides a composition comprising a chimeric costimulatory receptor (CCR), wherein the CCR comprises: i. an extracellular protein domain; ii. a hinge protein domain; iii. a transmembrane protein domain; iv. at least one intracellular protein domain.

[0051] In some embodiments, the present invention includes compositions comprising a chimeric costimulatory receptor (CCR), wherein the CCR comprises: i. an extracellular protein domain; ii. a hinge protein domain; iii. a transmembrane protein domain; iv. at least one intracellular protein domain; The extracellular protein domain comprises an scFv binding domain.

[0052] In some embodiments, the present invention provides a composition comprising a chimeric costimulatory receptor (CCR), wherein the CCR comprises: i. an extracellular protein domain; ii. a hinge protein domain; iii. a transmembrane protein domain; iv. at least one intracellular protein domain; The extracellular protein domain comprises an scFv binding domain, and the scFv binding domain is selected from the group consisting of an anti-CD19 domain, an anti-CD20 domain, an anti-CD22 domain, an anti-CD24 domain, an anti-CD33 domain, an anti-CD38 domain, an anti-CD39 domain, an anti-CD73 domain, an anti-CD123 domain, an anti-CD138 domain, an anti-CD228 domain, an anti-LRRC15 domain, an anti-CEA domain, an anti-FRα domain, an anti-EPCAM domain, an anti-PD-L1 domain, an anti-PSMA domain, an anti-gp100 domain, an anti-MUC1 domain, an anti-MCSP domain, an anti-EGFR domain, an anti-GD2 domain, an anti-TROP-2 domain, an anti-GPC3 domain, an anti-MICA domain, an anti-MICB domain, an anti-VISTA domain, an anti-ULBP domain, an anti-HER2 domain, an anti-MCM5 domain, an anti-FAP domain, an anti-5T4 domain, an anti-LFA-1 domain, an anti-B7-H3 domain, and an anti-MUC16 domain.

[0053] In some embodiments, the present invention includes compositions comprising a chimeric costimulatory receptor (CCR), wherein the CCR comprises: i. an extracellular protein domain; ii. a hinge protein domain; iii. a transmembrane protein domain; iv. at least one intracellular protein domain; The extracellular protein domain is the PD-1 domain.

[0054] In some embodiments, the present invention includes compositions comprising a chimeric costimulatory receptor (CCR), wherein the CCR comprises: i. an extracellular protein domain; ii. a hinge protein domain; iii. a transmembrane protein domain; iv. at least one intracellular protein domain; The intracellular protein domain is selected from the group consisting of a CD28 domain, a CD134 (OX40) domain, a CD278 (ICOS) domain, a CD137 (4-1BB) domain, a CD27 domain, an IL-2Rβ domain, an IL-2Rγ domain, an IL-18R1 domain, an IL-7Rα domain, an IL-12R1 domain, an IL-12R2 domain, an IL-15Rα domain, an IL-21R domain, and combinations thereof.

[0055] In some embodiments, the present invention includes compositions comprising a chimeric costimulatory receptor (CCR), wherein the CCR comprises: i. an extracellular protein domain; ii. a hinge protein domain; iii. a transmembrane protein domain; iv. at least one intracellular protein domain; The intracellular protein domain is selected from the group consisting of a CD28 domain, a CD134 (OX40) domain, a CD278 (ICOS) domain, a CD137 (4-1BB) domain, a CD27 domain, an IL-2Rβ domain, an IL-2Rγ domain, an IL-18R1 domain, an IL-7Rα domain, an IL-12R1 domain, an IL-12R2 domain, an IL-15Rα domain, an IL-21R domain, and combinations thereof; and the transmembrane protein domain is selected from the group consisting of a CD3α domain, a CD3β domain, a CDζ domain, , CD3ε domain, CD4 domain, CD5 domain, CD8α domain, CD9 domain, CD16 domain, CD22 domain, CD27 domain, CD28 domain, CD33 domain, CD37 domain, CD45 domain, CD64 domain, CD80 domain, CD86 domain, CD134 domain, CD137 domain, CD154 domain, IgG1 domain, IgG4 domain, IgD domain, IL-2Rα domain, IL-2Rβ domain, and IL-2Rγ domain.

[0056] In some embodiments, the present invention includes compositions comprising a chimeric costimulatory receptor (CCR), wherein the CCR comprises: i. an extracellular protein domain; ii. a hinge protein domain; iii. a transmembrane protein domain; iv. at least one intracellular protein domain; The intracellular protein domain is selected from the group consisting of a CD28 domain, a CD134 (OX40) domain, a CD278 (ICOS) domain, a CD137 (4-1BB) domain, a CD27 domain, an IL-2Rβ domain, an IL-2Rγ domain, an IL-18R1 domain, an IL-7Rα domain, an IL-12R1 domain, an IL-12R2 domain, an IL-15Rα domain, an IL-21R domain, and combinations thereof. The transmembrane protein domain is selected from the group consisting of a CD3α domain, a CD3β domain, a CDζ domain, a CD3ε domain, a CD4 domain, a CD5 domain, a CD8α domain, a CD9 domain, a CD16 domain, a CD22 domain, a CD27 domain, a CD28 domain, a CD33 domain, a CD37 domain, a CD45 domain, a CD64 domain, a CD80 domain, a CD86 domain, a CD13 4 domain, CD137 domain, CD154 domain, IgG1 domain, IgG4 domain, IgD domain, IL-2Rα domain, IL-2Rβ domain, and IL-2Rγ domain; and the hinge protein domain is selected from the group consisting of CD3α domain, CD3β domain, CDζ domain, CD3ε domain, CD4 domain, CD5 domain, CD8α domain, CD9 domain, CD16 domain, CD22 domain, CD27 domain, CD28 domain, CD33 domain, CD37 domain, CD45 domain, CD64 domain, CD80 domain, CD86 domain, CD134 domain, CD137 domain, CD154 domain, IgG1 domain, IgG4 domain, IgD domain, IL-2Rα domain, IL-2Rβ domain, and IL-2Rγ domain.

[0057] In some embodiments, the present invention includes compositions comprising a chimeric costimulatory receptor (CCR), wherein the CCR comprises: i. an extracellular protein domain; ii. a hinge protein domain; iii. a transmembrane protein domain; iv. at least one intracellular protein domain; The intracellular protein domain is selected from the group consisting of a CD28 domain, a CD134 (OX40) domain, a CD278 (ICOS) domain, a CD137 (4-1BB) domain, a CD27 domain, an IL-2Rβ domain, an IL-2Rγ domain, an IL-18R1 domain, an IL-7Rα domain, an IL-12R1 domain, an IL-12R2 domain, an IL-15Rα domain, an IL-21R domain, and combinations thereof. The transmembrane protein domain is selected from the group consisting of a CD3α domain, a CD3β domain, a CDζ domain, a CD3ε domain, a CD4 domain, a CD5 domain, a CD8α domain, a CD9 domain, a CD16 domain, a CD22 domain, a CD27 domain, a CD28 domain, a CD33 domain, a CD37 domain, a CD45 domain, a CD64 domain, a CD80 domain, a CD86 domain, a CD134 domain, a CD13 the hinge protein domain is selected from the group consisting of a CD3α domain, a CD3β domain, a CDζ domain, a CD3ε domain, a CD4 domain, a CD5 domain, a CD8α domain, a CD9 domain, a CD16 domain, a CD22 domain, a CD27 domain, a CD28 domain, a CD33 domain, a CD37 domain, a CD45 domain, a CD64 domain, a CD80 domain, a CD86 domain, a CD134 domain, a CD137 domain, a CD154 domain, an IgG1 domain, an IgG4 domain, an IgD domain, an IL-2Rα domain, an IL-2Rβ domain, and an IL-2Rγ domain; and the composition further comprises tumor-infiltrating lymphocytes.

[0058] In some embodiments, the present invention includes compositions comprising a chimeric costimulatory receptor (CCR), wherein the CCR comprises: i. an extracellular protein domain; ii. a hinge protein domain; iii. a transmembrane protein domain; iv. at least one intracellular protein domain; The intracellular protein domain is selected from the group consisting of a CD28 domain, a CD134 (OX40) domain, a CD278 (ICOS) domain, a CD137 (4-1BB) domain, a CD27 domain, an IL-2Rβ domain, an IL-2Rγ domain, an IL-18R1 domain, an IL-7Rα domain, an IL-12R1 domain, an IL-12R2 domain, an IL-15Rα domain, an IL-21R domain, and combinations thereof. The transmembrane protein domain is selected from the group consisting of a CD3α domain, a CD3β domain, a CDζ domain, a CD3ε domain, a CD4 domain, a CD5 domain, a CD8α domain, a CD9 domain, a CD16 domain, a CD22 domain, a CD27 domain, a CD28 domain, a CD33 domain, a CD37 domain, a CD45 domain, a CD64 domain, a CD80 domain, a CD86 domain, a CD134 domain, a CD13 the hinge protein domain is selected from the group consisting of a CD3α domain, a CD3β domain, a CDζ domain, a CD3ε domain, a CD4 domain, a CD5 domain, a CD8α domain, a CD9 domain, a CD16 domain, a CD22 domain, a CD27 domain, a CD28 domain, a CD33 domain, a CD37 domain, a CD45 domain, a CD64 domain, a CD80 domain, a CD86 domain, a CD134 domain, a CD137 domain, a CD154 domain, an IgG1 domain, an IgG4 domain, an IgD domain, an IL-2Rα domain, an IL-2Rβ domain, and an IL-2Rγ domain; and the composition further comprises bone marrow-infiltrating lymphocytes.

[0059] In some embodiments, the present invention includes compositions comprising a chimeric costimulatory receptor (CCR), wherein the CCR comprises: i. an extracellular protein domain; ii. a hinge protein domain; iii. a transmembrane protein domain; iv. at least one intracellular protein domain; The intracellular protein domain is selected from the group consisting of a CD28 domain, a CD134 (OX40) domain, a CD278 (ICOS) domain, a CD137 (4-1BB) domain, a CD27 domain, an IL-2Rβ domain, an IL-2Rγ domain, an IL-18R1 domain, an IL-7Rα domain, an IL-12R1 domain, an IL-12R2 domain, an IL-15Rα domain, an IL-21R domain, and combinations thereof; and the transmembrane protein domain is selected from the group consisting of a CD3α domain, a CD3β domain, a CDζ domain, a CD3ε domain, a CD4 domain, a CD5 domain, a CD8α domain, a CD9 domain, a CD16 domain, a CD22 domain, a CD27 domain, a CD28 domain, a CD33 domain, a CD37 domain, a CD45 domain, a CD64 domain, a CD80 domain, a CD86 domain, a CD134 domain, a CD1 the hinge protein domain is selected from the group consisting of a CD3α domain, a CD3β domain, a CDζ domain, a CD3ε domain, a CD4 domain, a CD5 domain, a CD8α domain, a CD9 domain, a CD16 domain, a CD22 domain, a CD27 domain, a CD28 domain, a CD33 domain, a CD37 domain, a CD45 domain, a CD64 domain, a CD80 domain, a CD86 domain, a CD134 domain, a CD137 domain, a CD154 domain, an IgG1 domain, an IgG4 domain, an IgD domain, an IL-2Rα domain, an IL-2Rβ domain, and an IL-2Rγ domain; and the composition further comprises peripheral blood lymphocytes.

[0060] In some embodiments, the present invention includes methods of treating cancer by administering to a patient in need of cancer treatment a population of tumor-infiltrating lymphocytes (TILs), bone marrow-infiltrating lymphocytes (MILs), or peripheral blood lymphocytes (PBLs), wherein the TILs, MILs, or PBLs are genetically modified to express a chemokine receptor.

[0061] In some embodiments, the invention includes a method of treating cancer by administering to a patient in need of cancer treatment a population of tumor-infiltrating lymphocytes (TILs), bone marrow-infiltrating lymphocytes (MILs), or peripheral blood lymphocytes (PBLs), wherein the TILs, MILs, or PBLs are genetically modified to express a chemokine receptor, and the cancer is treated by administering the TIL population, the method comprising: (a) obtaining and / or receiving a first population of TILs from a tumor excised from a patient by processing a tumor sample obtained from the patient into a plurality of tumor fragments or tumor digests; (b) adding the first TIL population to the closed system; (c) performing a first expansion by culturing the first TIL population in a first cell culture medium comprising IL-2 and optionally an OKT-3 antibody, and antigen-presenting cells (APCs) to produce a second TIL population, wherein the first expansion is performed in a sealed container that provides a first gas-permeable surface area, and the first expansion is performed for about 3 to 14 days to obtain the second TIL population, and the transition from step (b) to step (c) occurs without opening the system. (d) genetically modifying a second population of TILs to express a chemokine receptor; (e) performing a second expansion of the second TIL population in a second cell culture medium comprising IL-2, OKT-3 antibody, and APC to produce a third TIL population, wherein the second expansion is performed for about 3 to 14 days to obtain the third TIL population, the third TIL population being a therapeutic TIL population, and wherein the second expansion is performed in a sealed container providing a second gas permeable surface area; (f) harvesting the therapeutic TIL population obtained from step (e); (g) transferring the harvested TIL population from step (f) into an infusion bag, wherein the transition from step (e) to (f) occurs without opening the system; (h) cryopreserving the infusion bag containing the harvested TIL population from step (f) using a cryopreservation process; (i) administering a therapeutically effective dose of the third population of TILs to the patient from the infusion bag of step (g).

[0062] In some embodiments, the invention includes methods of treating cancer by administering to a patient in need of cancer treatment a population of tumor-infiltrating lymphocytes (TILs), bone marrow-infiltrating lymphocytes (MILs), or peripheral blood lymphocytes (PBLs), wherein the TILs, MILs, or PBLs are genetically modified to express a chemokine receptor, and the cancer is treated by administering the TIL population, the method comprising: wherein the chemokine receptor is a protein selected from the group consisting of CXCR1, CXCR2, CXCR3, CXCR4, CXCR5, CXCR6, CXCR7 (ACKR3), CCR1, CCR2, CCR3, CCR4, CCR5, CCR6, CCR7, CCR8, CCR9, CCR10, CCR11, CX3CR1, and combinations thereof.

[0063] In some embodiments, the invention includes methods of treating cancer by administering to a patient in need of cancer treatment a population of tumor-infiltrating lymphocytes (TILs), bone marrow-infiltrating lymphocytes (MILs), or peripheral blood lymphocytes (PBLs), wherein the TILs, MILs, or PBLs are genetically modified to express a chemokine receptor, and the cancer is treated by administering the TIL population, the method comprising: the chemokine receptor is a protein selected from the group consisting of CXCR1, CXCR2, CXCR3, CXCR4, CXCR5, CXCR6, CXCR7 (ACKR3), CCR1, CCR2, CCR3, CCR4, CCR5, CCR6, CCR7, CCR8, CCR9, CCR10, CCR11, CX3CR1, and combinations thereof; and step (d) further comprises genetically modifying the TILs to express the chemokine receptor using a lentivirus or retrovirus.

[0064] In some embodiments, the present invention includes methods of treating cancer by administering to a patient in need of cancer treatment a population of tumor infiltrating lymphocytes (TILs), bone marrow infiltrating lymphocytes (MILs), or peripheral blood lymphocytes (PBLs), wherein the TILs, MILs, or PBLs are genetically modified to express a chemokine receptor, and the cancer is treated by administering the TIL population, the method comprising: 9, CCR10, CCR11, CX3CR1, and combinations thereof, and step (d) further comprises genetically modifying the TILs using a lentivirus or retrovirus to express a chemokine receptor, and the TILs, MILs, or PBLs are further genetically modified to stably or transiently reduce expression of a gene selected from the group consisting of PD-1, LAG-3, TIM-3, CTLA-4, TIGIT, CISH, TGFβR2, PKA, CBL-B, BAFF(BR3), SOCS1, ANKRD11, BCOR, and combinations thereof.

[0065] In some embodiments, the present invention includes methods of treating cancer by administering to a patient in need of cancer treatment a population of tumor-infiltrating lymphocytes (TILs), bone marrow-infiltrating lymphocytes (MILs), or peripheral blood lymphocytes (PBLs), wherein the TILs, MILs, or PBLs are genetically modified to express a chemokine receptor, and the cancer is a solid tumor cancer that is treated by administration of the TILs.

[0066] In any of the foregoing embodiments, the cancer is selected from the group consisting of sarcoma, pancreatic cancer, liver cancer, glioblastoma, gastrointestinal cancer, melanoma, ovarian cancer, endometrial cancer, thyroid cancer, colorectal cancer, cervical cancer, lung cancer, non-small cell lung cancer, small cell lung cancer, mesothelioma, bladder cancer, breast cancer, cancer caused by human papillomavirus, head and neck cancer, renal cancer, and renal cell carcinoma, and the patient is human.

[0067] In any of the foregoing embodiments, the cancer is further treated with a PD-1 or PD-L1 inhibitor in combination with TILs, wherein the PD-1 or PD-L1 inhibitor is selected from the group consisting of nivolumab, pembrolizumab, cemiplimab, tislelizumab, sintilimab, toripalimab, dostarimab, durvalumab, avelumab, atezolizumab, retifanlimab, and fragments, variants, and biosimilars thereof.

[0068] In any of the preceding embodiments, the cancer is non-small cell lung cancer and the patient is 1) a predefined tumor proportion score (TPS) of PD-L1 of <1%; 2) PD-L1 TPS score of 1% to 49%, or 3) have at least one of the predetermined absences of one or more driver mutations.

[0069] In any of the foregoing embodiments, the cancer is non-small cell lung cancer and the patient has a TPS of PD-L1 of <1%.

[0070] In any of the foregoing embodiments, the patient has a cancer that is not indicated for treatment with an EGFR inhibitor, a BRAF inhibitor, an ALK inhibitor, a c-Ros inhibitor, a RET inhibitor, an ERBB2 inhibitor, a BRCA inhibitor, a MAP2K1 inhibitor, a PIK3CA inhibitor, a CDKN2A inhibitor, a PTEN inhibitor, a UMD inhibitor, an NRAS inhibitor, a KRAS inhibitor, an NF1 inhibitor, a MET inhibitor, a TP53 inhibitor, a CREBBP inhibitor, a KMT2C inhibitor, a KMT2D mutation, an ARID1A mutation, an RB1 inhibitor, an ATM inhibitor, a SETD2 inhibitor, a FLT3 inhibitor, a PTPN11 inhibitor, an FGFR1 inhibitor, an EP300 inhibitor, a MYC inhibitor, an EZH2 inhibitor, a JAK2 inhibitor, an FBXW7 inhibitor, a CCND3 inhibitor, and a GNA11 inhibitor.

[0071] In any of the foregoing embodiments, the patient has the absence of one or more driver mutations, wherein the one or more driver mutations are an EGFR mutation, an EGFR insertion, an EGFR exon 20, a KRAS mutation, a BRAF mutation, a BRAF V600 mutation, ALK mutation, c-ROS mutation (ROS1 mutation), ROS1 fusion, RET mutation, RET fusion, ERBB2 mutation, ERBB2 amplification, BRCA mutation, MAP2K1 mutation, PIK3CA, CDKN2A, PTEN mutation, UMD mutation, NRAS mutation, KRAS mutation, NF1 mutation, MET mutation, MET splice and / or altered MET signaling, TP53 mutation, CREBBP mutation, KMT2C mutation, KMT2D mutation, ARID1A mutation, RB1 mutation, ATM mutation, SETD2 mutation, FLT3 mutation, PTPN11 mutation, FGFR1 mutation, EP300 mutation, MYC mutation, EZH2 mutation, JAK2 mutation, FBXW7 mutation, CCND3 mutation, and GNA11 mutation.

[0072] In any of the foregoing embodiments, the cancer is refractory or resistant to treatment with a chemotherapeutic agent or chemotherapy regimen.

[0073] In any of the foregoing embodiments, the cancer is refractory or resistant to treatment with a VEGF-A inhibitor, and the VEGF-A inhibitor is selected from the group consisting of bevacizumab, ranibizumab, icrucumab, and fragments, variants, and biosimilars thereof.

[0074] In any of the foregoing embodiments, the cancer is refractory or resistant to treatment with a PD-1 or PD-L1 inhibitor, and the PD-1 or PD-L1 inhibitor is selected from the group consisting of nivolumab, pembrolizumab, cemiplimab, tislelizumab, sintilimab, toripalimab, dostarimab, durvalumab, avelumab, atezolizumab, retifanlimab, and fragments, variants, and biosimilars thereof.

[0075] In any of the foregoing embodiments, the cancer is refractory or resistant to treatment with a CTLA-4 inhibitor, and the CTLA-4 inhibitor is selected from the group consisting of ipilimumab, tremelimumab, zalifrelimab, and fragments, variants, and biosimilars thereof.

[0076] In any of the foregoing embodiments, IL-2 is initially present in the first cell culture medium and the second cell culture medium at an initial concentration of 1000 IU / mL to 6000 IU / mL.

[0077] In any of the foregoing embodiments, the OKT-3 antibody is initially present in the second cell culture medium at an initial concentration of about 30 ng / mL.

[0078] In any of the preceding 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.

[0079] In any of the preceding 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.

[0080] In any of the foregoing embodiments, the method may further comprise treating the patient with a non-myeloablative lymphodepletion regimen prior to administering the third population of TILs to the patient.

[0081] In any of the foregoing embodiments, the method may further include treating the patient with a non-myeloablative lymphodepletion regimen prior to administering the third population of TILs to the patient, the non-myeloablative lymphodepletion regimen including cyclophosphamide at 60 mg / m 2 / day for 2 days, followed by fludarabine at 25 mg / m 2 / day for 5 days.

[0082] In any of the foregoing embodiments, the method may further include treating the patient with a non-myeloablative lymphodepletion regimen prior to administering the third population of TILs to the patient, the non-myeloablative lymphodepletion regimen including cyclophosphamide at 60 mg / m 2 / day and fludarabine at a dose of 25 mg / m 2 / day for 2 days, followed by fludarabine at 25 mg / m 2 / day for 3 days.

[0083] In any of the foregoing embodiments, the method may further include treating the patient with an IL-2 regimen starting the day after the third population of TILs is administered to the patient.

[0084] In any of the foregoing embodiments, the method may further comprise treating the patient with an IL-2 regimen beginning on the same day that the third population of TILs is administered to the patient.

[0085] In any of the foregoing embodiments, the method may further include treating the patient with an IL-2 regimen, wherein the IL-2 regimen is a high-dose IL-2 regimen comprising 600,000 or 720,000 IU / kg aldesleukin, or a fragment, variant, or biosimilar thereof, administered as a 15-minute bolus intravenous infusion every 8 hours until tolerated.

[0086] In any of the foregoing embodiments, the method may further include treating the patient with an IL-2 regimen, wherein the IL-2 regimen includes administration of bempegaldesleukin, or a fragment, variant, or biosimilar thereof.

[0087] In any of the foregoing embodiments, the method may further include treating the patient with an IL-2 regimen, wherein the IL-2 regimen includes administration of THOR-707, or a fragment, variant, or biosimilar thereof.

[0088] In any of the foregoing embodiments, the method may further include treating the patient with an IL-2 regimen, wherein the IL-2 regimen includes administration of nembareukin alfa, or a fragment, variant, or biosimilar thereof.

[0089] In any of the foregoing embodiments, the method may further include treating the patient with an IL-2 regimen, wherein the IL-2 regimen comprises administering an antibody, or a fragment, variant, or biosimilar thereof, comprising a heavy chain selected from the group consisting of SEQ ID NO:29 and SEQ ID NO:38, and a light chain selected from the group consisting of SEQ ID NO:37 and SEQ ID NO:39.

[0090] In any of the foregoing embodiments, a therapeutically effective population of TILs is administered, and is about 2×10 9 ~Approx. 15×10 10 Includes TILs.

[0091] In any of the foregoing embodiments, the first expansion is performed for a period of 11 days or less.

[0092] In any of the foregoing embodiments, the second expansion is performed over a period of no more than 11 days.

[0093] In some embodiments, the present invention provides compositions comprising tumor-infiltrating lymphocytes (TILs), bone marrow-infiltrating lymphocytes (MILs), or peripheral blood lymphocytes (PBLs) that have been genetically modified to express a chemokine receptor.

[0094] In some embodiments, the present invention relates to the composition of claim 94, wherein the chemokine receptor is a protein selected from the group consisting of CXCR1, CXCR2, CXCR3, CXCR4, CXCR5, CXCR6, CXCR7 (ACKR3), CCR1, CCR2, CCR3, CCR4, CCR5, CCR6, CCR7, CCR8, CCR9, CCR10, CCR11, XCR1, CX3CR1, and combinations thereof.

[0095] In some embodiments, the composition, TIL, MIL, or PBL of claim 94 or 95 is further genetically modified to stably or transiently reduce expression of a gene selected from the group consisting of PD-1, LAG-3, TIM-3, CTLA-4, TIGIT, CISH, TGFβR2, PKA, CBL-B, BAFF(BR3), and combinations thereof.

[0096] In some embodiments, a composition comprising a chemokine receptor is provided, wherein the composition further comprises tumor-infiltrating lymphocytes, bone marrow-infiltrating lymphocytes, or peripheral blood lymphocytes. [Brief explanation of the drawings]

[0097] [Figure 1] An exemplary Gen2 (Process 2A) chart providing an overview of steps A-F. [Figure 2A] 1 is a process flow diagram of an embodiment of Gen2 (Process 2A) for TIL fabrication. [Figure 2B] 1 is a process flow diagram of an embodiment of Gen2 (Process 2A) for TIL fabrication. [Figure 2C] 1 is a process flow diagram of an embodiment of Gen2 (Process 2A) for TIL fabrication. [Figure 3] 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 Gen2 (Process 2A) for TIL fabrication. [Figure 6] Detailed comparison of Process 1C and Gen2 (Process 2A) embodiments for TIL fabrication. [Figure 7] 1. Exemplary Gen3 TIL fabrication process. [Figure 8A]A comparison of embodiments of the 2A process (an approximately 22 day process) and the Gen3 process (an approximately 14-16 day process) for TIL fabrication is shown. [Figure 8B] An exemplary Process Gen3 chart providing an overview of steps A-F (approximately a 14-16 day process). [Figure 8C] A chart providing three exemplary Gen3 processes along with an overview of steps A-F (approximately 14- to 16-day processes) for each of the three process variations. [Figure 8D] An exemplary modified Gen2-like process (approximately a 22-day process) providing an overview of steps A-F. [Figure 9] 1 provides an experimental flow chart for the comparison between Gen2 (Process 2A) and Gen3 processes. [Figure 10] 1 shows a comparison of various Gen2 (Process 2A) and Gen3.1 process embodiments. [Figure 11] 1 is a table illustrating various features of embodiments of the Gen2, Gen2.1, and Gen3.0 processes. [Figure 12] Summary of media conditions for an embodiment of the Gen3 process, designated Gen3.1. [Figure 13] 1 is a table illustrating various features of embodiments of the Gen2, Gen2.1, and Gen3.0 processes. [Figure 14] 1 is a table comparing various features of embodiments of the Gen2 and Gen3.0 processes. [Figure 15] 1 is a table providing media use in various embodiments of the described expansion process. [Figure 16] Schematic of an exemplary embodiment of the Gen3 process (16-day process). [Figure 17] Schematic of an exemplary embodiment of a method for expanding T cells from hematopoietic malignancies using the Gen3 expansion platform. [Figure 18]Structures IA and IB are provided. The cylinders refer to individual polypeptide binding domains. Structures IA and IB comprise three linearly linked TNFRSF-binding domains 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 IgG1-Fc (comprising the CH3 and CH2 domains), which is then used to link two of the trivalent proteins together via disulfide bonds (small oblong ellipses), stabilizing the structure and providing an agonist that can bring together the six receptor and intracellular signaling domains of the signaling protein to form a signaling complex. The TNFRSF-binding domains shown as cylinders can be, for example, scFv domains comprising VH and VL chains connected by a linker that may contain hydrophilic residues and Gly and Ser sequences for flexibility, and Glu and Lys for solubility. [Figure 19] Schematic of an exemplary embodiment of the Gen3 process (16-day process). [Figure 20] 1 provides a process overview of an exemplary embodiment of the Gen3.1 process (16-day process). [Figure 21] Schematic of an exemplary embodiment of the Gen3.1 process (16-17 day process). [Figure 22] Schematic of an exemplary embodiment of the Gen3 process (16-day process). [Figure 23] 1 is a comparison table of an exemplary Gen2 process and an exemplary Gen3 process. [Figure 24] Schematic of an exemplary embodiment of the Gen3 process (16-17 day process) showing the preparation timeline. [Figure 25] Schematic of an exemplary embodiment of the Gen3 process (14-16 day process). [Figure 26A] Schematic of an exemplary embodiment of the Gen3 process (16-day process). [Figure 26B] Schematic of an exemplary embodiment of the Gen3 process (16-day process). [Figure 27]Schematic of an exemplary embodiment of the Gen3 process (16-day process). [Figure 28] Comparison of Gen2, Gen2.1, and Gen3 process (16-day process) embodiments. [Figure 29] Comparison of Gen2, Gen2.1, and Gen3 process (16-day process) embodiments. [Figure 30] Components of a Gen3 embodiment. [Figure 31] Flowchart comparison of Gen3 embodiments (Gen3.0, Gen3.1 control, Gen3.1 test). [Figure 32] Components of an exemplary embodiment of the Gen3 process (16-17 day process) are shown. [Figure 33] Approval criteria table [Figure 34] Diagram of an exemplary scFv CCR construct. [Figure 35] Exemplary PD-1 switch CCR design. [Figure 36] Exemplary PD-1 switch CCR designs with alternative CD28 signaling domains. [Figure 37] Exemplary CCR construct designs. [Figure 38] 1. Exemplary vector design for lentiviral expression of CCR in TILs of anti-TROP-2 (VL-linker-VH) CCR comprising IgG4 hinge and transmembrane domains, IL-2Rβ intracellular domain, and an embodiment of the invention. [Figure 39] 1. Exemplary vector design for lentiviral expression of CCR in TILs of anti-FAP (VL-linker-VH) CCR comprising CD8α hinge and transmembrane domains, IL-2Rβ intracellular domain, and an embodiment of the invention. [Figure 40] 14A and 14B show exemplary vector designs for lentiviral expression of CCRs in TILs of anti-PD-L1 (VL-linker-VH) CCRs comprising the CD8α hinge and transmembrane domains and the IL-2Rβ intracellular domain using the 38A1 antibody and embodiments of the invention. [Figure 41]1. Exemplary vector designs for retroviral expression of CXCR1 in TILs and embodiments of the present invention. [Figure 42] Exemplary vector designs for retroviral expression of CCR8 in TILs and embodiments of the present invention. [Figure 43] Flow cytometry analysis of cervical cancer tumor digests. EPCAM phycoerythrin (PE) / TROP-2 PE. [Figure 44] Flow cytometry analysis of EPCAM allophycocyanin (APC) / TROP-2 PE in cervical cancer tumor digests. [Figure 45] EPCAM / TROP-2 expression in head and neck squamous cell carcinoma digesta. [Figure 46] EPCAM / TROP-2 expression in non-small cell lung cancer tumor digesta. [Figure 47] Cell frequency distribution in TIL preparations from Gen2 REP preparations. Nine different TILs were thawed and stained for characterization on two different days, and PBMCs were used as controls. [Figure 48] Cell frequency distribution in TIL preparations from Gen2 REP preparations. Nine different TILs were thawed and stained for characterization on two different days, and PBMCs were used as controls. [Figure 49] Flow cytometry results showing chemokine receptors on CD8+ TILs. [Figure 50] Flow cytometry results showing chemokine receptors on CD4+ TILs. [Figure 51] Illustrative embodiments of chimeric costimulatory receptors of the invention. Six CCR constructs are shown that use the PD-1 or anti-PD-1 (38A1) scFv extracellular domain (ECD). TM refers to the transmembrane domain, and ICN refers to the intracellular domain. [Figure 52] 1. Map of the pQCXIX vector backbone, an embodiment of a different CCR and chemokine receptor vector of the invention. [Figure 53]Expression of CCR constructs "CCR4" and "CCR5" (as shown in Figure 51) in HEK reporter cells. [Figure 54] 1A-1C are exemplary embodiments of chimeric costimulatory receptors of the present invention. [Figure 55] Domain map of the amino acid sequences of two CCRs, including SP-(38A1 scFv)-(CD28 hinge and transmembrane)-(IL-2Rβ intracellular)-T2A-SP-(19H9 scFv)-(CD28 hinge and transmembrane)-(IL-2Rγ intracellular), using both the 38A1 and 19H9 PD-L1 domains (SEQ ID NO: 658) described herein. [Figure 56] Domain map of the amino acid sequences of two CCRs, including SP-(38A1 scFv)-(CD28 hinge and transmembrane)-(IL-18R1 intracellular)-T2A-SP-(19H9 scFv)-(CD28 hinge and transmembrane)-(IL-18RAP intracellular), using both the 38A1 and 19H9 PD-L1 domains (SEQ ID NO: 659) described herein. [Figure 57] Domain map of the amino acid sequences of two CCRs, including SP-(anti-TROP-2 scFv)-(CD8 hinge)-(IL-2Rβ transmembrane and intracellular)-T2A-SP-(anti-TROP-2 scFv)-(CD8 hinge)-(IL-2Rγ transmembrane and intracellular) (SEQ ID NO: 660). [Figure 58] Domain map of the amino acid sequences of two CCRs, including SP-(anti-TROP-2 scFv)-(CD8 hinge)-(IL-18R1-transmembrane and intracellular)-T2A-SP-(anti-TROP-2 scFv)-(CD8 hinge)-(IL-18RAP-transmembrane and intracellular) (SEQ ID NO: 661). [Figure 59] Domain map of the amino acid sequences, which are the amino acid sequences of two CCRs comprising SP-(cAR47A6.4 scFv)-(CD28 hinge-transmembrane)-(IL-2Rβ intracellular)-T2A-SP-(KM4097 scFv)-(CD28 hinge and transmembrane)-(IL-2Rγ intracellular) (SEQ ID NO: 662). [Figure 60]Domain map of the amino acid sequences, which are the amino acid sequences of two CCRs comprising SP-(cAR47A6.4 scFv)-(CD28 hinge-transmembrane)-(IL-18R1 intracellular)-T2A-SP-(KM4097scFv)-(CD28 hinge-transmembrane)-(IL-18RAP intracellular) (SEQ ID NO: 663). [Figure 61] 1. Map of the pLenti vector, an embodiment of a different CCR and chemokine receptor vector of the present invention. [Figure 62] (A) HEK-IL-18 reporter cells transfected with bi-epitope CCR8 (38A1 scFv-CD28™-IL-18R1-T2A-19H9 scFv-CD28™-IL-18RAP) and incubated with biotin-conjugated PD-L1 protein after streptavidin fluorescent staining, and (B) HEK-IL-18 reporter cells transfected with CCR12 (cAR47A6.4 scFv-CD28™-IL-18R1-T2A-KM4097 scFv-CD28™-IL-18RAP) and incubated with biotin-conjugated TROP2 protein after streptavidin fluorescent staining. Expression of both CCR8 and CCR12 is demonstrated by these results. [Figure 63] hPD-L1 Raji cells were incubated with the indicated (x-axis) concentrations of 38A1-IgG4-HA (hemagglutinin) antibody targeting PD-L1, in the presence of the competing hPD-L1-binding antibody 19H9. After 2 hours of incubation, cells were washed and stained with anti-HA-APC (allophycocyanin). The x-axis indicates the titrated concentration of 38A1-IgG4-HA antibody, and the y-axis indicates the % of PD-L1 positive staining cells of total hPD-L1 Raji cells. [Figure 64]hPD-L1 Raji cells were incubated with the indicated (x-axis) concentrations of 19H9-IgG4-Flag antibody targeting PD-L1, in the presence of the competing hPD-L1-binding antibody 38A1. After 2 hours of incubation, cells were washed and stained with anti-Flag-AF488. In Figure 64, the x-axis shows the concentration of the titrated 19H9-IgG4-Flag antibody, and the y-axis shows the % of cells that stained positive for PD-L1 of total hPD-L1 Raji cells. [Figure 65] Flow cytometry results with staining for the indicated antibodies on each axis. [Figure 66] The effect of AKT inhibitor (AKTi) treatment on TIL expansion and viability at two different concentrations of the pan-AKT inhibitor ipatasertib (0.3 μM and 1 μM) was added either before and during REP (blue bars) or only during the REP phase (purple bars). The fold expansion and viability of TILs at the end of the 22-day expansion process are shown. The frequencies of CD8+, CD4+, and CD4+(Foxp3+) cells after the expansion process with cryopreserved cells are also shown. [Figure 67] Experimental design to evaluate the blocking efficacy of two PD-L1 antibodies (38A1 and 19H9). [Figure 68] Results of an experiment to evaluate the blocking efficacy of two PD-L1 antibodies (38A1 and 19H9). [Figure 69] T cell subsets in control and AKT inhibitor (AKTi)-treated TILs. The frequencies of TCM (CD45RA-CCR7+), TEM (CD45RA-CCR7-), and TEMRA (CD45+CCR7-) cells in CD8+ and CD4+ TILs after treatment are shown. * indicates p<0.05. [Figure 70] Cytokine and chemokine receptor expression on control and AKT inhibitor (AKTi)-treated TILs. Cryopreserved control or AKTi-treated TILs were analyzed by flow cytometry. Representative histograms and frequencies of IL-7R+ and CXCR3+ CD8+ TILs. * indicates p<0.05, ** indicates p<0.01. [Figure 71]Distribution of CD69 and CD39 mono- and double-positive populations in control and AKT inhibitor (AKTi)-treated CD8+ TILs assessed by flow cytometry. * indicates p<0.05, ** indicates p<0.01, and *** indicates p<0.001. [Figure 72] Expression of inhibitory receptors and transcription factors in CD69-CD39- and CD69+CD39+CD8+ TILs; frequencies of PD1, LAG-3, TIM-3, and TIGIT, as well as Tbet, Eomes, BATF, and TOX in CD69-CD39- and CD69+CD39+ cells. * indicates p<0.05, ** indicates p<0.01, and **** indicates p<0.0001. Representative histograms and frequencies of CD62L expression on CD69-CD39- and CD69+CD39+CD8+ TILs are shown. [Figure 73] Marker expression in control and AKT inhibitor (AKTi)-treated TILs after overnight stimulation. Cryopreserved control and 1 μM AKTi-treated TILs, both pre- and post-REP, were stimulated overnight with anti-CD3 / CD28 beads at a bead-to-cell ratio of 1:5. Frequencies of CD69-CD39- and CD69+CD39+ cells, and transcription factor expression on CD8+ TILs. * indicates p<0.05, ** indicates p<0.01, and *** indicates p<0.001. [Figure 74] Cytokine expression in control and AKT inhibitor (AKTi)-treated CD8+ TILs, * indicates p<0.05. [Figure 75]Results of allogeneic cytotoxicity assay. The left panel shows cryopreserved control and TILs treated with 1 μM of the AKT inhibitor (ipatasertib) both before and during REP co-cultured with KILR® THP-1 cells (Eurofins DiscoverX, Fremont, CA, USA) at a 10:1 effector-to-target cell ratio for 24 hours to measure cytotoxicity in an allogeneic setting. The right panel shows results from control and AKT inhibitor (AKTi)-treated TILs stimulated every 5 days with anti-CD3 / CD28 beads at a 1:1 bead-to-cell ratio. Three days after the third stimulation, cells were washed, beads were removed, and cells were co-cultured with KILR THP-1 cells at a 10:1 effector-to-target cell ratio for 24 hours. [Figure 76] Expansion, viability, and T cell distribution data for control TILs (gray bars) and decitabine-treated TILs with increasing concentrations of decitabine are shown. Treatment was added either during the REP stage only (blue bars) or before and during the REP stage (green bars). Panel A shows the fold expansion and viability of TILs at the end of the 22-day expansion process. Panel B shows the frequency of CD8+, CD4+, and CD4+(Foxp3+) cells by flow cytometry after the expansion process on cryopreserved cells. *P<0.05, **P<0.01. [Figure 77] T cell subsets in control and decitabine-treated TILs. Frequencies of TCM (CD45RA-CCR7+), TEM (CD45RA-CCR7-), and TEMRA (CD45+CCR7-) cells are shown in panel A (CD8+) and panel B (CD4+) TILs after expansion. *P<0.05, **P<0.01. [Figure 78]Expression of surface markers on decitabine-treated TILs. Control cryopreserved TILs or decitabine-treated cryopreserved TILs were thawed and stained for flow cytometry analysis. Panel A shows the expression of CD25, ICOS, CD28, and IL-7R on CD8+ TILs. Panel B shows the expression of inhibitory receptors PD-1 and TIGIT on CD8+ TILs. Similar results were observed for CD4+ TILs. *P<0.05, **P<0.01, ***P<0.001, ****P<0.0001. [Figure 79] Expression of transcription factors in decitabine-treated TILs. Control or decitabine-treated cryopreserved TILs were thawed and stained for flow cytometry analysis. Expression of Eomes, KLF2, BATF, and T-bet in CD8+ TILs is shown. *P<0.05, **P<0.01. [Figure 80] Cytokine expression in control and decitabine-treated TILs after in vitro stimulation. Cryopreserved control and decitabine-treated TILs were stimulated overnight with anti-CD3 / CD28 beads at a bead-to-cell ratio of 1:5. Expression of IFN-γ (IFNγ), TNF-α (TNFα), and granzyme B (GZMB) on CD8+ TILs is shown. *P<0.05, **P<0.01. [Figure 81] Cytotoxicity of control and decitabine-treated TILs. In panel A, cryopreserved control TILs and TILs treated with 100 nM DAC in the REP were cocultured with KILR® THP-1 cells (Eurofins DiscoverX, Fremont, CA, USA) at an effector:target cell ratio of 10:1 for 24 hours to measure cytotoxicity in an allogeneic setting. In panel B, control and decitabine-treated TILs were stimulated every 5 days with TransActtm (Miltenyi Biotec, Germany). One day after the third stimulation, cells were washed and cocultured with KILR THP-1 cells at an effector:target cell ratio of 10:1 for 24 hours to measure cytotoxicity. *P<0.05. [Figure 82]Control TILs and decitabine-treated TILs were stimulated every 5 days with TransActtm (Miltenyi Biotec, Germany). One day after the third stimulation, cells were washed and stained for flow cytometry analysis. The expression of IL-7R, PD-1, and TIM3 in TILs after repeated stimulation is shown in panel A, and the expression levels of transcription factors in TILs after repeated stimulation are shown in panel B. *P<0.05, **P<0.01. [Figure 83] Vector design using pLenti backbone of CCR7.2 biepitope CCR targeting PD-L1, which is also a different CCR and chemokine receptor vector embodiment of the present invention. [Figure 84] Vector design using pLenti backbone of CCR8.2 biepitope CCR targeting PD-L1, which is also a different CCR and chemokine receptor vector embodiment of the present invention. [Figure 85] Vector design using the pLenti backbone of the CCR11.2 biepitope CCR targeting TROP-2, which is also a different CCR and chemokine receptor vector embodiment of the present invention. [Figure 86] Vector design using the pLenti backbone of the CCR12.2 biepitope CCR targeting TROP-2, which is also a different CCR and chemokine receptor vector embodiment of the present invention. [Figure 87]

[0023] Figure 1 shows the results of a HeKIL-18 reporter line experiment using PD-L1-targeting CCRs (CCR8 and CCR8.2) demonstrating enhanced IL-18 signaling through the use of alternative transmembrane (TM) domains. CCR8 is a biepitope CCR with the general structure 38A1scFv-CD28TM-IL-18R1-IC-T2A-19H9scFv-CD28TM-IL-18RAP-IC described herein. CCR8.2 is a biepitope CCR with the general structure 38A1scFv-IL-18R1TM-IL-18R1-IC-T2A-19H9scFV-IL-18RAPTM-IL-18RAP-IC. [Figure 88]

[0023] Figure 1 shows the results of a HeKIL-18 reporter line experiment using PD-L1-targeting CCRs (CCR12 and CCR12.2) demonstrating the enhancement of IL-18 signaling through the use of alternative transmembrane (TM) domains. CCR12 is a biepitope CCR with the general structure cAR47A6.4scFv-CD28TM-IL-18R1-IC-T2A-KM4097scFv-CD28TM-IL-18RAP-IC as described herein. CCR12.2 is a biepitope CCR with the general structure cAR47A6.4scFv-IL-18R1TM-IL-18R1-IC-T2A-KM4097scFv-IL-18RAPTM-IL-18RAP-IC. [Figure 89] Results of a PD-L1 targeted CCR experiment (shown in Figure 87) shown compared to different concentrations of IL-18 control. [Figure 90] Results of a TROP-2 targeted CCR experiment (shown in Figure 88) shown compared to different concentrations of IL-18 control. [Figure 91] Primarily, these are exemplary CCR designs for constructs with the 4-1BB (CD137) intracellular domain, which is also an embodiment of the present invention. EC refers to extracellular, TM refers to transmembrane, SP refers to signal peptide, and IC refers to intracellular. [Figure 92] Vector design using pLenti backbone for CCR13 CCR targeting FAS, also a different CCR and chemokine receptor vector embodiment of the invention. [Figure 93] Vector design using pLenti backbone of CCR14 CCR targeting PD-1, also a different CCR and chemokine receptor vector embodiment of the invention. [Figure 94] Vector design using the pLenti backbone of the CCR15 CCR targeting TGFβRII, which is also a different CCR and chemokine receptor vector embodiment of the invention. [Figure 95]Vector design using pLenti backbone of CCR14 CCR (with CD28 intracellular domain) targeting PD-1, also a different CCR and chemokine receptor vector embodiment of the invention. [Figure 96] The CCR constructs were inserted into the pLenti-IRES-GFP lentiviral plasmid. TILs were transduced with the lentivirus, allowed to rest for 2 days, and then expanded using the REP expansion process for 11 days. Surface expression of the CCR constructs shown here was detected by flow cytometry. [Figure 97] Expansion, survival and killing efficacy of TILs after REP expressing CCR. [Figure 98] 1 is an exemplary CCR design for a construct with an LTBR intracellular domain, which is also an embodiment of the present invention. EC refers to extracellular, TM refers to transmembrane, SP refers to signal peptide, and IC refers to intracellular. [Figure 99] Vector design using the pLenti backbone of the CCR17 CCR targeting FAS, which is also a different CCR and chemokine receptor vector embodiment of the invention. [Figure 100] Vector design using the pLenti backbone of the CCR18 CCR targeting PD-1, which is also a different CCR and chemokine receptor vector embodiment of the present invention. [Figure 101] Vector design using the pLenti backbone of the CCR19 CCR targeting TGFβRII, which is also a different CCR and chemokine receptor vector embodiment of the invention.

[0098] Brief Description of Sequence Listing SEQ ID NO: 1 is the amino acid sequence of the heavy chain of muromonab.

[0099] SEQ ID NO: 2 is the amino acid sequence of the light chain of muromonab.

[0100] SEQ ID NO: 3 is the amino acid sequence of recombinant human IL-2 protein.

[0101] SEQ ID NO: 4 is the amino acid sequence of aldesleukin.

[0102] SEQ ID NO: 5 is an IL-2 form.

[0103] SEQ ID NO: 6 is the amino acid sequence of nemvaleukin alpha.

[0104] SEQ ID NO: 7 is an IL-2 form.

[0105] SEQ ID NO: 8 is a mucin domain polypeptide.

[0106] SEQ ID NO: 9 is the amino acid sequence of recombinant human IL-4 protein.

[0107] SEQ ID NO: 10 is the amino acid sequence of recombinant human IL-7 protein.

[0108] SEQ ID NO: 11 is the amino acid sequence of recombinant human IL-15 protein.

[0109] SEQ ID NO: 12 is the amino acid sequence of recombinant human IL-21 protein.

[0110] SEQ ID NO: 13 is the IL-2 sequence.

[0111] SEQ ID NO: 14 is the IL-2 mutein sequence.

[0112] SEQ ID NO: 15 is the sequence of an IL-2 mutein.

[0113] SEQ ID NO: 16 is HCDR1_IL-2 of IgG.IL2R67A.H1.

[0114] SEQ ID NO: 17 is HCDR2 of IgG.IL2R67A.H1.

[0115] SEQ ID NO: 18 is the HCDR3 of IgG.IL2R67A.H1.

[0116] SEQ ID NO: 19 is HCDR1_IL-2 Kabat of IgG.IL2R67A.H1.

[0117] SEQ ID NO: 20 is the HCDR2 Kabat of IgG.IL2R67A.H1.

[0118] SEQ ID NO: 21 is the HCDR3 Kabat of IgG.IL2R67A.H1.

[0119] SEQ ID NO: 22 is the HCDR1_IL-2 clotia of IgG.IL2R67A.H1.

[0120] SEQ ID NO: 23 is the HCDR2 clone of IgG.IL2R67A.H1.

[0121] SEQ ID NO: 24 is the HCDR3 clone of IgG.IL2R67A.H1.

[0122] SEQ ID NO: 25 is HCDR1_IL-2 IMGT of IgG.IL2R67A.H1.

[0123] SEQ ID NO: 26 is HCDR2 IMGT of IgG.IL2R67A.H1.

[0124] SEQ ID NO: 27 is the HCDR3 IMGT of IgG.IL2R67A.H1.

[0125] SEQ ID NO: 28 is the V H It's a chain.

[0126] SEQ ID NO: 29 is the heavy chain of IgG.IL2R67A.H1.

[0127] SEQ ID NO: 30 is the LCDR1 Kabat of IgG.IL2R67A.H1.

[0128] SEQ ID NO: 31 is the LCDR2 Kabat of IgG.IL2R67A.H1.

[0129] SEQ ID NO: 32 is the LCDR3 Kabat of IgG.IL2R67A.H1.

[0130] SEQ ID NO: 33 is the LCDR1 chothia of IgG.IL2R67A.H1.

[0131] SEQ ID NO: 34 is the LCDR2 chothia of IgG.IL2R67A.H1.

[0132] SEQ ID NO: 35 is the LCDR3 chothia of IgG.IL2R67A.H1.

[0133] SEQ ID NO: 36 is V L It's a chain.

[0134] SEQ ID NO: 37 is the light chain.

[0135] SEQ ID NO: 38 is the light chain.

[0136] SEQ ID NO: 39 is the light chain.

[0137] SEQ ID NO: 40 is the amino acid sequence of human 4-1BB.

[0138] SEQ ID NO: 41 is the amino acid sequence of mouse 4-1BB.

[0139] SEQ ID NO: 42 is the heavy chain of the 4-1BB agonist monoclonal antibody utomilumab (PF-05082566).

[0140] SEQ ID NO: 43 is the light chain of the 4-1BB agonist monoclonal antibody utomilumab (PF-05082566).

[0141] SEQ ID NO: 44 represents the heavy chain variable region (V) of the 4-1BB agonist monoclonal antibody utomilumab (PF-05082566). H )

[0142] SEQ ID NO: 45 is the light chain variable region (V) of the 4-1BB agonist monoclonal antibody utomilumab (PF-05082566). L )

[0143] SEQ ID NO: 46 is the heavy chain CDR1 of the 4-1BB agonist monoclonal antibody utomilumab (PF-05082566).

[0144] SEQ ID NO: 47 is the heavy chain CDR2 of the 4-1BB agonist monoclonal antibody utomilumab (PF-05082566).

[0145] SEQ ID NO: 48 is the heavy chain CDR3 of the 4-1BB agonist monoclonal antibody utomilumab (PF-05082566).

[0146] SEQ ID NO: 49 is the light chain CDR1 of the 4-1BB agonist monoclonal antibody utomilumab (PF-05082566).

[0147] SEQ ID NO: 50 is the light chain CDR2 of the 4-1BB agonist monoclonal antibody utomilumab (PF-05082566).

[0148] SEQ ID NO: 51 is the light chain CDR3 of the 4-1BB agonist monoclonal antibody utomilumab (PF-05082566).

[0149] SEQ ID NO: 52 is the heavy chain of the 4-1BB agonist monoclonal antibody urelumab (BMS-663513).

[0150] SEQ ID NO: 53 is the light chain of the 4-1BB agonist monoclonal antibody urelumab (BMS-663513).

[0151] SEQ ID NO: 54 is the heavy chain variable region (VH) of the 4-1BB agonist monoclonal antibody urelumab (BMS-663513).

[0152] SEQ ID NO: 55 is the light chain variable region (VL) of the 4-1BB agonist monoclonal antibody urelumab (BMS-663513).

[0153] SEQ ID NO: 56 is the heavy chain CDR1 of the 4-1BB agonist monoclonal antibody urelumab (BMS-663513).

[0154] SEQ ID NO: 57 is the heavy chain CDR2 of the 4-1BB agonist monoclonal antibody urelumab (BMS-663513).

[0155] SEQ ID NO: 58 is the heavy chain CDR3 of the 4-1BB agonist monoclonal antibody urelumab (BMS-663513).

[0156] SEQ ID NO: 59 is the light chain CDR1 of the 4-1BB agonist monoclonal antibody urelumab (BMS-663513).

[0157] SEQ ID NO: 60 is the light chain CDR2 of the 4-1BB agonist monoclonal antibody urelumab (BMS-663513).

[0158] SEQ ID NO: 61 is the light chain CDR3 of the 4-1BB agonist monoclonal antibody urelumab (BMS-663513).

[0159] SEQ ID NO: 62 is the Fc domain of the TNFRSF agonist fusion protein.

[0160] SEQ ID NO: 63 is the linker of the TNFRSF agonist fusion protein or scFv.

[0161] SEQ ID NO: 64 is the linker of the TNFRSF agonist fusion protein or scFv.

[0162] SEQ ID NO: 65 is the linker of the TNFRSF agonist fusion protein or scFv.

[0163] SEQ ID NO: 66 is the linker of the TNFRSF agonist fusion protein or scFv.

[0164] SEQ ID NO: 67 is the linker of the TNFRSF agonist fusion protein or scFv.

[0165] SEQ ID NO: 68 is the linker of the TNFRSF agonist fusion protein or scFv.

[0166] SEQ ID NO: 69 is the linker of the TNFRSF agonist fusion protein or scFv.

[0167] SEQ ID NO: 70 is the linker of the TNFRSF agonist fusion protein or scFv.

[0168] SEQ ID NO: 71 is the linker of the TNFRSF agonist fusion protein or scFv.

[0169] SEQ ID NO: 72 is the linker of the TNFRSF agonist fusion protein or scFv.

[0170] SEQ ID NO: 73 is the Fc domain of the TNFRSF agonist fusion protein.

[0171] SEQ ID NO: 74 is the linker of the TNFRSF agonist fusion protein or scFv.

[0172] SEQ ID NO: 75 is the linker of the TNFRSF agonist fusion protein or scFv.

[0173] SEQ ID NO: 76 is the linker of the TNFRSF agonist fusion protein or scFv.

[0174] SEQ ID NO: 77 is the 4-1BB ligand (4-1BBL) amino acid sequence.

[0175] SEQ ID NO: 78 is the soluble portion of the 4-1BBL polypeptide.

[0176] SEQ ID NO: 79 is the heavy chain variable region (V) of 4-1BB agonist antibody 4B4-1-1 version 1 H )

[0177] SEQ ID NO: 80 represents the light chain variable region (V) of 4-1BB agonist antibody 4B4-1-1 version 1 L )

[0178] SEQ ID NO: 81 is the heavy chain variable region (V H )

[0179] SEQ ID NO: 82 is the light chain variable region (V) of 4-1BB agonist antibody 4B4-1-1 version 2 L )

[0180] SEQ ID NO: 83 identifies the heavy chain variable region (V H )

[0181] SEQ ID NO: 84 identifies the light chain variable region (V L )

[0182] SEQ ID NO: 85 is the amino acid sequence of human OX40.

[0183] SEQ ID NO: 86 is the amino acid sequence of mouse OX40.

[0184] SEQ ID NO: 87 is the heavy chain of the OX40 agonist monoclonal antibody tabolixizumab (MEDI-0562).

[0185] SEQ ID NO: 88 is the light chain of the OX40 agonist monoclonal antibody tabolixizumab (MEDI-0562).

[0186] SEQ ID NO: 89 is the heavy chain variable region (V) of the OX40 agonist monoclonal antibody tabolixizumab (MEDI-0562). H )

[0187] SEQ ID NO: 90 is the light chain variable region (V) of the OX40 agonist monoclonal antibody tabolixizumab (MEDI-0562). L )

[0188] SEQ ID NO: 91 is the heavy chain CDR1 of the OX40 agonist monoclonal antibody tabolixizumab (MEDI-0562).

[0189] SEQ ID NO: 92 is the heavy chain CDR2 of the OX40 agonist monoclonal antibody tabolixizumab (MEDI-0562).

[0190] SEQ ID NO: 93 is the heavy chain CDR3 of the OX40 agonist monoclonal antibody tabolixizumab (MEDI-0562).

[0191] SEQ ID NO: 94 is the light chain CDR1 of the OX40 agonist monoclonal antibody tabolixizumab (MEDI-0562).

[0192] SEQ ID NO: 95 is the light chain CDR2 of the OX40 agonist monoclonal antibody tabolixizumab (MEDI-0562).

[0193] SEQ ID NO: 96 is the light chain CDR3 of the OX40 agonist monoclonal antibody tabolixizumab (MEDI-0562).

[0194] SEQ ID NO: 97 is the heavy chain of the OX40 agonist monoclonal antibody 11D4.

[0195] SEQ ID NO: 98 is the light chain of the OX40 agonist monoclonal antibody 11D4.

[0196] SEQ ID NO: 99 is the heavy chain variable region (V) of the OX40 agonist monoclonal antibody 11D4. H )

[0197] SEQ ID NO: 100 is the light chain variable region (V) of the OX40 agonist monoclonal antibody 11D4. L )

[0198] SEQ ID NO: 101 is the heavy chain CDR1 of the OX40 agonist monoclonal antibody 11D4.

[0199] SEQ ID NO: 102 is the heavy chain CDR2 of the OX40 agonist monoclonal antibody 11D4.

[0200] SEQ ID NO: 103 is the heavy chain CDR3 of the OX40 agonist monoclonal antibody 11D4.

[0201] SEQ ID NO: 104 is the light chain CDR1 of the OX40 agonist monoclonal antibody 11D4.

[0202] SEQ ID NO: 105 is the light chain CDR2 of the OX40 agonist monoclonal antibody 11D4.

[0203] SEQ ID NO: 106 is the light chain CDR3 of the OX40 agonist monoclonal antibody 11D4.

[0204] SEQ ID NO: 107 is the heavy chain of the OX40 agonist monoclonal antibody 18D8.

[0205] SEQ ID NO: 108 is the light chain of the OX40 agonist monoclonal antibody 18D8.

[0206] SEQ ID NO: 109 is the heavy chain variable region (V) of the OX40 agonist monoclonal antibody 18D8. H )

[0207] SEQ ID NO: 110 is the light chain variable region (V) of the OX40 agonist monoclonal antibody 18D8. L )

[0208] SEQ ID NO: 111 is the heavy chain CDR1 of the OX40 agonist monoclonal antibody 18D8.

[0209] SEQ ID NO: 112 is the heavy chain CDR2 of the OX40 agonist monoclonal antibody 18D8.

[0210] SEQ ID NO: 113 is the heavy chain CDR3 of the OX40 agonist monoclonal antibody 18D8.

[0211] SEQ ID NO: 114 is the light chain CDR1 of the OX40 agonist monoclonal antibody 18D8.

[0212] SEQ ID NO: 115 is the light chain CDR2 of the OX40 agonist monoclonal antibody 18D8.

[0213] SEQ ID NO: 116 is the light chain CDR3 of the OX40 agonist monoclonal antibody 18D8.

[0214] SEQ ID NO: 117 is the heavy chain variable region (V) of the OX40 agonist monoclonal antibody Hu119-122. H )

[0215] SEQ ID NO: 118 is the light chain variable region (V) of the OX40 agonist monoclonal antibody Hu119-122. L )

[0216] SEQ ID NO: 119 is the heavy chain CDR1 of the OX40 agonist monoclonal antibody Hu119-122.

[0217] SEQ ID NO: 120 is the heavy chain CDR2 of the OX40 agonist monoclonal antibody Hu119-122.

[0218] SEQ ID NO: 121 is the heavy chain CDR3 of the OX40 agonist monoclonal antibody Hu119-122.

[0219] SEQ ID NO: 122 is the light chain CDR1 of the OX40 agonist monoclonal antibody Hu119-122.

[0220] SEQ ID NO: 123 is the light chain CDR2 of the OX40 agonist monoclonal antibody Hu119-122.

[0221] SEQ ID NO: 124 is the light chain CDR3 of the OX40 agonist monoclonal antibody Hu119-122.

[0222] SEQ ID NO: 125 is the heavy chain variable region (V) of the OX40 agonist monoclonal antibody Hu106-222. H )

[0223] SEQ ID NO: 126 is the light chain variable region (V) of the OX40 agonist monoclonal antibody Hu106-222. L )

[0224] SEQ ID NO: 127 is the heavy chain CDR1 of the OX40 agonist monoclonal antibody Hu106-222.

[0225] SEQ ID NO: 128 is the heavy chain CDR2 of the OX40 agonist monoclonal antibody Hu106-222.

[0226] SEQ ID NO: 129 is the heavy chain CDR3 of the OX40 agonist monoclonal antibody Hu106-222.

[0227] SEQ ID NO: 130 is the light chain CDR1 of the OX40 agonist monoclonal antibody Hu106-222.

[0228] SEQ ID NO: 131 is the light chain CDR2 of the OX40 agonist monoclonal antibody Hu106-222.

[0229] SEQ ID NO: 132 is the light chain CDR3 of the OX40 agonist monoclonal antibody Hu106-222.

[0230] SEQ ID NO: 133 is the OX40 ligand (OX40L) amino acid sequence.

[0231] SEQ ID NO: 134 is the soluble portion of the OX40L polypeptide.

[0232] SEQ ID NO: 135 is an alternative soluble portion of the OX40L polypeptide.

[0233] SEQ ID NO: 136 is the heavy chain variable region (V H )

[0234] SEQ ID NO: 137 is the light chain variable region (V) of OX40 agonist monoclonal antibody 008. L )

[0235] SEQ ID NO: 138 is the heavy chain variable region (V) of OX40 agonist monoclonal antibody 011. H )

[0236] SEQ ID NO: 139 is the light chain variable region (V) of the OX40 agonist monoclonal antibody 011. L )

[0237] SEQ ID NO: 140 is the heavy chain variable region (V) of the OX40 agonist monoclonal antibody 021. H )

[0238] SEQ ID NO: 141 is the light chain variable region (V) of the OX40 agonist monoclonal antibody 021. L )

[0239] SEQ ID NO: 142 is the heavy chain variable region (V) of the OX40 agonist monoclonal antibody 023. H )

[0240] SEQ ID NO: 143 is the light chain variable region (V) of the OX40 agonist monoclonal antibody 023. L )

[0241] SEQ ID NO: 144 is the heavy chain variable region (V H )

[0242] SEQ ID NO: 145 is the light chain variable region (V) of the OX40 agonist monoclonal antibody L )

[0243] SEQ ID NO: 146 is the heavy chain variable region (V H )

[0244] SEQ ID NO: 147 is the light chain variable region (V) of the OX40 agonist monoclonal antibody L )

[0245] SEQ ID NO: 148 is the heavy chain variable region (V H )

[0246] SEQ ID NO: 149 is the heavy chain variable region (V) of a humanized OX40 agonist monoclonal antibody. H )

[0247] SEQ ID NO: 150 is the light chain variable region (V) of a humanized OX40 agonist monoclonal antibody. L )

[0248] SEQ ID NO: 151 is the light chain variable region (V) of a humanized OX40 agonist monoclonal antibody. L )

[0249] SEQ ID NO: 152 is the heavy chain variable region (V H )

[0250] SEQ ID NO: 153 is the heavy chain variable region (V) of a humanized OX40 agonist monoclonal antibody. H )

[0251] SEQ ID NO: 154 is the light chain variable region (V) of a humanized OX40 agonist monoclonal antibody. L )

[0252] SEQ ID NO: 155 is the light chain variable region (V) of a humanized OX40 agonist monoclonal antibody. L)

[0253] SEQ ID NO: 156 is the heavy chain variable region (V H )

[0254] SEQ ID NO: 157 is the light chain variable region (V) of the OX40 agonist monoclonal antibody L )

[0255] SEQ ID NO: 158 is the heavy chain amino acid sequence of the PD-1 inhibitor nivolumab.

[0256] SEQ ID NO: 159 is the light chain amino acid sequence of the PD-1 inhibitor nivolumab.

[0257] SEQ ID NO: 160 represents the heavy chain variable region (V) of the PD-1 inhibitor nivolumab H ) amino acid sequence.

[0258] SEQ ID NO: 161 represents the light chain variable region (V) of the PD-1 inhibitor nivolumab L ) amino acid sequence.

[0259] SEQ ID NO: 162 is the heavy chain CDR1 amino acid sequence of the PD-1 inhibitor nivolumab.

[0260] SEQ ID NO: 163 is the heavy chain CDR2 amino acid sequence of the PD-1 inhibitor nivolumab.

[0261] SEQ ID NO: 164 is the heavy chain CDR3 amino acid sequence of the PD-1 inhibitor nivolumab.

[0262] SEQ ID NO: 165 is the light chain CDR1 amino acid sequence of the PD-1 inhibitor nivolumab.

[0263] SEQ ID NO: 166 is the light chain CDR2 amino acid sequence of the PD-1 inhibitor nivolumab.

[0264] SEQ ID NO: 167 is the light chain CDR3 amino acid sequence of the PD-1 inhibitor nivolumab.

[0265] SEQ ID NO: 168 is the heavy chain amino acid sequence of the PD-1 inhibitor pembrolizumab.

[0266] SEQ ID NO: 169 is the light chain amino acid sequence of the PD-1 inhibitor pembrolizumab.

[0267] SEQ ID NO: 170 represents the heavy chain variable region (V) of the PD-1 inhibitor pembrolizumab H ) amino acid sequence.

[0268] SEQ ID NO: 171 is the light chain variable region (V) of the PD-1 inhibitor pembrolizumab L ) amino acid sequence.

[0269] SEQ ID NO: 172 is the heavy chain CDR1 amino acid sequence of the PD-1 inhibitor pembrolizumab.

[0270] SEQ ID NO: 173 is the heavy chain CDR2 amino acid sequence of the PD-1 inhibitor pembrolizumab.

[0271] SEQ ID NO: 174 is the heavy chain CDR3 amino acid sequence of the PD-1 inhibitor pembrolizumab.

[0272] SEQ ID NO: 175 is the light chain CDR1 amino acid sequence of the PD-1 inhibitor pembrolizumab.

[0273] SEQ ID NO: 176 is the light chain CDR2 amino acid sequence of the PD-1 inhibitor pembrolizumab.

[0274] SEQ ID NO: 177 is the light chain CDR3 amino acid sequence of the PD-1 inhibitor pembrolizumab.

[0275] SEQ ID NO: 178 is the heavy chain amino acid sequence of the PD-L1 inhibitor durvalumab.

[0276] SEQ ID NO: 179 is the light chain amino acid sequence of the PD-L1 inhibitor durvalumab.

[0277] SEQ ID NO: 180 represents the heavy chain variable region (V) of the PD-L1 inhibitor durvalumab H ) amino acid sequence.

[0278] SEQ ID NO: 181 represents the light chain variable region (V) of the PD-L1 inhibitor durvalumab L ) amino acid sequence.

[0279] SEQ ID NO: 182 is the heavy chain CDR1 amino acid sequence of the PD-L1 inhibitor durvalumab.

[0280] SEQ ID NO: 183 is the heavy chain CDR2 amino acid sequence of the PD-L1 inhibitor durvalumab.

[0281] SEQ ID NO: 184 is the heavy chain CDR3 amino acid sequence of the PD-L1 inhibitor durvalumab.

[0282] SEQ ID NO: 185 is the light chain CDR1 amino acid sequence of the PD-L1 inhibitor durvalumab.

[0283] SEQ ID NO: 186 is the light chain CDR2 amino acid sequence of the PD-L1 inhibitor durvalumab.

[0284] SEQ ID NO: 187 is the light chain CDR3 amino acid sequence of the PD-L1 inhibitor durvalumab.

[0285] SEQ ID NO: 188 is the heavy chain amino acid sequence of the PD-L1 inhibitor avelumab.

[0286] SEQ ID NO: 189 is the light chain amino acid sequence of the PD-L1 inhibitor avelumab.

[0287] SEQ ID NO: 190 represents the heavy chain variable region (V) of the PD-L1 inhibitor avelumab H ) amino acid sequence.

[0288] SEQ ID NO: 191 represents the light chain variable region (V) of the PD-L1 inhibitor avelumab L ) amino acid sequence.

[0289] SEQ ID NO: 192 is the heavy chain CDR1 amino acid sequence of the PD-L1 inhibitor avelumab.

[0290] SEQ ID NO: 193 is the heavy chain CDR2 amino acid sequence of the PD-L1 inhibitor avelumab.

[0291] SEQ ID NO: 194 is the heavy chain CDR3 amino acid sequence of the PD-L1 inhibitor avelumab.

[0292] SEQ ID NO: 195 is the light chain CDR1 amino acid sequence of the PD-L1 inhibitor avelumab.

[0293] SEQ ID NO: 196 is the light chain CDR2 amino acid sequence of the PD-L1 inhibitor avelumab.

[0294] SEQ ID NO: 197 is the light chain CDR3 amino acid sequence of the PD-L1 inhibitor avelumab.

[0295] SEQ ID NO: 198 is the heavy chain amino acid sequence of the PD-L1 inhibitor atezolizumab.

[0296] SEQ ID NO: 199 is the light chain amino acid sequence of the PD-L1 inhibitor atezolizumab.

[0297] SEQ ID NO: 200 represents the heavy chain variable region (V) of the PD-L1 inhibitor atezolizumab H ) amino acid sequence.

[0298] SEQ ID NO: 201 is the light chain variable region (V) of the PD-L1 inhibitor atezolizumab L ) amino acid sequence.

[0299] SEQ ID NO: 202 is the heavy chain CDR1 amino acid sequence of the PD-L1 inhibitor atezolizumab.

[0300] SEQ ID NO: 203 is the heavy chain CDR2 amino acid sequence of the PD-L1 inhibitor atezolizumab.

[0301] SEQ ID NO: 204 is the heavy chain CDR3 amino acid sequence of the PD-L1 inhibitor atezolizumab.

[0302] SEQ ID NO: 205 is the light chain CDR1 amino acid sequence of the PD-L1 inhibitor atezolizumab.

[0303] SEQ ID NO: 206 is the light chain CDR2 amino acid sequence of the PD-L1 inhibitor atezolizumab.

[0304] SEQ ID NO: 207 is the light chain CDR3 amino acid sequence of the PD-L1 inhibitor atezolizumab.

[0305] SEQ ID NO: 208 is the heavy chain amino acid sequence of the CTLA-4 inhibitor ipilimumab.

[0306] SEQ ID NO: 209 is the light chain amino acid sequence of the CTLA-4 inhibitor ipilimumab.

[0307] SEQ ID NO: 210 represents the heavy chain variable region (V) of the CTLA-4 inhibitor ipilimumab H ) amino acid sequence.

[0308] SEQ ID NO: 211 represents the light chain variable region (V) of the CTLA-4 inhibitor ipilimumab L ) amino acid sequence.

[0309] SEQ ID NO: 212 is the heavy chain CDR1 amino acid sequence of the CTLA-4 inhibitor ipilimumab.

[0310] SEQ ID NO: 213 is the heavy chain CDR2 amino acid sequence of the CTLA-4 inhibitor ipilimumab.

[0311] SEQ ID NO: 214 is the heavy chain CDR3 amino acid sequence of the CTLA-4 inhibitor ipilimumab.

[0312] SEQ ID NO: 215 is the light chain CDR1 amino acid sequence of the CTLA-4 inhibitor ipilimumab.

[0313] SEQ ID NO: 216 is the light chain CDR2 amino acid sequence of the CTLA-4 inhibitor ipilimumab.

[0314] SEQ ID NO: 217 is the light chain CDR3 amino acid sequence of the CTLA-4 inhibitor ipilimumab.

[0315] SEQ ID NO: 218 is the heavy chain amino acid sequence of the CTLA-4 inhibitor tremelimumab.

[0316] SEQ ID NO: 219 is the light chain amino acid sequence of the CTLA-4 inhibitor tremelimumab.

[0317] SEQ ID NO: 220 represents the heavy chain variable region (V) of the CTLA-4 inhibitor tremelimumab H ) amino acid sequence.

[0318] SEQ ID NO: 221 represents the light chain variable region (V) of the CTLA-4 inhibitor tremelimumab L ) amino acid sequence.

[0319] SEQ ID NO: 222 is the heavy chain CDR1 amino acid sequence of the CTLA-4 inhibitor tremelimumab.

[0320] SEQ ID NO: 223 is the heavy chain CDR2 amino acid sequence of the CTLA-4 inhibitor tremelimumab.

[0321] SEQ ID NO: 224 is the heavy chain CDR3 amino acid sequence of the CTLA-4 inhibitor tremelimumab.

[0322] SEQ ID NO: 225 is the light chain CDR1 amino acid sequence of the CTLA-4 inhibitor tremelimumab.

[0323] SEQ ID NO: 226 is the light chain CDR2 amino acid sequence of the CTLA-4 inhibitor tremelimumab.

[0324] SEQ ID NO: 227 is the light chain CDR3 amino acid sequence of the CTLA-4 inhibitor tremelimumab.

[0325] SEQ ID NO: 228 is the heavy chain amino acid sequence of the CTLA-4 inhibitor zalifrelimab.

[0326] SEQ ID NO: 229 is the light chain amino acid sequence of the CTLA-4 inhibitor zalifrelimab.

[0327] SEQ ID NO: 230 is the heavy chain variable region (V) of the CTLA-4 inhibitor zalifrelimab H ) amino acid sequence.

[0328] SEQ ID NO: 231 is the light chain variable region (V) of the CTLA-4 inhibitor zalifrelimab L ) amino acid sequence.

[0329] SEQ ID NO: 232 is the heavy chain CDR1 amino acid sequence of the CTLA-4 inhibitor zalifrelimab.

[0330] SEQ ID NO: 233 is the heavy chain CDR2 amino acid sequence of the CTLA-4 inhibitor zalifrelimab.

[0331] SEQ ID NO: 234 is the heavy chain CDR3 amino acid sequence of the CTLA-4 inhibitor zalifrelimab.

[0332] SEQ ID NO: 235 is the light chain CDR1 amino acid sequence of the CTLA-4 inhibitor zalifrelimab.

[0333] SEQ ID NO: 236 is the light chain CDR2 amino acid sequence of the CTLA-4 inhibitor zalifrelimab.

[0334] SEQ ID NO: 237 is the light chain CDR3 amino acid sequence of the CTLA-4 inhibitor zalifrelimab.

[0335] SEQ ID NO: 238 is the amino acid sequence of the scFv linker.

[0336] SEQ ID NO: 239 is the amino acid sequence of the scFv linker.

[0337] SEQ ID NO: 240 is the amino acid sequence of the scFv linker.

[0338] SEQ ID NO: 241 is the amino acid sequence of the scFv linker.

[0339] SEQ ID NO: 242 is the amino acid sequence of the scFv linker.

[0340] SEQ ID NO: 243 is the amino acid sequence of the scFv linker.

[0341] SEQ ID NO: 244 is the amino acid sequence of the PD-1 extracellular domain.

[0342] SEQ ID NO: 245 is the amino acid sequence of the PD-1 extracellular and transmembrane domains.

[0343] SEQ ID NO: 246 is the amino acid sequence of the PD-1 extracellular domain and the CD28 transmembrane domain.

[0344] SEQ ID NO: 247 is the nucleotide sequence of the PD-1 extracellular and transmembrane domains.

[0345] SEQ ID NO: 248 is the nucleotide sequence of the PD-1 extracellular domain and the CD28 transmembrane domain.

[0346] SEQ ID NO: 249 is the amino acid sequence of scFv-Fc antibody 38A1.

[0347] SEQ ID NO: 250 is the amino acid sequence of the scFv antibody 38A1 variable heavy chain.

[0348] SEQ ID NO: 251 is the amino acid sequence of the variable light chain of scFv antibody 38A1.

[0349] SEQ ID NO: 252 is the amino acid sequence of the scFv antibody 38A1 variable heavy chain CDR1.

[0350] SEQ ID NO: 253 is the amino acid sequence of the scFv antibody 38A1 variable heavy chain CDR2.

[0351] SEQ ID NO: 254 is the amino acid sequence of the scFv antibody 38A1 variable heavy chain CDR3.

[0352] SEQ ID NO: 255 is the amino acid sequence of the scFv antibody 38A1 variable light chain CDR1.

[0353] SEQ ID NO: 256 is the amino acid sequence of the scFv antibody 38A1 variable light chain CDR2.

[0354] SEQ ID NO: 257 is the amino acid sequence of the scFv antibody 38A1 variable light chain CDR3.

[0355] SEQ ID NO: 258 is the amino acid sequence of scFv-Fc antibody 19H9.

[0356] SEQ ID NO: 259 is the amino acid sequence of the scFv antibody 19H9 variable heavy chain.

[0357] SEQ ID NO: 260 is the amino acid sequence of the scFv antibody 19H9 variable light chain.

[0358] SEQ ID NO: 261 is the amino acid sequence of the scFv antibody 19H9 variable heavy chain CDR1.

[0359] SEQ ID NO: 262 is the amino acid sequence of the scFv antibody 19H9 variable heavy chain CDR2.

[0360] SEQ ID NO: 263 is the amino acid sequence of the scFv antibody 19H9 variable heavy chain CDR3.

[0361] SEQ ID NO: 264 is the amino acid sequence of the scFv antibody 19H9 variable light chain CDR1.

[0362] SEQ ID NO: 265 is the amino acid sequence of the scFv antibody 19H9 variable light chain CDR2.

[0363] SEQ ID NO: 266 is the amino acid sequence of the scFv antibody 19H9 variable light chain CDR3.

[0364] SEQ ID NO: 267 is the anti-CEA variable heavy chain amino acid sequence.

[0365] SEQ ID NO: 268 is the anti-CEA variable light chain amino acid sequence.

[0366] SEQ ID NO: 269 is the anti-CEA heavy chain CDR1 amino acid sequence.

[0367] SEQ ID NO: 270 is the anti-CEA heavy chain CDR2 amino acid sequence.

[0368] SEQ ID NO: 271 is the anti-CEA heavy chain CDR3 amino acid sequence.

[0369] SEQ ID NO: 272 is the anti-CEA light chain CDR1 amino acid sequence.

[0370] SEQ ID NO: 273 is the anti-CEA light chain CDR2 amino acid sequence.

[0371] SEQ ID NO: 274 is the anti-CEA light chain CDR3 amino acid sequence.

[0372] SEQ ID NO: 275 is the anti-CD73 variable heavy chain amino acid sequence.

[0373] SEQ ID NO: 276 is the anti-CD73 variable light chain amino acid sequence.

[0374] SEQ ID NO: 277 is the anti-CD73 heavy chain CDR1 amino acid sequence.

[0375] SEQ ID NO: 278 is the anti-CD73 heavy chain CDR2 amino acid sequence.

[0376] SEQ ID NO: 279 is the anti-CD73 heavy chain CDR3 amino acid sequence.

[0377] SEQ ID NO: 280 is the anti-CD73 light chain CDR1 amino acid sequence.

[0378] SEQ ID NO: 281 is the anti-CD73 light chain CDR2 amino acid sequence.

[0379] SEQ ID NO: 282 is the anti-CD73 light chain CDR3 amino acid sequence.

[0380] SEQ ID NO: 283 is the anti-CD73 variable heavy chain amino acid sequence.

[0381] SEQ ID NO: 284 is the anti-CD73 variable light chain amino acid sequence.

[0382] SEQ ID NO: 285 is the anti-CD73 heavy chain CDR1 amino acid sequence.

[0383] SEQ ID NO: 286 is the anti-CD73 heavy chain CDR2 amino acid sequence.

[0384] SEQ ID NO: 287 is the anti-CD73 heavy chain CDR3 amino acid sequence.

[0385] SEQ ID NO: 288 is the anti-CD73 light chain CDR1 amino acid sequence.

[0386] SEQ ID NO: 289 is the anti-CD73 light chain CDR2 amino acid sequence.

[0387] SEQ ID NO: 290 is the anti-CD73 light chain CDR3 amino acid sequence.

[0388] SEQ ID NO: 291 is the anti-TROP-2 variable heavy chain amino acid sequence.

[0389] SEQ ID NO: 292 is the anti-TROP-2 variable heavy chain amino acid sequence.

[0390] SEQ ID NO: 293 is the anti-TROP-2 variable heavy chain amino acid sequence.

[0391] SEQ ID NO: 294 is the anti-TROP-2 variable heavy chain amino acid sequence.

[0392] SEQ ID NO: 295 is the anti-TROP-2 variable heavy chain amino acid sequence.

[0393] SEQ ID NO: 296 is the anti-TROP-2 variable heavy chain amino acid sequence.

[0394] SEQ ID NO: 297 is the anti-TROP-2 variable light chain amino acid sequence.

[0395] SEQ ID NO: 298 is the anti-TROP-2 variable light chain amino acid sequence.

[0396] SEQ ID NO: 299 is the anti-TROP-2 variable light chain amino acid sequence.

[0397] SEQ ID NO: 300 is the anti-TROP-2 variable light chain amino acid sequence.

[0398] SEQ ID NO: 301 is the anti-TROP-2 heavy chain CDR1 amino acid sequence.

[0399] SEQ ID NO: 302 is the anti-TROP-2 heavy chain CDR2 amino acid sequence.

[0400] SEQ ID NO: 303 is the anti-TROP-2 heavy chain CDR3 amino acid sequence.

[0401] SEQ ID NO: 304 is the anti-TROP-2 light chain CDR1 amino acid sequence.

[0402] SEQ ID NO: 305 is the anti-TROP-2 light chain CDR2 amino acid sequence.

[0403] SEQ ID NO: 306 is the anti-TROP-2 light chain CDR3 amino acid sequence.

[0404] SEQ ID NO: 307 is the amino acid sequence of the anti-TROP-2 antibody m7E6 variable heavy chain.

[0405] SEQ ID NO: 308 is the amino acid sequence of the anti-TROP-2 antibody m7E6 variable light chain.

[0406] SEQ ID NO: 309 is the amino acid sequence of the anti-TROP-2 antibody h7E6 variable heavy chain.

[0407] SEQ ID NO: 310 is the amino acid sequence of the variable light chain of anti-TROP-2 antibody m7E6 and h7E6_SVG.

[0408] SEQ ID NO: 311 is the amino acid sequence of the variable heavy chain of anti-TROP-2 antibody h7E6_SVGL and h7E6_SVG.

[0409] SEQ ID NO: 312 is the amino acid sequence of the anti-TROP-2 antibody h7E6_SVGL variable light chain.

[0410] SEQ ID NO: 313 is the amino acid sequence of the anti-TROP-2 antibody m6G11 variable heavy chain.

[0411] SEQ ID NO: 314 is the amino acid sequence of the variable light chain of anti-TROP-2 antibody m6G11.

[0412] SEQ ID NO: 315 is the amino acid sequence of the anti-TROP-2 antibody h6G11 variable heavy chain.

[0413] SEQ ID NO: 316 is the amino acid sequence of the anti-TROP-2 antibody h6G11 variable light chain.

[0414] SEQ ID NO: 317 is the amino acid sequence of the anti-TROP-2 antibody h6G11-FKG_SF variable heavy chain.

[0415] SEQ ID NO: 318 is the amino acid sequence of the anti-TROP-2 antibody h6G11-FKG_SF variable light chain.

[0416] SEQ ID NO: 319 is the amino acid sequence of anti-TROP-2 antibody variable heavy chain CDR1.

[0417] SEQ ID NO: 320 is the amino acid sequence of the anti-TROP-2 antibody variable heavy chain CDR2.

[0418] SEQ ID NO: 321 is the amino acid sequence of the anti-TROP-2 antibody variable heavy chain CDR3.

[0419] SEQ ID NO: 322 is the amino acid sequence of anti-TROP-2 antibody variable light chain CDR1.

[0420] SEQ ID NO: 323 is the amino acid sequence of the anti-TROP-2 antibody variable light chain CDR2.

[0421] SEQ ID NO: 324 is the amino acid sequence of the anti-TROP-2 antibody variable light chain CDR3.

[0422] SEQ ID NO: 325 is the nucleotide sequence encoding the anti-TROP-2 antibody m7E6 variable heavy chain.

[0423] SEQ ID NO: 326 is the nucleotide sequence encoding the variable light chain of anti-TROP-2 antibody m7E6.

[0424] SEQ ID NO: 327 is the nucleotide sequence encoding the anti-TROP-2 antibody h7E6 variable heavy chain.

[0425] SEQ ID NO: 328 is the nucleotide sequence encoding the variable light chain of anti-TROP-2 antibody m7E6.

[0426] SEQ ID NO: 329 is the nucleotide sequence encoding the anti-TROP-2 antibody h7E6_SVGL variable heavy chain.

[0427] SEQ ID NO: 330 is the nucleotide sequence encoding the anti-TROP-2 antibody h7E6_SVGL variable light chain.

[0428] SEQ ID NO: 331 is the nucleotide sequence encoding the variable heavy chain of the anti-TROP-2 antibody m6G11.

[0429] SEQ ID NO: 332 is the nucleotide sequence encoding the variable light chain of the anti-TROP-2 antibody m6G11.

[0430] SEQ ID NO: 333 is the nucleotide sequence encoding the variable heavy chain of the anti-TROP-2 antibody h6G11.

[0431] SEQ ID NO: 334 is the nucleotide sequence encoding the variable light chain of the anti-TROP-2 antibody h6G11.

[0432] SEQ ID NO: 335 is the nucleotide sequence encoding the anti-TROP-2 antibody h6G11-FKG_SF variable heavy chain.

[0433] SEQ ID NO: 336 is the nucleotide sequence encoding the anti-TROP-2 antibody h6G11-FKG_SF variable light chain.

[0434] SEQ ID NO: 337 is the anti-TROP-2 sacituzumab variable heavy chain amino acid sequence.

[0435] SEQ ID NO: 338 is the anti-TROP-2 sacituzumab variable light chain amino acid sequence.

[0436] SEQ ID NO: 339 is the anti-TROP-2 sacituzumab heavy chain CDR1 amino acid sequence.

[0437] SEQ ID NO: 340 is the anti-TROP-2 sacituzumab heavy chain CDR2 amino acid sequence.

[0438] SEQ ID NO: 341 is the anti-TROP-2 sacituzumab heavy chain CDR3 amino acid sequence.

[0439] SEQ ID NO: 342 is the anti-TROP-2 sacituzumab light chain CDR1 amino acid sequence.

[0440] SEQ ID NO: 343 is the anti-TROP-2 sacituzumab light chain CDR2 amino acid sequence.

[0441] SEQ ID NO: 344 is the anti-TROP-2 sacituzumab light chain CDR3 amino acid sequence.

[0442] SEQ ID NO: 345 is the amino acid sequence of the anti-EPCAM scFv antibody 3-17I scFv.

[0443] SEQ ID NO: 346 is the amino acid sequence of the anti-EPCAM scFv antibody 7-F17 scFv.

[0444] SEQ ID NO: 347 is the amino acid sequence of the anti-EPCAM scFv antibody 12-C15 scFv.

[0445] SEQ ID NO: 348 is the amino acid sequence of the anti-EPCAM scFv antibody 16-G5 scFv.

[0446] SEQ ID NO: 349 is the amino acid sequence of the anti-EPCAM scFv antibody 17-C20 scFv.

[0447] SEQ ID NO: 350 is the amino acid sequence of the anti-EPCAM scFv antibody 24-G6 scFv.

[0448] SEQ ID NO: 351 is the anti-EPCAM antibody variable heavy chain amino acid sequence.

[0449] SEQ ID NO: 352 is the anti-EPCAM antibody variable light chain amino acid sequence.

[0450] SEQ ID NO: 353 is the anti-EPCAM antibody variable light chain amino acid sequence.

[0451] SEQ ID NO: 354 is the anti-EPCAM antibody variable light chain amino acid sequence.

[0452] SEQ ID NO: 355 is the anti-EPCAM antibody variable light chain amino acid sequence.

[0453] SEQ ID NO: 356 is the anti-EPCAM antibody variable light chain amino acid sequence.

[0454] SEQ ID NO: 357 is the anti-EPCAM antibody variable light chain amino acid sequence.

[0455] SEQ ID NO: 358 is the anti-EPCAM antibody heavy chain CDR1 amino acid sequence.

[0456] SEQ ID NO: 359 is the anti-EPCAM antibody heavy chain CDR2 amino acid sequence.

[0457] SEQ ID NO: 360 is the anti-EPCAM antibody heavy chain CDR3 amino acid sequence.

[0458] SEQ ID NO: 361 is the anti-EPCAM antibody light chain CDR1 amino acid sequence.

[0459] SEQ ID NO: 362 is the anti-EPCAM antibody light chain CDR2 amino acid sequence.

[0460] SEQ ID NO: 363 is the anti-EPCAM antibody light chain CDR3 amino acid sequence.

[0461] SEQ ID NO: 364 is the nucleotide sequence encoding the anti-EPCAM scFv antibody 3-17I scFv.

[0462] SEQ ID NO: 365 is the nucleotide sequence encoding the anti-EPCAM scFv antibody 7-F17 scFv.

[0463] SEQ ID NO: 366 is the nucleotide sequence encoding the anti-EPCAM scFv antibody 12-C15 scFv.

[0464] SEQ ID NO: 366 is the nucleotide sequence encoding the anti-EPCAM scFv antibody 16-G5 scFv.

[0465] SEQ ID NO: 367 is the nucleotide sequence encoding the anti-EPCAM scFv antibody 17-C20 scFv.

[0466] SEQ ID NO: 368 is the nucleotide sequence encoding the anti-EPCAM scFv antibody 24-G6 scFv.

[0467] SEQ ID NO: 369 is the nucleotide sequence encoding the anti-EPCAM scFv variable heavy chain.

[0468] SEQ ID NO: 370 is the nucleotide sequence encoding the anti-EPCAM scFv variable light chain.

[0469] SEQ ID NO: 371 is the nucleotide sequence encoding the anti-EPCAM scFv variable light chain.

[0470] SEQ ID NO: 372 is the nucleotide sequence encoding the anti-EPCAM scFv variable light chain.

[0471] SEQ ID NO: 373 is the nucleotide sequence encoding the anti-EPCAM scFv variable light chain.

[0472] SEQ ID NO: 374 is the nucleotide sequence encoding the anti-EPCAM scFv variable light chain.

[0473] SEQ ID NO: 375 is the nucleotide sequence encoding the anti-EPCAM scFv variable light chain.

[0474] SEQ ID NO: 376 is the nucleotide sequence encoding the anti-EPCAM scFv variable light chain.

[0475] SEQ ID NO: 377 is the anti-EPCAM antibody variable heavy chain amino acid sequence.

[0476] SEQ ID NO: 378 is the anti-EPCAM antibody variable light chain amino acid sequence.

[0477] SEQ ID NO: 379 is the anti-EPCAM antibody heavy chain CDR1 amino acid sequence.

[0478] SEQ ID NO: 380 is the anti-EPCAM antibody heavy chain CDR2 amino acid sequence.

[0479] SEQ ID NO: 381 is the anti-EPCAM antibody heavy chain CDR3 amino acid sequence.

[0480] SEQ ID NO: 382 is the anti-EPCAM antibody light chain CDR1 amino acid sequence.

[0481] SEQ ID NO: 383 is the anti-EPCAM antibody light chain CDR2 amino acid sequence.

[0482] SEQ ID NO: 384 is the anti-EPCAM antibody light chain CDR3 amino acid sequence.

[0483] SEQ ID NO: 385 is the amino acid sequence of the anti-tissue factor antibody TF260 variable heavy chain.

[0484] SEQ ID NO: 386 is the amino acid sequence of the anti-tissue factor antibody TF260 variable light chain.

[0485] SEQ ID NO: 387 is the amino acid sequence of the anti-tissue factor antibody TF196 variable heavy chain.

[0486] SEQ ID NO: 388 is the amino acid sequence of the anti-tissue factor antibody TF196 variable light chain.

[0487] SEQ ID NO: 389 is the amino acid sequence of the anti-tissue factor antibody TF278 variable heavy chain.

[0488] SEQ ID NO: 390 is the amino acid sequence of the anti-tissue factor antibody TF278 variable light chain.

[0489] SEQ ID NO: 391 is the amino acid sequence of the anti-tissue factor antibody TF277 variable heavy chain.

[0490] SEQ ID NO: 392 is the amino acid sequence of the anti-tissue factor antibody TF277 variable light chain.

[0491] SEQ ID NO: 393 is the amino acid sequence of the anti-tissue factor antibody TF392 variable heavy chain.

[0492] SEQ ID NO: 394 is the amino acid sequence of the anti-tissue factor antibody TF392 variable light chain.

[0493] SEQ ID NO: 395 is the amino acid sequence of the anti-tissue factor antibody TF9 variable heavy chain.

[0494] SEQ ID NO: 396 is the amino acid sequence of the anti-tissue factor antibody TF9 variable light chain.

[0495] SEQ ID NO: 397 is the anti-tissue factor antibody TF260 heavy chain CDR1 amino acid sequence.

[0496] SEQ ID NO: 398 is the anti-tissue factor antibody TF260 heavy chain CDR2 amino acid sequence.

[0497] SEQ ID NO: 399 is the anti-tissue factor antibody TF260 heavy chain CDR3 amino acid sequence.

[0498] SEQ ID NO: 400 is the anti-tissue factor antibody TF260 light chain CDR1 amino acid sequence.

[0499] SEQ ID NO: 401 is the anti-tissue factor antibody TF260 light chain CDR2 amino acid sequence.

[0500] SEQ ID NO: 402 is the anti-tissue factor antibody TF260 light chain CDR3 amino acid sequence.

[0501] SEQ ID NO: 403 is the anti-tissue factor antibody TF196 heavy chain CDR1 amino acid sequence.

[0502] SEQ ID NO: 404 is the anti-tissue factor antibody TF196 heavy chain CDR2 amino acid sequence.

[0503] SEQ ID NO: 405 is the anti-tissue factor antibody TF196 heavy chain CDR3 amino acid sequence.

[0504] SEQ ID NO: 406 is the anti-tissue factor antibody TF196 light chain CDR1 amino acid sequence.

[0505] SEQ ID NO: 407 is the anti-tissue factor antibody TF196 light chain CDR2 amino acid sequence.

[0506] SEQ ID NO: 408 is the anti-tissue factor antibody TF196 light chain CDR3 amino acid sequence.

[0507] SEQ ID NO: 409 is the anti-tissue factor antibody TF9 heavy chain CDR1 amino acid sequence.

[0508] SEQ ID NO: 410 is the anti-tissue factor antibody TF9 heavy chain CDR2 amino acid sequence.

[0509] SEQ ID NO: 411 is the anti-tissue factor antibody TF9 heavy chain CDR3 amino acid sequence.

[0510] SEQ ID NO: 412 is the anti-tissue factor antibody TF9 light chain CDR1 amino acid sequence.

[0511] SEQ ID NO: 413 is the anti-tissue factor antibody TF9 light chain CDR2 amino acid sequence.

[0512] SEQ ID NO: 414 is the anti-tissue factor antibody TF9 light chain CDR3 amino acid sequence.

[0513] SEQ ID NO: 415 is the amino acid sequence of the anti-tissue factor antibody variable heavy chain.

[0514] SEQ ID NO: 416 is the amino acid sequence of the anti-tissue factor antibody variable light chain.

[0515] SEQ ID NO: 417 is the amino acid sequence of the anti-tissue factor antibody variable heavy chain.

[0516] SEQ ID NO: 418 is the amino acid sequence of the anti-tissue factor antibody variable light chain.

[0517] SEQ ID NO: 419 is the amino acid sequence of the anti-tissue factor antibody variable heavy chain.

[0518] SEQ ID NO: 420 is the amino acid sequence of the anti-tissue factor antibody variable light chain.

[0519] SEQ ID NO: 421 is the amino acid sequence of the anti-tissue factor antibody variable heavy chain.

[0520] SEQ ID NO: 422 is the amino acid sequence of the anti-tissue factor antibody variable light chain.

[0521] SEQ ID NO: 423 is the amino acid sequence of the anti-tissue factor antibody variable heavy chain.

[0522] SEQ ID NO: 424 is the amino acid sequence of the anti-tissue factor antibody variable light chain.

[0523] SEQ ID NO: 425 is the nucleotide sequence encoding the anti-tissue factor antibody TF260 variable heavy chain.

[0524] SEQ ID NO: 426 is the nucleotide sequence encoding the anti-tissue factor antibody TF260 variable light chain.

[0525] SEQ ID NO: 427 is the nucleotide sequence encoding the anti-tissue factor antibody TF196 variable heavy chain.

[0526] SEQ ID NO: 428 is the nucleotide sequence encoding the variable light chain of the anti-tissue factor antibody TF196.

[0527] SEQ ID NO: 429 is the nucleotide sequence encoding the anti-tissue factor antibody TF278 variable heavy chain.

[0528] SEQ ID NO: 430 is the nucleotide sequence encoding the anti-tissue factor antibody TF278 variable light chain.

[0529] SEQ ID NO: 431 is the nucleotide sequence encoding the anti-tissue factor antibody TF277 variable heavy chain.

[0530] SEQ ID NO: 432 is the nucleotide sequence encoding the anti-tissue factor antibody TF277 variable light chain.

[0531] SEQ ID NO: 433 is the nucleotide sequence encoding the anti-tissue factor antibody TF392 variable heavy chain.

[0532] SEQ ID NO: 434 is the nucleotide sequence encoding the anti-tissue factor antibody TF392 variable light chain.

[0533] SEQ ID NO: 435 is the nucleotide sequence encoding the anti-tissue factor antibody TF9 variable heavy chain.

[0534] SEQ ID NO: 436 is the nucleotide sequence encoding the anti-tissue factor antibody TF9 variable light chain.

[0535] SEQ ID NO: 437 is the amino acid sequence of an anti-LFA-1 or anti-CD11a antibody variable heavy chain.

[0536] SEQ ID NO: 438 is the amino acid sequence of the anti-LFA-1 or anti-CD11a antibody variable light chain.

[0537] SEQ ID NO: 439 is the amino acid sequence of an anti-LFA-1 or anti-CD11a antibody variable heavy chain.

[0538] SEQ ID NO: 440 is the amino acid sequence of the anti-LFA-1 or anti-CD11a antibody variable light chain.

[0539] SEQ ID NO: 441 is the anti-LFA-1 or anti-CD11a antibody heavy chain CDR1 amino acid sequence.

[0540] SEQ ID NO: 442 is the anti-LFA-1 or anti-CD11a antibody heavy chain CDR2 amino acid sequence.

[0541] SEQ ID NO: 443 is the anti-LFA-1 or anti-CD11a antibody heavy chain CDR3 amino acid sequence.

[0542] SEQ ID NO: 444 is the anti-LFA-1 or anti-CD11a antibody light chain CDR1 amino acid sequence.

[0543] SEQ ID NO: 445 is the anti-LFA-1 or anti-CD11a antibody light chain CDR2 amino acid sequence.

[0544] SEQ ID NO: 446 is the anti-LFA-1 or anti-CD11a antibody light chain CDR3 amino acid sequence.

[0545] SEQ ID NO: 447 is the amino acid sequence of the sibrotuzumab-based anti-FAP scFv.

[0546] SEQ ID NO: 448 is the amino acid sequence of the anti-FAP antibody sibrotuzumab variable heavy chain.

[0547] SEQ ID NO: 449 is the amino acid sequence of the variable light chain of the anti-FAP antibody sibrotuzumab.

[0548] SEQ ID NO: 450 is the amino acid sequence of the anti-FAP antibody FAP5 variable heavy chain.

[0549] SEQ ID NO: 451 is the amino acid sequence of the anti-FAP antibody FAP5 variable light chain.

[0550] SEQ ID NO: 452 is the nucleotide sequence encoding the variable heavy chain of the anti-FAP antibody sibrotuzumab.

[0551] SEQ ID NO: 453 is the nucleotide sequence encoding the variable light chain of the anti-FAP antibody sibrotuzumab.

[0552] SEQ ID NO: 454 is the amino acid sequence of the anti-VISTA antibody 1B8 variable heavy chain.

[0553] SEQ ID NO: 455 is the amino acid sequence of the anti-VISTA antibody 1B8 variable light chain.

[0554] SEQ ID NO: 456 is the amino acid sequence of the anti-VISTA antibody 1B8 heavy chain CDR1.

[0555] SEQ ID NO: 457 is the amino acid sequence of the anti-VISTA antibody 1B8 heavy chain CDR2.

[0556] SEQ ID NO: 458 is the amino acid sequence of the anti-VISTA antibody 1B8 heavy chain CDR3.

[0557] SEQ ID NO: 459 is the amino acid sequence of the anti-VISTA antibody 1B8 light chain CDR1.

[0558] SEQ ID NO: 460 is the amino acid sequence of the anti-VISTA antibody 1B8 light chain CDR2.

[0559] SEQ ID NO: 461 is the amino acid sequence of the anti-VISTA antibody 1B8 light chain CDR3.

[0560] SEQ ID NO: 462 is the amino acid sequence of the variable heavy chain of anti-VISTA antibody 2C12.

[0561] SEQ ID NO: 463 is the amino acid sequence of the variable light chain of anti-VISTA antibody 2C12.

[0562] SEQ ID NO: 464 is the amino acid sequence of the heavy chain CDR1 of anti-VISTA antibody 2C12.

[0563] SEQ ID NO: 465 is the amino acid sequence of the heavy chain CDR2 of anti-VISTA antibody 2C12.

[0564] SEQ ID NO: 466 is the amino acid sequence of the heavy chain CDR3 of anti-VISTA antibody 2C12.

[0565] SEQ ID NO: 467 is the amino acid sequence of the light chain CDR1 of anti-VISTA antibody 2C12.

[0566] SEQ ID NO: 468 is the amino acid sequence of the light chain CDR2 of anti-VISTA antibody 2C12.

[0567] SEQ ID NO: 469 is the amino acid sequence of the light chain CDR3 of anti-VISTA antibody 2C12.

[0568] SEQ ID NO: 470 is the amino acid sequence of the anti-VISTA antibody 1A12 variable heavy chain.

[0569] SEQ ID NO: 471 is the amino acid sequence of the variable light chain of anti-VISTA antibody 1A12.

[0570] SEQ ID NO: 472 is the amino acid sequence of the heavy chain CDR1 of anti-VISTA antibody 1A12.

[0571] SEQ ID NO: 473 is the amino acid sequence of the heavy chain CDR2 of anti-VISTA antibody 1A12.

[0572] SEQ ID NO: 474 is the amino acid sequence of the anti-VISTA antibody 1A12 heavy chain CDR3.

[0573] SEQ ID NO: 475 is the amino acid sequence of the anti-VISTA antibody 1A12 light chain CDR1.

[0574] SEQ ID NO: 476 is the amino acid sequence of the anti-VISTA antibody 1A12 light chain CDR2.

[0575] SEQ ID NO: 477 is the amino acid sequence of the anti-VISTA antibody 1A12 light chain CDR3.

[0576] SEQ ID NO: 478 is the amino acid sequence of the anti-VISTA antibody 3C5 variable heavy chain.

[0577] SEQ ID NO: 479 is the amino acid sequence of the anti-VISTA antibody 3C5 variable light chain.

[0578] SEQ ID NO: 480 is the amino acid sequence of the anti-VISTA antibody 3C5 heavy chain CDR1.

[0579] SEQ ID NO: 481 is the amino acid sequence of the anti-VISTA antibody 3C5 heavy chain CDR2.

[0580] SEQ ID NO: 482 is the amino acid sequence of the anti-VISTA antibody 3C5 heavy chain CDR3.

[0581] SEQ ID NO: 483 is the amino acid sequence of the anti-VISTA antibody 3C5 light chain CDR1.

[0582] SEQ ID NO: 484 is the amino acid sequence of the anti-VISTA antibody 3C5 light chain CDR2.

[0583] SEQ ID NO: 485 is the amino acid sequence of the anti-VISTA antibody 3C5 light chain CDR3.

[0584] SEQ ID NO: 486 is the amino acid sequence of the anti-LRRC15 antibody huM25 variable heavy chain.

[0585] SEQ ID NO: 487 is the amino acid sequence of the anti-LRRC15 antibody huM25 variable light chain.

[0586] SEQ ID NO: 488 is the amino acid sequence of the anti-LRRC15 antibody huM25 heavy chain CDR1.

[0587] SEQ ID NO: 489 is the amino acid sequence of the anti-LRRC15 antibody huM25 heavy chain CDR2.

[0588] SEQ ID NO: 490 is the amino acid sequence of the anti-LRRC15 antibody huM25 heavy chain CDR3.

[0589] SEQ ID NO: 491 is the amino acid sequence of the anti-LRRC15 antibody huM25 light chain CDR1.

[0590] SEQ ID NO: 492 is the amino acid sequence of the anti-LRRC15 antibody huM25 light chain CDR2.

[0591] SEQ ID NO: 493 is the amino acid sequence of the anti-LRRC15 antibody huM25 light chain CDR3.

[0592] SEQ ID NO: 494 is the amino acid sequence of the anti-LRRC15 antibody huAD208.4.1 variable heavy chain.

[0593] SEQ ID NO: 495 is the amino acid sequence of the anti-LRRC15 antibody huAD208.4.1 variable light chain.

[0594] SEQ ID NO: 496 is the amino acid sequence of the anti-LRRC15 antibody huAD208.4.1 heavy chain CDR1.

[0595] SEQ ID NO: 497 is the amino acid sequence of the anti-LRRC15 antibody huAD208.4.1 heavy chain CDR2.

[0596] SEQ ID NO: 498 is the amino acid sequence of the anti-LRRC15 antibody huAD208.4.1 heavy chain CDR3.

[0597] SEQ ID NO: 499 is the amino acid sequence of the anti-LRRC15 antibody huAD208.4.1 light chain CDR1.

[0598] SEQ ID NO:500 is the amino acid sequence of the anti-LRRC15 antibody huAD208.4.1 light chain CDR2.

[0599] SEQ ID NO: 501 is the amino acid sequence of the anti-LRRC15 antibody huAD208.4.1 light chain CDR3.

[0600] SEQ ID NO: 502 is the amino acid sequence of the anti-LRRC15 antibody huAD208.12.1 variable heavy chain.

[0601] SEQ ID NO: 503 is the amino acid sequence of the anti-LRRC15 antibody huAD208.12.1 variable light chain.

[0602] SEQ ID NO: 504 is the amino acid sequence of the anti-LRRC15 antibody huAD208.12.1 heavy chain CDR1.

[0603] SEQ ID NO: 505 is the amino acid sequence of the anti-LRRC15 antibody huAD208.12.1 heavy chain CDR2.

[0604] SEQ ID NO: 506 is the amino acid sequence of the anti-LRRC15 antibody huAD208.12.1 heavy chain CDR3.

[0605] SEQ ID NO: 507 is the amino acid sequence of the anti-LRRC15 antibody huAD208.12.1 light chain CDR1.

[0606] SEQ ID NO: 508 is the amino acid sequence of the anti-LRRC15 antibody huAD208.12.1 light chain CDR2.

[0607] SEQ ID NO: 509 is the amino acid sequence of the anti-LRRC15 antibody huAD208.12.1 light chain CDR3.

[0608] SEQ ID NO:510 is the amino acid sequence of the anti-LRRC15 antibody huAD208.14.1 variable heavy chain.

[0609] SEQ ID NO:511 is the amino acid sequence of the variable light chain of the anti-LRRC15 antibody huAD208.14.1.

[0610] SEQ ID NO: 512 is the amino acid sequence of the anti-LRRC15 antibody huAD208.14.1 heavy chain CDR1.

[0611] SEQ ID NO: 513 is the amino acid sequence of the anti-LRRC15 antibody huAD208.14.1 heavy chain CDR2.

[0612] SEQ ID NO:514 is the amino acid sequence of the anti-LRRC15 antibody huAD208.14.1 heavy chain CDR3.

[0613] SEQ ID NO: 515 is the amino acid sequence of the anti-LRRC15 antibody huAD208.14.1 light chain CDR1.

[0614] SEQ ID NO: 516 is the amino acid sequence of the anti-LRRC15 antibody huAD208.14.1 light chain CDR2.

[0615] SEQ ID NO: 517 is the amino acid sequence of the anti-LRRC15 antibody huAD208.14.1 light chain CDR3.

[0616] SEQ ID NO: 518 is the amino acid sequence of the anti-LRRC15 antibody hu139.10 variable heavy chain.

[0617] SEQ ID NO: 519 is the amino acid sequence of the anti-LRRC15 antibody hu139.10 variable light chain.

[0618] SEQ ID NO: 520 is the amino acid sequence of the anti-LRRC15 antibody hu139.10 heavy chain CDR1.

[0619] SEQ ID NO: 521 is the amino acid sequence of the anti-LRRC15 antibody hu139.10 heavy chain CDR2.

[0620] SEQ ID NO: 522 is the amino acid sequence of the anti-LRRC15 antibody hu139.10 heavy chain CDR3.

[0621] SEQ ID NO: 523 is the amino acid sequence of the anti-LRRC15 antibody hu139.10 light chain CDR1.

[0622] SEQ ID NO: 524 is the amino acid sequence of the anti-LRRC15 antibody hu139.10 light chain CDR2.

[0623] SEQ ID NO: 525 is the amino acid sequence of the anti-LRRC15 antibody hu139.10 light chain CDR3.

[0624] SEQ ID NO: 526 is the amino acid sequence of the anti-LRRC15 antibody muAD210.40.9 variable heavy chain.

[0625] SEQ ID NO: 527 is the amino acid sequence of the anti-LRRC15 antibody muAD210.40.9 variable light chain.

[0626] SEQ ID NO: 528 is the amino acid sequence of the anti-LRRC15 antibody muAD210.40.9 heavy chain CDR1.

[0627] SEQ ID NO: 529 is the amino acid sequence of the anti-LRRC15 antibody muAD210.40.9 heavy chain CDR2.

[0628] SEQ ID NO: 530 is the amino acid sequence of the anti-LRRC15 antibody muAD210.40.9 heavy chain CDR3.

[0629] SEQ ID NO: 531 is the amino acid sequence of the anti-LRRC15 antibody muAD210.40.9 light chain CDR1.

[0630] SEQ ID NO: 532 is the amino acid sequence of the anti-LRRC15 antibody muAD210.40.9 light chain CDR2.

[0631] SEQ ID NO: 533 is the amino acid sequence of the anti-LRRC15 antibody muAD210.40.9 light chain CDR3.

[0632] SEQ ID NO: 534 is the amino acid sequence of the anti-LRRC15 antibody muAD209.9.1 variable heavy chain.

[0633] SEQ ID NO: 535 is the amino acid sequence of the anti-LRRC15 antibody muAD209.9.1 variable light chain.

[0634] SEQ ID NO: 536 is the amino acid sequence of the anti-LRRC15 antibody muAD209.9.1 heavy chain CDR1.

[0635] SEQ ID NO: 537 is the amino acid sequence of the anti-LRRC15 antibody muAD209.9.1 heavy chain CDR2.

[0636] SEQ ID NO: 538 is the amino acid sequence of the anti-LRRC15 antibody muAD209.9.1 heavy chain CDR3.

[0637] SEQ ID NO: 539 is the amino acid sequence of the anti-LRRC15 antibody muAD209.9.1 light chain CDR1.

[0638] SEQ ID NO: 540 is the amino acid sequence of the anti-LRRC15 antibody muAD209.9.1 light chain CDR2.

[0639] SEQ ID NO: 541 is the amino acid sequence of the anti-LRRC15 antibody muAD209.9.1 light chain CDR3.

[0640] SEQ ID NO: 542 is the amino acid sequence of the anti-B7-H3 antibody hBRCA84D variable heavy chain.

[0641] SEQ ID NO: 543 is the amino acid sequence of the anti-B7-H3 antibody hBRCA84D variable light chain.

[0642] SEQ ID NO: 544 is the amino acid sequence of the anti-B7-H3 antibody hBRCA84D heavy chain CDR1.

[0643] SEQ ID NO: 545 is the amino acid sequence of the anti-B7-H3 antibody hBRCA84D heavy chain CDR2.

[0644] SEQ ID NO: 546 is the amino acid sequence of the anti-B7-H3 antibody hBRCA84D heavy chain CDR3.

[0645] SEQ ID NO: 547 is the amino acid sequence of the anti-B7-H3 antibody hBRCA84D light chain CDR1.

[0646] SEQ ID NO: 548 is the amino acid sequence of the anti-B7-H3 antibody hBRCA84D light chain CDR2.

[0647] SEQ ID NO: 549 is the amino acid sequence of the anti-B7-H3 antibody hBRCA84D light chain CDR3.

[0648] SEQ ID NO: 550 is the amino acid sequence of the anti-B7-H3 antibody hBRCA84D variable heavy chain.

[0649] SEQ ID NO: 551 is the amino acid sequence of the anti-B7-H3 antibody hBRCA84D variable light chain.

[0650] SEQ ID NO: 552 is the amino acid sequence of the PD-1 transmembrane domain.

[0651] SEQ ID NO: 553 is the amino acid sequence of the CD28 transmembrane domain.

[0652] SEQ ID NO: 554 is the amino acid sequence of the CD27 transmembrane domain.

[0653] SEQ ID NO: 555 is the amino acid sequence of the CD8α transmembrane domain.

[0654] SEQ ID NO: 556 is the amino acid sequence of the CD8 alpha hinge domain.

[0655] SEQ ID NO:557 is the amino acid sequence of the IL-2Rβ hinge domain.

[0656] SEQ ID NO: 558 is the amino acid sequence of the IgG1 transmembrane and hinge domain.

[0657] SEQ ID NO: 559 is the amino acid sequence of the IgG1 hinge domain.

[0658] SEQ ID NO: 560 is the amino acid sequence of the IgG4 hinge domain.

[0659] SEQ ID NO: 561 is the amino acid sequence of the IgD hinge domain.

[0660] SEQ ID NO:562 is the nucleotide sequence encoding the PD-1 transmembrane domain.

[0661] SEQ ID NO:563 is the nucleotide sequence encoding the CD28 transmembrane domain.

[0662] SEQ ID NO:564 is the nucleotide sequence encoding the CD27 transmembrane domain.

[0663] SEQ ID NO: 565 is the nucleotide sequence encoding the CD8α transmembrane domain.

[0664] SEQ ID NO: 566 is the nucleotide sequence encoding the CD8 alpha hinge domain.

[0665] SEQ ID NO:567 is the nucleotide sequence encoding the IL-2Rβ hinge domain.

[0666] SEQ ID NO: 568 is the nucleotide sequence encoding the IgG1 transmembrane and hinge domains.

[0667] SEQ ID NO:569 is the nucleotide sequence encoding the IgG1 hinge domain.

[0668] SEQ ID NO: 570 is the nucleotide sequence encoding the IgG4 hinge domain.

[0669] SEQ ID NO:571 is the nucleotide sequence encoding the IgD hinge domain.

[0670] SEQ ID NO: 572 is the amino acid sequence of the CD28 intracellular domain.

[0671] SEQ ID NO: 573 is the amino acid sequence of the CD134 (OX40) intracellular domain.

[0672] SEQ ID NO: 574 is the amino acid sequence of the CD278 (ICOS) intracellular domain.

[0673] SEQ ID NO: 575 is the amino acid sequence of the CD137 (4-1BB) intracellular domain.

[0674] SEQ ID NO: 576 is the amino acid sequence of the CD27 intracellular domain.

[0675] SEQ ID NO: 577 is the amino acid sequence of the CD3 zeta intracellular domain.

[0676] SEQ ID NO:578 is the amino acid sequence of the IL-2Rβ intracellular domain.

[0677] SEQ ID NO:579 is the amino acid sequence of the IL-2Rγ intracellular domain.

[0678] SEQ ID NO:580 is the amino acid sequence of the IL-18R1 intracellular domain.

[0679] SEQ ID NO: 581 is the amino acid sequence of the IL-7Rα intracellular domain.

[0680] SEQ ID NO: 582 is the amino acid sequence of the IL-12R1 intracellular domain.

[0681] SEQ ID NO: 583 is the amino acid sequence of the IL-12R2 intracellular domain.

[0682] SEQ ID NO: 584 is the amino acid sequence of the IL-15Rα intracellular domain.

[0683] SEQ ID NO: 585 is the amino acid sequence of the IL-21R intracellular domain.

[0684] SEQ ID NO: 586 is the amino acid sequence of the LTBR intracellular domain.

[0685] SEQ ID NO: 587 is the amino acid sequence of the linker.

[0686] SEQ ID NO:588 is the nucleotide sequence encoding the CD28 intracellular domain.

[0687] SEQ ID NO: 589 is the nucleotide sequence encoding the CD134 (OX40) intracellular domain.

[0688] SEQ ID NO: 590 is the nucleotide sequence encoding the CD278 (ICOS) intracellular domain.

[0689] SEQ ID NO: 591 is the nucleotide sequence encoding the CD137 (4-1BB) intracellular domain.

[0690] SEQ ID NO:592 is the nucleotide sequence encoding the CD27 intracellular domain.

[0691] SEQ ID NO:593 is the nucleotide sequence encoding the CD3 zeta intracellular domain.

[0692] SEQ ID NO:594 is the nucleotide sequence encoding the IL-2Rβ intracellular domain.

[0693] SEQ ID NO:595 is the nucleotide sequence encoding the IL-2Rγ intracellular domain.

[0694] SEQ ID NO:596 is the nucleotide sequence encoding the IL-18R1 intracellular domain.

[0695] SEQ ID NO:597 is the nucleotide sequence encoding the IL-7Rα intracellular domain.

[0696] SEQ ID NO:598 is the nucleotide sequence encoding the IL-12R1 intracellular domain.

[0697] SEQ ID NO:599 is the nucleotide sequence encoding the IL-12R2 intracellular domain.

[0698] SEQ ID NO:600 is the nucleotide sequence encoding the IL-15Rα intracellular domain.

[0699] SEQ ID NO:601 is the nucleotide sequence encoding the IL-21R intracellular domain.

[0700] SEQ ID NO: 602 is the nucleotide sequence encoding the LTBR intracellular domain.

[0701] SEQ ID NO:603 is the nucleotide sequence encoding the linker.

[0702] SEQ ID NO:604 is the nucleotide sequence of the EF-1 promoter.

[0703] SEQ ID NO:605 is the nucleotide sequence for the CMV promoter.

[0704] SEQ ID NO: 606 is the nucleotide sequence of the MSCV promoter.

[0705] SEQ ID NO: 607 is the nucleotide sequence of the NFAT promoter.

[0706] SEQ ID NO: 608 is the amino acid sequence of the T2A self-cleaving peptide (from thosea asigna virus 2A).

[0707] SEQ ID NO: 609 is the amino acid sequence of the P2A self-cleaving peptide (from porcine teschovirus-1 2A).

[0708] SEQ ID NO: 610 is the amino acid sequence of the E2A self-cleaving peptide (derived from Equine rhinitis A virus).

[0709] SEQ ID NO: 611 is the amino acid sequence of the F2A self-cleaving peptide (derived from foot-and-mouth disease virus).

[0710] SEQ ID NO: 612 is the amino acid sequence of the linker.

[0711] SEQ ID NO:613 is the nucleotide sequence encoding the T2A self-cleaving peptide.

[0712] SEQ ID NO:614 is the nucleotide sequence encoding the P2A self-cleaving peptide.

[0713] SEQ ID NO:615 is the nucleotide sequence encoding the E2A self-cleaving peptide.

[0714] SEQ ID NO: 616 is the nucleotide sequence encoding the F2A self-cleaving peptide.

[0715] SEQ ID NO:617 is the nucleotide sequence encoding the IRES domain.

[0716] SEQ ID NO: 618 is (anti-TROP2-V L )-(linker)-(anti-TROP2-V H 1 is the nucleotide sequence of a vector encoding a CCR comprising IgG4 hinge and transmembrane)-(IL-2Rβ).

[0717] SEQ ID NO: 619 is (anti-FAP-V L )-(linker)-(anti-FAP-V H 1 is the nucleotide sequence of a vector encoding a CCR comprising CD8α hinge and transmembrane)-(IL-18R1).

[0718] SEQ ID NO: 620 is a recombinant antibody prepared using the 38A1 anti-PD-L1 domain described herein (anti-PD-L1-V L )-(linker)-(anti-PD-L1-V H 1 is the nucleotide sequence of a vector encoding a CCR comprising CD8α hinge and transmembrane)-CD27.

[0719] SEQ ID NO: 621 is the nucleotide sequence of a vector encoding two CCRs, comprising SP-(38A1 scFv)-(CD28 hinge and transmembrane)-(IL-2Rβ intracellular)-T2A-SP-(19H9 scFv)-(CD28 hinge and transmembrane)-(IL-2Rγ intracellular), using both the 38A1 and 19H9 PD-L1 domains described herein, where SP refers to the signal peptide.

[0720] SEQ ID NO: 622 is the nucleotide sequence of a vector encoding two CCRs, comprising SP-(38A1 scFv)-(CD28 hinge and transmembrane)-(IL-18R1 intracellular)-T2A-SP-(19H9 scFv)-(CD28 hinge and transmembrane)-(IL-18RAP intracellular), using both the 38A1 and 19H9 PD-L1 domains described herein, where SP refers to the signal peptide.

[0721] SEQ ID NO: 623 is the nucleotide sequence of a vector encoding two CCRs comprising SP-(anti-TROP-2 scFv)-(CD8 hinge)-(IL-2Rβ transmembrane and intracellular)-T2A-SP-(anti-TROP-2 scFv)-(CD8 hinge)-(IL-2Rγ transmembrane and intracellular), where SP refers to the signal peptide.

[0722] SEQ ID NO: 624 is the nucleotide sequence of a vector encoding two CCRs comprising SP-(anti-TROP-2 scFv)-(CD8 hinge)-(IL-18R1-transmembrane and intracellular)-T2A-SP-(anti-TROP-2 scFv)-(CD8 hinge)-(IL-18RAP-transmembrane and intracellular), where SP refers to the signal peptide.

[0723] SEQ ID NO: 625 is the nucleotide sequence of a vector encoding two CCRs comprising SP-(cAR47A6.4 scFv)-(CD28 hinge-transmembrane)-(IL-2Rβ intracellular)-T2A-SP-(KM4097 scFv)-(CD28 hinge and transmembrane)-(IL-2Rγ intracellular), where SP refers to the signal peptide.

[0724] SEQ ID NO: 626 is the nucleotide sequence for a vector encoding two CCRs comprising SP-(cAR47A6.4 scFv)-(CD28 hinge-transmembrane)-(IL-18R1 intracellular)-T2A-SP-(KM4097scFv)-(CD28 hinge-transmembrane)-(IL-18RAP intracellular). SP refers to the signal peptide.

[0725] SEQ ID NO: 627 is the amino acid sequence of the CXCR1 domain.

[0726] SEQ ID NO: 628 is the amino acid sequence of the CXCR2 variant 1 and 2 domains.

[0727] SEQ ID NO: 629 is the amino acid sequence of the CXCR3 variant 1 domain.

[0728] SEQ ID NO: 630 is the amino acid sequence of the CXCR3 variant 2 domain.

[0729] SEQ ID NO: 631 is the amino acid sequence of the CXCR4 variant 1 domain.

[0730] SEQ ID NO: 632 is the amino acid sequence of the CXCR4 variant 2 domain.

[0731] SEQ ID NO: 633 is the amino acid sequence of the CXCR4 variant 3 domain.

[0732] SEQ ID NO: 634 is the amino acid sequence of the CXCR4 variant 4 domain.

[0733] SEQ ID NO: 635 is the amino acid sequence of the CXCR4 variant 5 domain.

[0734] SEQ ID NO: 636 is the amino acid sequence of the CXCR5 variant 1 domain.

[0735] SEQ ID NO: 637 is the amino acid sequence of the CXCR5 variant 2 domain.

[0736] SEQ ID NO: 638 is the amino acid sequence of the CCR2 variant A domain.

[0737] SEQ ID NO: 639 is the amino acid sequence of the CCR2 variant B domain.

[0738] SEQ ID NO: 640 is the amino acid sequence of the CCR4 domain.

[0739] SEQ ID NO: 641 is the amino acid sequence of the CCR6 variant 1 and 2 domains.

[0740] SEQ ID NO: 642 is the amino acid sequence of the CCR7 variant 1 domain.

[0741] SEQ ID NO: 643 is the amino acid sequence of the CCR7 variant 2 domain.

[0742] SEQ ID NO: 644 is the amino acid sequence of the CCR7 variant 3, 4, and 5 domains.

[0743] SEQ ID NO: 645 is the amino acid sequence of the CCR8 domain.

[0744] SEQ ID NO: 646 is the nucleotide sequence encoding the CXCR1 domain.

[0745] SEQ ID NO: 647 is the nucleotide sequence encoding the CXCR2 variant 1 domain.

[0746] SEQ ID NO: 648 is the nucleotide sequence encoding the CXCR2 variant 2 domain.

[0747] SEQ ID NO: 649 is the nucleotide sequence encoding the CXCR3 variant 1 domain.

[0748] SEQ ID NO: 650 is the nucleotide sequence encoding the CXCR3 variant 2 domain.

[0749] SEQ ID NO: 651 is the nucleotide sequence encoding the CXCR4 variant 1 domain.

[0750] SEQ ID NO: 652 is the nucleotide sequence encoding the CXCR4 variant 2 domain.

[0751] SEQ ID NO: 653 is the nucleotide sequence encoding the CXCR4 variant 3 domain.

[0752] SEQ ID NO: 654 is the nucleotide sequence encoding the CXCR4 variant 4 domain.

[0753] SEQ ID NO: 655 is the nucleotide sequence encoding the CXCR4 variant 5 domain.

[0754] SEQ ID NO: 656 is the nucleotide sequence encoding the CXCR5 variant 1 domain.

[0755] SEQ ID NO: 657 is the nucleotide sequence encoding the CXCR5 variant 2 domain.

[0756] SEQ ID NO: 658 is the nucleotide sequence encoding the CCR2 variant A domain.

[0757] SEQ ID NO: 659 is the nucleotide sequence encoding the CCR2 variant B domain.

[0758] SEQ ID NO: 660 is the nucleotide sequence encoding the CCR4 domain.

[0759] SEQ ID NO: 661 is the nucleotide sequence encoding the CCR6 variant 1 domain.

[0760] SEQ ID NO: 662 is the nucleotide sequence encoding the CCR6 variant 2 domain.

[0761] SEQ ID NO: 663 is the nucleotide sequence encoding the CCR7 variant 1 domain.

[0762] SEQ ID NO: 664 is the nucleotide sequence encoding the CCR7 variant 2 domain.

[0763] SEQ ID NO: 665 is the nucleotide sequence encoding the CCR7 variant 3 domain.

[0764] SEQ ID NO: 666 is the nucleotide sequence encoding the CCR7 variant 4 domain.

[0765] SEQ ID NO: 667 is the nucleotide sequence encoding the CCR7 variant 5 domain.

[0766] SEQ ID NO: 668 is the nucleotide sequence encoding the CCR8 domain.

[0767] SEQ ID NO:669 is the nucleotide sequence of a vector encoding the CXCR1 chemokine receptor.

[0768] SEQ ID NO:670 is the nucleotide sequence of a vector encoding the CCR8 chemokine receptor.

[0769] SEQ ID NO: 671 is the amino acid sequence of two CCRs using both the 38A1 and 19H9 PD-L1 domains described herein, comprising SP-(38A1 scFv)-(CD28 hinge and transmembrane)-(IL-2Rβ intracellular)-T2A-SP-(19H9 scFv)-(CD28 hinge and transmembrane)-(IL-2Rγ intracellular), where SP refers to the signal peptide.

[0770] SEQ ID NO: 672 is the amino acid sequence of two CCRs using both the 38A1 and 19H9 PD-L1 domains described herein, comprising SP-(38A1 scFv)-(CD28 hinge and transmembrane)-(IL-18R1 intracellular)-T2A-SP-(19H9 scFv)-(CD28 hinge and transmembrane)-(IL-18RAP intracellular), where SP refers to the signal peptide.

[0771] SEQ ID NO: 673 is the amino acid sequence of two CCRs comprising SP-(anti-TROP-2 scFv)-(CD8 hinge)-(IL-2Rβ transmembrane and intracellular)-T2A-SP-(anti-TROP-2 scFv)-(CD8 hinge)-(IL-2Rγ transmembrane and intracellular), where SP refers to the signal peptide.

[0772] SEQ ID NO: 674 is the amino acid sequence of two CCRs comprising SP-(anti-TROP-2 scFv)-(CD8 hinge)-(IL-18R1-transmembrane and intracellular)-T2A-SP-(anti-TROP-2 scFv)-(CD8 hinge)-(IL-18RAP-transmembrane and intracellular), where SP refers to the signal peptide.

[0773] SEQ ID NO: 675 is the amino acid sequence of two CCRs comprising SP-(cAR47A6.4 scFv)-(CD28 hinge-transmembrane)-(IL-2Rβ intracellular)-T2A-SP-(KM4097 scFv)-(CD28 hinge and transmembrane)-(IL-2Rγ intracellular), where SP refers to the signal peptide.

[0774] SEQ ID NO: 676 is the amino acid sequence of two CCRs comprising SP-(cAR47A6.4 scFv)-(CD28 hinge-transmembrane)-(IL-18R1 intracellular)-T2A-SP-(KM4097scFv)-(CD28 hinge-transmembrane)-(IL-18RAP intracellular). SP refers to the signal peptide.

[0775] SEQ ID NO: 677 is the amino acid sequence of two CCRs comprising CCR7.2:chPD-L1-IL-2R (SP-38A1scFv-IL2Rβ12aaEC-TM-IL-2Rβ-IC-T2A-SP-19H9scFv-IL2Rγ12aaEC-TM-IL-2Rγ-IC). SP refers to signal peptide, EC refers to extracellular, TM refers to transmembrane, and IC refers to intracellular.

[0776] SEQ ID NO: 678 is the amino acid sequence of the two CCRs comprising CCR8.2:chPD-L1-IL-18R (SP-38A1scFv-IL-18R112aaEC-TM-IL-18R1-IC-T2A-SP-19H9scFv-IL-18RRAP12aaEC-TM-IL-18RAP-IC). SP refers to signal peptide, EC refers to extracellular, TM refers to transmembrane, and IC refers to intracellular.

[0777] SEQ ID NO: 679 is the amino acid sequence of two CCRs comprising CCR11.2:TROP2-IL-2R (SP-cAR47A6.4 scFv-IL2Rβ12aaEC-TM-IL-2Rβ-IC-T2A-SP-KM4097scFV-IL2Rγ12aaEC-TM-IL-2Rγ-IC). SP refers to signal peptide, EC refers to extracellular, TM refers to transmembrane, and IC refers to intracellular.

[0778] SEQ ID NO: 680 is the amino acid sequence of two CCRs comprising CCR12.2:TROP2-IL-18R (SP-cAR47A6.4 scFv-IL-18R112aaEC-TM-IL-18R1-IC-T2A-SP-KM4097scFv-IL-18RRAP12aaEC-TM-IL-18RAP-IC). SP refers to signal peptide, EC refers to extracellular, TM refers to transmembrane, and IC refers to intracellular.

[0779] SEQ ID NO:681 is the nucleotide sequence encoding CCR7.2.

[0780] SEQ ID NO: 682 is the nucleotide sequence encoding CCR8.2.

[0781] SEQ ID NO: 683 is the nucleotide sequence encoding CCR11.2.

[0782] SEQ ID NO:684 is the nucleotide sequence encoding CCR12.2.

[0783] SEQ ID NO: 685 is the nucleotide sequence of a vector encoding CCR7.2.

[0784] SEQ ID NO: 686 is the nucleotide sequence of a vector encoding CCR8.2.

[0785] SEQ ID NO: 687 is the nucleotide sequence of a vector encoding CCR11.2.

[0786] SEQ ID NO:688 is the nucleotide sequence of a vector encoding CCR12.2.

[0787] SEQ ID NO: 689 is the amino acid sequence of CCR13 (ch Fas-4-1BB).

[0788] SEQ ID NO: 690 is the amino acid sequence of CCR14 (ch PD-1-4-1BB).

[0789] SEQ ID NO: 691 is the amino acid sequence of CCR15 (TGFβRII-4-1BB).

[0790] SEQ ID NO: 692 is the amino acid sequence of CCR16(ch PD-1-CD28).

[0791] SEQ ID NO: 693 is the amino acid sequence of the FAS binding domain.

[0792] SEQ ID NO: 694 is the amino acid sequence of the TGFβRII binding domain.

[0793] SEQ ID NO:695 is the nucleotide sequence encoding CCR13 (ch Fas-4-1BB).

[0794] SEQ ID NO: 696 is the nucleotide sequence encoding CCR14 (ch PD-1-4-1BB).

[0795] SEQ ID NO: 697 is the nucleotide sequence encoding CCR15 (TGFβRII-4-1BB).

[0796] SEQ ID NO: 698 is the nucleotide sequence encoding CCR16 (ch PD-1-CD28).

[0797] SEQ ID NO:699 is the nucleotide sequence of a vector encoding CCR13 (ch Fas-4-1BB).

[0798] SEQ ID NO: 700 is the nucleotide sequence of a vector encoding CCR14 (ch PD-1-4-1BB).

[0799] SEQ ID NO: 701 is the nucleotide sequence of a vector encoding CCR15(ch TGFβRII-4-1BB).

[0800] SEQ ID NO: 702 is the nucleotide sequence of a vector encoding CCR16(ch PD-1-CD28).

[0801] SEQ ID NO: 703 is the amino acid sequence of CCR17(ch Fas-LTBR).

[0802] SEQ ID NO: 704 is the amino acid sequence of CCR18(ch PD-1-LTBR).

[0803] SEQ ID NO: 705 is the amino acid sequence of CCR19(ch TGFβRII-LTBR).

[0804] SEQ ID NO:706 is the nucleotide sequence encoding CCR17(ch Fas-LTBR).

[0805] SEQ ID NO: 707 is the nucleotide sequence encoding CCR18(ch PD-1-LTBR).

[0806] SEQ ID NO: 708 is the nucleotide sequence encoding CCR19(ch TGFβRII-LTBR).

[0807] SEQ ID NO:709 is the nucleotide sequence of a vector encoding CCR17(ch Fas-LTBR).

[0808] SEQ ID NO: 710 is the nucleotide sequence of a vector encoding CCR18(ch PD-1-LTBR).

[0809] SEQ ID NO: 711 is the nucleotide sequence of a vector encoding CCR19(ch TGFβRII-LTBR).

[0810] SEQ ID NO: 712 is the amino acid sequence of CCR20 (ch 19H9-4-1BB).

[0811] SEQ ID NO: 713 is the amino acid sequence of CCR21 (ch 19H9-LTBR).

[0812] SEQ ID NO: 714 is the amino acid sequence of CCR22 (ch 19H9-4-1BB version 2).

[0813] SEQ ID NO: 715 is the amino acid sequence of CCR23 (ch 19H9-LTBR version 2).

[0814] SEQ ID NO: 716 is the amino acid sequence of CCR24 (ch 19H9-LTBR-4-1BB).

[0815] SEQ ID NO: 717 is the amino acid sequence of CCR25 (ch 19H9-4-1BB-LTBR).

[0816] SEQ ID NO:718 is the nucleotide sequence encoding CCR20 (ch 19H9-4-1BB).

[0817] SEQ ID NO:719 is the nucleotide sequence encoding CCR21 (ch 19H9-LTBR).

[0818] SEQ ID NO: 720 is the nucleotide sequence encoding CCR22 (ch 19H9-4-1BB version 2).

[0819] SEQ ID NO: 721 is the nucleotide sequence encoding CCR23 (ch 19H9-LTBR version 2).

[0820] SEQ ID NO:722 is the nucleotide sequence encoding CCR24 (ch 19H9-LTBR-4-1BB).

[0821] SEQ ID NO: 723 is the nucleotide sequence encoding CCR25 (ch 19H9-4-1BB-LTBR). DETAILED DESCRIPTION OF THE INVENTION

[0822] I. Introduction Adoptive cell therapy utilizing TILs cultured ex vivo by rapid expansion protocols (REPs) has been successful in providing adoptive cell therapy to patients with cancers such as melanoma following host immunosuppression. Current TIL manufacturing and treatment processes are limited by duration, cost, sterility concerns, and other factors described herein. There is an urgent need to provide TIL manufacturing processes and therapies based on such processes that are suitable for use in treating patients who have few or no remaining viable treatment options. The present invention fulfills this need by providing manufacturing processes and products for use in generating TILs modified with CCRs or chemokine receptors, among other modifications described herein, to improve their efficacy, potency, safety, stemness, or other performance measures.

[0823] 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.

[0824] As used herein, the terms "co-administration," "co-administering," "administered in combination," "administering in combination," "simultaneous," and "concurrent" encompass administration of two or more active pharmaceutical ingredients (e.g., multiple TILs in preferred embodiments of the present invention) to a subject such that both active pharmaceutical ingredients and / or their metabolites are present in the subject at the same time. Simultaneous 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.

[0825] The term "in vivo" refers to events that take place inside a subject's body.

[0826] 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.

[0827] The term "ex vivo" refers to events involving the administration of a therapy or treatment to cells, tissues, and / or organs that have been removed from a subject's body. Suitably, the cells, tissues, and / or organs may be returned to the subject's body in a surgical or therapeutic manner.

[0828] 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.

[0829] As used herein, "tumor infiltrating lymphocytes" or "TILs" refers to a population of cells that are initially acquired as leukocytes that have left a subject's bloodstream and migrated to a tumor. TILs include CD8 + Cytotoxic T cells (lymphocytes), Th1 and Th17 CD4 + These include, but are not limited to, T cells, natural killer cells, dendritic cells, and M1 macrophages. TILs include both primary and secondary TILs. "Primary TILs" are those obtained from a patient tissue sample as outlined herein (sometimes referred to as "freshly harvested"), and "secondary TILs" are any expanded or propagated TIL cell populations discussed herein, including, but not limited to, bulk TILs and expanded TILs ("REP TILs" or "post-REP TILs"). TIL cell populations may include genetically modified TILs.

[0830] 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.

[0831] As used herein, "cryopreserved TILs" refers to TILs, either primary, bulk, or expanded (REP TILs), that are processed and stored at temperatures ranging from approximately -150°C to -60°C. General methods for cryopreservation are described elsewhere herein, including in the Examples. For clarity, "cryopreserved TILs" can be distinguished from frozen tissue samples that may be used as a source of primary TILs.

[0832] 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.

[0833] TILs can generally be defined either biochemically using cell surface markers or functionally by their ability to infiltrate tumors and achieve therapy. TILs can generally be classified by expression of one or more of the following biomarkers: CD4, CD8, T cell receptor (TCR) αβ, CD27, CD28, CD56, CCR7, CD45Ra, CD95, PD-1, and CD25. Additionally and alternatively, TILs can be functionally defined by their ability to infiltrate solid tumors upon reintroduction into patients.

[0834] 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.

[0835] The term "central memory T cells" refers to cells that are CD45R0+ and CCR7 (CCR7 高 ) and CD62L (CD62 高) is a subset of T cells that constitutively express the CD4 receptor. The surface phenotype of central memory T cells also includes TCR, CD3, CD127 (IL-7R), and IL-15R. Transcription factors of central memory T cells include BCL-6, BCL-6B, MBD2, and BMI1. After TCR triggering, central memory T cells primarily secrete IL-2 and CD40L as effector molecules. Central memory T cells predominate in the CD4 compartment in the blood and are proportionally enriched in lymph nodes and tonsils in humans.

[0836] The term "effector memory T cells" refers to T cells that are CD45R0+ like central memory T cells, but have lost constitutive expression of CCR7 (CCR7 低 ), heterogeneous or low CD62L expression (CD62L 低 ), refers to a subset of human or mammalian T cells. The surface phenotype of central memory T cells also includes TCR, CD3, CD127 (IL-7R), and IL-15R. Transcription factors of central memory T cells include BLIMP1. Effector memory T cells rapidly secrete high levels of inflammatory cytokines after antigen stimulation, including interferon gamma, IL-4, and IL-5. Effector memory T cells predominate in the CD8 compartment in the blood and are proportionally enriched in the lung, liver, and intestine in humans. CD8+ effector memory T cells carry large amounts of perforin.

[0837] The term "closed system" refers to a system that is closed to the external environment. Any closed system suitable for cell culture methods can be used in the methods of the present invention. Closed systems include, but are not limited to, sealed G containers. Once tumor segments are added to the closed system, the system is not opened to the external environment until the TILs are ready to be administered to a patient.

[0838] 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.

[0839] The terms "peripheral blood mononuclear cells" and "PBMCs" refer to peripheral blood cells with round nuclei, including lymphocytes (T cells, B cells, NK cells) and monocytes. When used as antigen-presenting cells (PBMCs are a type of antigen-presenting cell), the peripheral blood mononuclear cells are preferably irradiated allogeneic peripheral blood mononuclear cells.

[0840] The terms "peripheral blood lymphocytes" and "PBLs" refer to T cells expanded from peripheral blood. In some embodiments, PBLs are isolated from whole blood or apheresis products from a donor. In some embodiments, PBLs are isolated from whole blood or apheresis products from a donor by positive or negative selection of a T cell phenotype, such as a CD3+CD45+ T cell phenotype.

[0841] The term "anti-CD3 antibody" refers to an antibody or variant thereof, e.g., a monoclonal antibody, including a human, humanized, chimeric, or murine antibody, directed against the CD3 receptor in the T cell antigen receptor of mature T cells. Anti-CD3 antibodies include OKT-3, also known as muromonab. Anti-CD3 antibodies also include the UHCT1 clone, also known as T3 and CD3ε. Other anti-CD3 antibodies include, for example, otelixizumab, teplizumab, and visilizumab.

[0842] The term "OKT-3" (also referred to herein as "OKT3") refers to a monoclonal antibody, including a human, humanized, chimeric, or murine antibody against the CD3 receptor in the T cell antigen receptor of mature T cells, or a biosimilar or variant thereof, including commercially available forms such as OKT-3 (30 ng / mL, MACS GMP CD3 pure, Miltenyi Biotech, Inc., San Diego, CA, USA) and muromonab, or variants, conservative amino acid substitutions, glycoforms, or biosimilars thereof. The amino acid sequences of the heavy and light chains of muromonab are shown in Table 1 (SEQ ID NO: 1 and SEQ ID NO: 2). A hybridoma capable of producing OKT-3 has been deposited with the American Type Culture Collection and assigned ATCC accession number CRL8001. A hybridoma capable of producing OKT-3 has also been deposited with the European Collection of Authenticated Cell Cultures (ECACC) and assigned catalog number 86022706. [Table 1]

[0843] The term "IL-2" (also referred to herein as "IL2") refers to the T cell growth factor known as interleukin-2 and includes all forms of IL-2, including human and mammalian forms, their conservative amino acid substitutions, glycoforms, biosimilars, and variants. IL-2 is described, for example, in Nelson, J. Immunol. 2004, 172, 3983-88 and Malek, Annu. Rev. Immunol. 2008, 26, 453-79, the disclosures of which are incorporated herein by reference. The amino acid sequence of recombinant human IL-2 suitable for use in the present invention is shown in Table 2 (SEQ ID NO: 3). For example, the term IL-2 encompasses human recombinant IL-2 forms such as aldesleukin (PROLEUKIN, commercially available from multiple suppliers at 22 million IU per single-use vial), as well as the recombinant IL-2 form (catalog number CYT-209-b) marketed by CellGenix, Inc., Portsmouth, NH, USA (CELLGRO GMP) or ProSpec-Tany TechnoGene Ltd., East Brunswick, NJ, USA, and other commercial equivalents from other vendors. Aldesleukin (des-alanyl-1, serine-125 human IL-2) is a non-glycosylated human recombinant IL-2 form with a molecular weight of approximately 15 kDa. The amino acid sequence of aldesleukin suitable for use in the present invention is shown in Table 2 (SEQ ID NO: 4). The term IL-2 also refers to the pegylated IL-2 prodrug bempegaldesleukin (NKTR-214, an IL-2 prodrug in which an average of six lysine residues are replaced with [(2,7-bis{[methylpoly(oxyethylene)]carbamoyl}-9H-fluoren-9-yl)methoxy]carbonyl). 6The present invention also encompasses pegylated forms of IL-2 described herein, including pegylated human recombinant IL-2 such as SEQ ID NO: 4, which is available from Nektar Therapeutics (South San Francisco, CA, USA) or can be prepared by methods known in the art, such as the method described in Example 19 of International Patent Application Publication No. WO 2018 / 132496 A1 or Example 1 of U.S. Patent Application Publication No. US 2019 / 0275133 A1, the disclosures of which are incorporated herein by reference. Benpegaldesleukin (NKTR-214) and other pegylated IL-2 molecules suitable for use in the present invention are described in U.S. Patent Application Publication No. US 2014 / 0328791 A1 and International Patent Application Publication No. WO 2012 / 065086 A1, the disclosures of which are incorporated herein by reference. Alternative forms of conjugated IL-2 suitable for use in the present invention are described in U.S. Patent Nos. 4,766,106, 5,206,344, 5,089,261, and 4,902,502, the disclosures of which are incorporated herein by reference. Formulations of IL-2 suitable for use in the present invention are described in U.S. Patent No. 6,706,289, the disclosure of which is incorporated herein by reference.

[0844] In some embodiments, a suitable IL-2 form for use in the present invention is THOR-707, available from Synthorx, Inc. The preparation and properties of THOR-707 and additional alternative forms of IL-2 suitable for use in the present invention are described in U.S. Patent Application Publication Nos. US2020 / 0181220A1 and US2020 / 0330601A1, the disclosures of which are incorporated herein by reference. In some embodiments, a suitable IL-2 form for use in the present invention is an interleukin-2 (IL-2) complex comprising an isolated and purified IL-2 polypeptide and a conjugation moiety that binds to the isolated and purified IL-2 polypeptide at an amino acid position selected from K35, T37, R38, T41, F42, K43, F44, Y45, E61, E62, E68, K64, P65, V69, L72, and Y107, wherein the numbering of the amino acid residues corresponds to SEQ ID NO:5. In some embodiments, the amino acid position is selected from T37, R38, T41, F42, F44, Y45, E61, E62, E68, K64, P65, V69, L72, and Y107. In some embodiments, the amino acid position is selected from T37, R38, T41, F42, F44, Y45, E61, E62, E68, P65, V69, L72, and Y107. In some embodiments, the amino acid position is selected from T37, T41, F42, F44, Y45, P65, V69, L72, and Y107. In some embodiments, the amino acid position is selected from R38 and K64. In some embodiments, the amino acid position is selected from E61, E62, and E68. In some embodiments, the amino acid position is E62. In some embodiments, an amino acid residue selected from K35, T37, R38, T41, F42, K43, F44, Y45, E61, E62, E68, K64, P65, V69, L72, and Y107 is further mutated to lysine, cysteine, or histidine. In some embodiments, the amino acid residue is mutated to cysteine. In some embodiments, the amino acid residue is mutated to lysine. In some embodiments, K35, T37, R38, T41, F42, K43, F44, Y45, E61, E62, E68, K64, P65, V69, L72, and Y107 are ...and Y107 are further mutated to an unnatural amino acid. In some embodiments, the unnatural amino acid is selected from N6-azidoethoxy-L-lysine (AzK), N6-propargylethoxy-L-lysine (PraK), BCN-L-lysine, norbornene lysine, TCO-lysine, methyltetrazine lysine, allyloxycarbonyl lysine, 2-amino-8-oxononanoic acid, 2-amino-8-oxooctanoic acid, p-acetyl-L-phenylalanine, p-azidomethyl-L-phenylalanine (pAMF), p-iodo-L-phenylalanine, m-acetylphenylalanine, 2-amino-8-oxononanoic acid, p-propargyloxyphenylalanine, p-propargyl-phenylalanine, 3-methyl-phenylalanine, L-dopa, fluorinated phenylalanine, isopropyl and selenocysteine, or selenocysteine. In some embodiments, the IL-2 complex has a reduced affinity for the IL-2 receptor alpha (IL-2Rα) subunit compared to a wild-type IL-2 polypeptide. In some embodiments, the reduced affinity is about a 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 95%, 99%, or greater than a 99% reduction in binding affinity for IL-2Rα compared to a wild-type IL-2 polypeptide. In some embodiments, the reduced affinity is about a 1-fold, 2-fold, 3-fold, 4-fold, 5-fold, 6-fold, 7-fold, 8-fold, 9-fold, 10-fold, 30-fold, 50-fold, 100-fold, 200-fold, 300-fold, 500-fold, or greater reduction in binding affinity for IL-2Rα compared to a wild-type IL-2 polypeptide.1000-fold or more. In some embodiments, the conjugated moiety impairs or blocks the binding of IL-2 to IL-2Rα. In some embodiments, the conjugated moiety comprises a water-soluble polymer. In some embodiments, the additional conjugated moiety comprises a water-soluble polymer. In some embodiments, each of the water-soluble polymers independently comprises polyethylene glycol (PEG), poly(propylene glycol) (PPG), copolymers of ethylene glycol and propylene glycol, poly(oxyethylated polyol), poly(olefinic alcohol), poly(vinylpyrrolidone), poly(hydroxyalkyl methacrylamide), poly(hydroxyalkyl methacrylate), poly(saccharide), poly(α-hydroxy acid), poly(vinyl alcohol), polyphosphazene, polyoxazoline (POZ), poly(N-acryloylmorpholine), or a combination thereof. In some embodiments, each of the water-soluble polymers independently comprises PEG. In some embodiments, the PEG is linear PEG or branched PEG. In some embodiments, each of the water-soluble polymers independently comprises a polysaccharide. In some embodiments, the polysaccharide comprises dextran, polysialic acid (PSA), hyaluronic acid (HA), amylose, heparin, heparan sulfate (HS), dextrin, or hydroxyethyl starch (HES). In some embodiments, each of the water soluble polymers independently comprises a glycan. In some embodiments, each of the water soluble polymers independently comprises a polyamine. In some embodiments, the conjugation moiety comprises a protein. In some embodiments, the additional conjugation moieties comprise a protein. In some embodiments, each of the proteins independently comprises albumin, transferrin, or transthyretin. In some embodiments, each of the proteins independently comprises an Fc portion. In some embodiments, each of the proteins independently comprises an Fc portion of an IgG. In some embodiments, the conjugation moiety comprises a polypeptide. In some embodiments, the additional conjugation moieties comprise a polypeptide. In some embodiments, each of the proteins independently comprises an XTEN peptide, a glycine-rich homoamino acid polymer (HAP),The conjugated moiety may comprise a PAS polypeptide, an elastin-like polypeptide (ELP), a CTP peptide, or a gelatin-like protein (GLK) polymer. In some embodiments, the isolated and purified IL-2 polypeptide is modified by glutamylation. In some embodiments, the conjugated moiety is directly attached to the isolated and purified IL-2 polypeptide. In some embodiments, the conjugated moiety is indirectly attached to the isolated and purified IL-2 polypeptide via a linker. In some embodiments, the linker comprises a homobifunctional linker. In some embodiments, the homobifunctional linker is selected from the group consisting of the Romant reagents dithiobis(succinimidyl propionate) DSP, 3'3'-dithiobis(sulfosuccinimidyl propionate) (DTSSP), disuccinimidyl suberate (DSS), bis(sulfosuccinimidyl) suberate (BS), disuccinimidyl tartrate (DST), disulfosuccinimidyl tartrate (sulfoDS T), ethylene glycobis(succinimidyl succinate) (EGS), disuccinimidyl glutarate (DSG), N,N'-disuccinimidyl carbonate (DSC), dimethyl adipimidate (DMA), dimethyl pimelimidate (DMP), dimethyl suberimidate (DMS), dimethyl-3,3'-dithiobispropionimidate (DTBP), 1,4-di-(3'-(2'-pyridyldithio) (e) propionamido) butane (DPDPB), bismaleimidohexane (BMH), halogenated aryl-containing compounds (DFDNB), such as 1,5-difluoro-2,4-dinitrobenzene or 1,3-difluoro-4,6-dinitrobenzene, 4,4'-difluoro-3,3'-dinitrophenyl sulfone (DFDNPS), bis-[β-(4-azidosalicylamido)ethyl] disulfide (BASED), formaldehyde, glutaraldehyde, 1,4-butanediol diglycidyl ether, adipic acid dihydrazide, carbohydrazide, o-toluidine, 3,3'-dimethylbenzidine, benzidine, α,α'-p-diaminodiphenyl, diiodo-p-xylene sulfonic acid, N,N'-ethylene-bis(iodoacetamide), or N,N'-hexamethylenebis(iodoacetamide). In some embodiments, the linker isIn some embodiments, the heterobifunctional linker includes N-succinimidyl 3-(2-pyridyldithio)propionate (sPDP), long-chain N-succinimidyl 3-(2-pyridyldithio)propionate (LC-sPDP), water-soluble long-chain N-succinimidyl 3-(2-pyridyldithio)propionate (sulfo-LC-sPDP), succinimidyloxycarbonyl-α-methyl-α-(2-pyridyldithio)toluene (sMPT), sulfosuccinimidyl-6-[α-methyl-α-(2-pyridyldithio)toluamide]hexamethyl. Succinimidyl-4-(N-maleimidomethyl)cyclohexane-1-carboxylate (sulfo-LC-sMPT), succinimidyl-4-(N-maleimidomethyl)cyclohexane-1-carboxylate (sMCC), sulfosuccinimidyl-4-(N-maleimidomethyl)cyclohexane-1-carboxylate (sulfo-sMCC), m-maleimidobenzoyl-N-hydroxysuccinimide ester (MB), m-maleimidobenzoyl-N-hydroxysulfosuccinimide ester (sulfo-MB), N-succinimidyl (4-iodoacetyl)aminobenzoate (sIAB), sulfosuccinimide Succinimidyl (4-iodoacetyl)aminobenzoate (sulfo-sIAB), succinimidyl-4-(p-maleimidophenyl)butyrate (sMPB), sulfosuccinimidyl-4-(p-maleimidophenyl)butyrate (sulfo-sMPB), N-(γ-maleimidobutyryloxy)succinimide ester (GMB), N-(γ-maleimidobutyryloxy)sulfosuccinimide ester (sulfo-GMB), succinimidyl 6-((iodoacetyl)amino)hexanoate (sIAX), succinimidyl 6-[6-(( (iodoacetyl)amino)hexanoyl)amino]hexanoate (slAXX), succinimidyl 4-(((iodoacetyl)amino)methyl)cyclohexane-1-carboxylate (sIAC), succinimidyl 6-(((((4-iodoacetyl)amino)methyl)cyclohexane-1-carbonyl)amino)hexanoate (sIACX), p-nitrophenyl iodoacetate (NPIA), carbonyl-reactive and sulfhydryl-reactive crosslinkers, such as 4-(4-N-maleimidophenyl)butyric acid hydrazide (MPBH),4-(N-maleimidomethyl)cyclohexane-1-carboxyl-hydrazide-8 (M2C2H), 3-(2-pyridyldithio)propionylhydrazide (PDPH), N-hydroxysuccinimidyl-4-azidosalicylate (NHs-AsA), N-hydroxysulfosuccinimidyl-4-azidosalicylate (sulfo-NHs-AsA), sulfosuccinimidyl-(4, -azidosalicylamido)hexanoate (sulfo-NHs-LC-AsA), sulfosuccinimidyl-2-(p-azidosalicylamido)ethyl-1,3'-dithiopropionate (sAsD), N-hydroxysuccinimidyl-4-azidobenzoate (HsAB), N-hydroxysulfosuccinimidyl-4-azidobenzoate (sulfo-HsAB), N-succinimidyl-6-(4'-azido-2'-nitrophenylamino)hexanoate (sANPAH), sulfo Succinimidyl-6-(4'-azido-2'-nitrophenylamino)hexanoate (sulfo-sANPAH), N-5-azido-2-nitrobenzoyloxysuccinimide (ANB-NO), sulfosuccinimidyl-2-(m-azido-o-nitrobenzamido)-ethyl-1,3'-dithiopropionate (sAND), N-succinimidyl-4(4-azidophenyl)1,3'-dithiopropionate (sADP), N-sulfosuccinimidyl(4-azidophenyl)- 1,3'-dithiopropionate (sulfo-sADP), sulfosuccinimidyl 4-(p-azidophenyl)butyrate (sulfo-sAPB), sulfosuccinimidyl 2-(7-azido-4-methylcoumarin-3-acetamido)ethyl-1,3'-dithiopropionate (sAED), sulfosuccinimidyl 7-azido-4-methylcoumarin-3-acetate (sulfo-sAMCA), p-nitrophenyl diazopyruvate (pNPDP), p-nitrophenyl diazo-3 ,3,3-trifluoropropionate (PNP-DTP), 1-(ρ-azidosalicylamido)-4-(iodoacetamido)butane (AsIB), N-[4-(ρ-azidosalicylamido)butyl]-3'-(2'-pyridyldithio)propionamide (APDP), benzophenone-4-iodoacetamide, p-azidobenzoylhydrazide (ABH), 4-(ρ-azidosalicylamido)butylamine (AsBA), or p-azidophenylglyoxal (APG). In some embodiments, the linker comprises a cleavable linker, optionally including a dipeptide linker. In some embodiments, the dipeptide linker comprises Val-Cit, Phe-Lys, Val-Ala, or Val-Lys.In some embodiments, the linker comprises a non-cleavable linker. In some embodiments, the linker optionally comprises a maleimide group, including maleimidocaproyl (mc), succinimidyl-4-(N-maleimidomethyl)cyclohexane-1-carboxylate (sMCC), or sulfosuccinimidyl-4-(N-maleimidomethyl)cyclohexane-1-carboxylate (sulfo-sMCC). In some embodiments, the linker further comprises a spacer. In some embodiments, the spacer comprises p-aminobenzyl alcohol (PAB), p-aminobenzyloxycarbonyl (PABC), a derivative, or an analog thereof. In some embodiments, the conjugated moiety is capable of extending the serum half-life of the IL-2 conjugate. In some embodiments, the additional conjugated moiety is capable of extending the serum half-life of the IL-2 conjugate. In some embodiments, the IL-2 form suitable for use in the present invention is a fragment of any of the IL-2 forms described herein. In some embodiments, IL-2 forms suitable for use in the present invention are pegylated as disclosed in U.S. Patent Application Publication Nos. US2020 / 0181220A1 and US2020 / 0330601A1. In some embodiments, IL-2 forms suitable for use in the present invention are IL-2 conjugates comprising an IL-2 polypeptide comprising N6-azidoethoxy-L-lysine (AzK) covalently attached to a conjugation moiety comprising polyethylene glycol (PEG), wherein the IL-2 polypeptide comprises an amino acid sequence having at least 80% sequence identity to SEQ ID NO:5, and wherein AzK substitutes an amino acid at position K35, F42, F44, K43, E62, P65, R38, T41, E68, Y45, V69, or L72 relative to amino acid position within SEQ ID NO:5. In some embodiments, the IL-2 polypeptide comprises an N-terminal deletion of one residue relative to SEQ ID NO:5. In some embodiments, forms of IL-2 suitable for use in the present invention lack IL-2R alpha chain association but retain normal binding to the intermediate affinity IL-2R beta-gamma signaling complex.In some embodiments, a suitable IL-2 form for use in the present invention is an IL-2 conjugate comprising an IL-2 polypeptide comprising N6-azidoethoxy-L-lysine (AzK) covalently attached to a conjugation moiety comprising polyethylene glycol (PEG), wherein the IL-2 polypeptide comprises an amino acid sequence having at least 90% sequence identity to SEQ ID NO:5, and wherein AzK substitutes an amino acid at position K35, F42, F44, K43, E62, P65, R38, T41, E68, Y45, V69, or L72 relative to amino acid position in SEQ ID NO:5. In some embodiments, a suitable IL-2 form for use in the present invention is an IL-2 conjugate comprising an IL-2 polypeptide comprising N6-azidoethoxy-L-lysine (AzK) covalently attached to a conjugation moiety comprising polyethylene glycol (PEG), wherein the IL-2 polypeptide comprises an amino acid sequence having at least 95% sequence identity to SEQ ID NO:5, and wherein AzK substitutes an amino acid at position K35, F42, F44, K43, E62, P65, R38, T41, E68, Y45, V69, or L72 relative to amino acid position in SEQ ID NO:5. In some embodiments, a suitable IL-2 form for use in the present invention is an IL-2 conjugate comprising an IL-2 polypeptide comprising N6-azidoethoxy-L-lysine (AzK) covalently attached to a conjugation moiety comprising polyethylene glycol (PEG), wherein the IL-2 polypeptide comprises an amino acid sequence having at least 98% sequence identity to SEQ ID NO:5, and wherein AzK substitutes an amino acid at position K35, F42, F44, K43, E62, P65, R38, T41, E68, Y45, V69, or L72 relative to amino acid position in SEQ ID NO:5.

[0845] In some embodiments, a form of IL-2 suitable for use in the present invention is nembareukin alfa, also known as ALKS-4230 (SEQ ID NO: 6), available from Alkermes, Inc. Nembareukin alfa is a nucleotide analogue of IL-2 that is linked via a peptidyl linker ( 60 GG 61 ) and fused to human interleukin-2 fragment (62-132) via a peptidyl linker ( 133 GSGGGS138 Human interleukin-2 receptor α-chain fragment (139-303) fused via a nucleotide sequence (Cys), produced in Chinese hamster ovary (CHO) cells, and glycosylated. 125 >Ser 51 ; human interleukin-2 (IL-2) (4-74)-peptide (62-132) fused via a G2 peptide linker (60-61) and human interleukin-2 receptor alpha chain (IL2R subunit alpha, IL2Rα, IL2RA) (1-165)-peptide (139-303) fused via a GSG3S peptide linker (133-138), produced in Chinese hamster ovary (CHO) cells, and alpha-glycosylated human interleukin-2 (IL-2) (75-133)-peptide [Cys 125(51)>Ser]-mutant (1-59). The amino acid sequence of nemvaleukin alfa is set forth in SEQ ID NO: 6. In some embodiments, nemvaleukin alfa exhibits the following post-translational modifications: disulfide bridges at the following positions: 31-116, 141-285, 184-242, 269-301, 166-197, or 166-199, 168-199, or 168-197 (using the numbering of SEQ ID NO: 6), and glycosylation sites at the following positions: N187, N206, T212, using the numbering of SEQ ID NO: 6. The preparation and properties of nemvaleukin alfa, as well as additional alternative forms of IL-2 suitable for use in the present invention, are described in U.S. Patent Application Publication No. 2021 / 0038684A1 and U.S. Patent No. 10,183,979, the disclosures of which are incorporated herein by reference. In some embodiments, an IL-2 form suitable for use in the present invention is a protein having at least 80%, at least 90%, at least 95%, or at least 90% sequence identity to SEQ ID NO:6. In some embodiments, an IL-2 form suitable for use in the present invention has the amino acid sequence set forth in SEQ ID NO:6 or conservative amino acid substitutions thereof. In some embodiments, an IL-2 form suitable for use in the present invention is a fusion protein comprising amino acids 24-452 of SEQ ID NO:7, or a variant, fragment, or derivative thereof. In some embodiments, an IL-2 form suitable for use in the present invention is a fusion protein comprising an amino acid sequence having at least 80%, at least 90%, at least 95%, or at least 90% sequence identity to amino acids 24-452 of SEQ ID NO:7, or a variant, fragment, or derivative thereof. Other IL-2 forms suitable for use in the present invention are described in U.S. Pat. No. 10,183,979, the disclosure of which is incorporated herein by reference.Optionally, in some embodiments, a form of IL-2 suitable for use in the present invention is a fusion protein comprising a first fusion partner linked to a second fusion partner by a mucin domain polypeptide linker, wherein the first fusion partner is IL-1Rα or a protein having at least 98% amino acid sequence identity to IL-1Rα and having receptor antagonist activity for IL-Rα, the second fusion partner comprises all or a portion of an immunoglobulin comprising an Fc region, and the mucin domain polypeptide linker comprises SEQ ID NO:8 or an amino acid sequence having at least 90% sequence identity to SEQ ID NO:8, and wherein the half-life of the fusion protein is improved compared to the fusion of the first fusion partner with the second fusion partner in the absence of the mucin domain polypeptide linker. [Table 2]

[0846] In some embodiments, IL-2 forms suitable for use in the present invention comprise a heavy chain variable region (V) comprising complementarity determining regions HCDR1, HCDR2, and HCDR3. H ) and a light chain variable region (V L ) and V H or V L and an IL-2 molecule or a fragment thereof grafted onto the CDR of a heavy chain variable region (V) comprising complementarity determining regions HCDR1, HCDR2, and HCDR3. H ) and a light chain variable region (V L ) and V H or V Land an IL-2 molecule or fragment thereof grafted onto the CDRs of: H or V L or a fragment thereof grafted onto the CDR of the antibody cytokine grafted protein, wherein the IL-2 molecule is a mutein, and the antibody cytokine grafted protein preferentially expands T effector cells over regulatory T cells, and the antibody further comprises an IgG class heavy chain and an IgG class light chain selected from the group consisting of an IgG class light chain comprising SEQ ID NO: 39 and an IgG class heavy chain comprising SEQ ID NO: 38, an IgG class light chain comprising SEQ ID NO: 37 and an IgG class heavy chain comprising SEQ ID NO: 29, an IgG class light chain comprising SEQ ID NO: 39 and an IgG class heavy chain comprising SEQ ID NO: 29, an IgG class light chain comprising SEQ ID NO: 37 and an IgG class heavy chain comprising SEQ ID NO: 38.

[0847] In one embodiment, the IL-2 molecule or fragment thereof is V H In one embodiment, the IL-2 molecule or fragment thereof is a mutein. H In one embodiment, the IL-2 molecule or fragment thereof is a mutein. H In one embodiment, the IL-2 molecule or fragment thereof is a mutein. L In one embodiment, the IL-2 molecule or a fragment thereof is grafted onto LCDR1 of V L In one embodiment, the IL-2 molecule or a fragment thereof is grafted onto LCDR2 of V LThe LCDR3 of the IL-2 molecule is grafted onto the LCDR3 of the IL-2 molecule, and the IL-2 molecule is a mutein.

[0848] The insertion of the IL-2 molecule can be at or near the N-terminal region of the CDR, the middle region of the CDR, or at or near the C-terminal region of the CDR. In some embodiments, the antibody cytokine graft protein comprises an IL-2 molecule incorporated into the CDR, where the IL2 sequence does not frameshift the CDR sequence. In some embodiments, the antibody cytokine graft protein comprises an IL-2 molecule incorporated into the CDR, where the IL-2 sequence replaces all or part of the CDR sequence. The replacement with the IL-2 molecule can be at or near the N-terminal region of the CDR, the middle region of the CDR, or the C-terminal region of the CDR. The replacement with the IL-2 molecule can be as little as one or two amino acids of the CDR sequence, or the entire CDR sequence.

[0849] In some embodiments, the IL-2 molecule is directly grafted onto the CDR without a peptide linker and without additional amino acids between the CDR sequence and the IL-2 sequence. In some embodiments, the IL-2 molecule is indirectly grafted onto the CDR using a peptide linker with one or more additional amino acids between the CDR sequence and the IL-2 sequence.

[0850] In some embodiments, the IL-2 molecule described herein is an IL-2 mutein. In some cases, the IL-2 mutein comprises an R67A substitution. In some embodiments, the IL-2 mutein comprises the amino acid sequence of SEQ ID NO: 14 or SEQ ID NO: 15. In some embodiments, the IL-2 mutein comprises the amino acid sequence of Table 1 of U.S. Patent Application Publication No. US2020 / 0270334A1, the disclosure of which is incorporated herein by reference.

[0851] In some embodiments, the antibody cytokine transplant protein comprises an HCDR1 selected from the group consisting of SEQ ID NO: 16, SEQ ID NO: 19, SEQ ID NO: 22, and SEQ ID NO: 25. In some embodiments, the antibody cytokine transplant protein comprises an HCDR1 selected from the group consisting of SEQ ID NO: 7, SEQ ID NO: 10, SEQ ID NO: 13, and SEQ ID NO: 16. In some embodiments, the antibody cytokine transplant protein comprises an HCDR2 selected from the group consisting of SEQ ID NO: 17, SEQ ID NO: 20, SEQ ID NO: 23, and SEQ ID NO: 26. In some embodiments, the antibody cytokine transplant protein comprises an HCDR3 selected from the group consisting of SEQ ID NO: 18, SEQ ID NO: 21, SEQ ID NO: 24, and SEQ ID NO: 27. In some embodiments, the antibody cytokine transplant protein comprises a V comprising the amino acid sequence of SEQ ID NO: 28. H In one embodiment, the antibody cytokine transplant protein comprises a heavy chain comprising the amino acid sequence of SEQ ID NO: 29. In one embodiment, the antibody cytokine transplant protein comprises a V region comprising the amino acid sequence of SEQ ID NO: 36. L In one embodiment, the antibody cytokine transplant protein comprises a light chain comprising the amino acid sequence of SEQ ID NO: 37. In one embodiment, the antibody cytokine transplant protein comprises a V region comprising the amino acid sequence of SEQ ID NO: 28. H V comprising the region and the amino acid sequence of SEQ ID NO: 36 LIn one embodiment, the antibody cytokine transplant protein comprises a heavy chain region comprising the amino acid sequence of SEQ ID NO:29 and a light chain region comprising the amino acid sequence of SEQ ID NO:37. In one embodiment, the antibody cytokine transplant protein comprises a heavy chain region comprising the amino acid sequence of SEQ ID NO:29 and a light chain region comprising the amino acid sequence of SEQ ID NO:39. In one embodiment, the antibody cytokine transplant protein comprises a heavy chain region comprising the amino acid sequence of SEQ ID NO:38 and a light chain region comprising the amino acid sequence of SEQ ID NO:37. In one embodiment, the antibody cytokine transplant protein comprises a heavy chain region comprising the amino acid sequence of SEQ ID NO:38 and a light chain region comprising the amino acid sequence of SEQ ID NO:39. In one embodiment, the antibody cytokine transplant protein comprises IgG.IL2F71A.H1 or IgG.IL2R67A.H1 of U.S. Patent Application Publication No. 2020 / 0270334A1, or a variant, derivative, or fragment thereof, or conservative amino acid substitutions thereof, or a protein having at least 80%, at least 90%, at least 95%, or at least 98% sequence identity thereto. In certain embodiments, the antibody component of the antibody cytokine transplant proteins described herein comprises immunoglobulin sequences, framework sequences, or CDR sequences of palivizumab. In some embodiments, the antibody cytokine transplant proteins described herein have a longer serum half-life than wild-type IL-2 molecules, such as, but not limited to, aldesleukin or equivalent molecules. In certain embodiments, the antibody cytokine transplant proteins described herein have a sequence set forth in Table 3. [Table 3-1] [Table 3-2]

[0852] The term "IL-4" (also referred to herein as "IL4") refers to the cytokine known as interleukin 4, which is produced by Th2 T cells, as well as eosinophils, basophils, and mast cells. IL-4 regulates the differentiation of naive helper T cells (Th0 cells) into Th2 T cells. Steinke and Borish, Respir. Res. 2001, 2, 66-70. Upon activation by IL-4, Th2 T cells subsequently produce additional IL-4 in a positive feedback loop. IL-4 also stimulates B cell proliferation and class II MHC expression, inducing class switching from B cells to IgE and IgG1 expression. Recombinant human IL-4 suitable for use in the present invention is commercially available from several suppliers, including ProSpec-Tany TechnoGene Ltd., East Brunswick, NJ, USA (catalog number CYT-211) and ThermoFisher Scientific, Inc., Waltham, MA, USA (human IL-15 recombinant protein, catalog number Gibco CTP0043). The amino acid sequence of recombinant human IL-4 suitable for use in the present invention is shown in Table 2 (SEQ ID NO: 9).

[0853] The term "IL-4" (also referred to herein as "IL4") refers to the cytokine known as interleukin 4, which is produced by Th2 T cells, as well as eosinophils, basophils, and mast cells. IL-4 regulates the differentiation of naive helper T cells (Th0 cells) into Th2 T cells. Steinke and Borish, Respir. Res. 2001, 2, 66-70. Upon activation by IL-4, Th2 T cells subsequently produce additional IL-4 in a positive feedback loop. IL-4 also stimulates B cell proliferation and class II MHC expression, inducing class switching from B cells to IgE and IgG1 expression. Recombinant human IL-4 suitable for use in the present invention is commercially available from several suppliers, including ProSpec-Tany TechnoGene Ltd., East Brunswick, NJ, USA (catalog number CYT-211) and ThermoFisher Scientific, Inc., Waltham, MA, USA (human IL-15 recombinant protein, catalog number Gibco CTP0043). The amino acid sequence of recombinant human IL-4 suitable for use in the present invention is shown in Table 2 (SEQ ID NO: 5).

[0854] The term "IL-7" (also referred to herein as "IL7") refers to a glycosylated tissue-derived cytokine known as interleukin-7, which can be obtained from stromal and epithelial cells, as well as dendritic cells. Fry and Mackall, Blood 2002, 99, 3892-904. IL-7 can stimulate T cell development. IL-7 binds to the IL-7 receptor, a heterodimer consisting of the IL-7 receptor alpha and the common gamma chain receptor, which is a series of signals important for T cell development in the thymus and survival in the periphery. Recombinant human IL-7 suitable for use in the present invention is commercially available from several suppliers, including ProSpec-Tany TechnoGene Ltd., East Brunswick, NJ, USA (catalog number CYT-254) and ThermoFisher Scientific, Inc., Waltham, MA, USA (human IL-15 recombinant protein, catalog number Gibco PHC0071). The amino acid sequence of a recombinant human IL-7 suitable for use in the present invention is shown in Table 2 (SEQ ID NO: 6).

[0855] The term "IL-15" (also referred to herein as "IL15") refers to the T-cell growth factor known as interleukin-15 and includes all forms of IL-2, including human and mammalian forms, conservative amino acid substitutions, glycoforms, biosimilars, and variants. IL-15 is described, for example, in Fehniger and Caligiuri, Blood 2001, 97, 14-32, the disclosure of which is incorporated herein by reference. IL-15 shares β and γ signaling receptor subunits with IL-2. Recombinant human IL-15 is a single, non-glycosylated polypeptide chain containing 114 amino acids (and an N-terminal methionine) with a molecular weight of 12.8 kDa. Recombinant human IL-15 is commercially available from several suppliers, including ProSpec-Tany TechnoGene Ltd., East Brunswick, NJ, USA (catalog number CYT-230-b) and ThermoFisher Scientific, Inc., Waltham, MA, USA (human IL-15 recombinant protein, catalog number 34-8159-82). The amino acid sequence of recombinant human IL-15 suitable for use in the present invention is shown in Table 2 (SEQ ID NO: 7).

[0856] The term "IL-21" (also referred to herein as "IL21") refers to the pleiotropic cytokine protein known as interleukin-21, and includes all forms of IL-21, including human and mammalian forms, conservative amino acid substitutions, glycoforms, biosimilars, and variants. IL-21 is described, for example, in Spolski and Leonard, Nat. Rev. Drug. Disc. 2014, 13, 379-95, the disclosure of which is incorporated herein by reference. IL-21 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 suppliers, 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).

[0857] When an "antitumor effective amount," "tumor inhibiting effective amount," or "therapeutic amount" is indicated, the exact amount of the composition of the present invention to be administered can be determined by a physician, taking into account individual differences in the age, weight, tumor size, extent of infection or metastasis, and health status of the patient (subject). Generally, 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 , 10 7 ~10 11 , 10 7 ~10 10 , 10 8 ~10 11 , 10 8 ~10 10 , 10 9 ~10 11 , or 10 9 ~10 10The TIL (optionally including genetically engineered TIL) compositions may be administered at doses of 1000-15000 cells (including all integer values ​​within those ranges). TIL (optionally including genetically modified cytotoxic lymphocytes) compositions may also be administered multiple times at these doses. TIL (optionally including genetically engineered TIL) can be administered by using injection techniques commonly known in immunotherapy (see, e.g., Rosenberg et al., New Eng. J. Med. 1988, 319, 1676). Optimal dosages and treatment regimes for a particular patient can be readily determined by one skilled in the medical arts by monitoring the patient for signs of disease and adjusting treatment accordingly.

[0858] The terms "hematological malignancies," "blood system malignancies," or terms of related meaning, refer to cancers and tumors of mammalian hematopoietic and lymphatic tissues, including, but not limited to, blood, bone marrow, lymph nodes, and lymphatic tissues. Hematological malignancies are also referred to as "liquid tumors." Hematological malignancies may include, but are not limited to, acute lymphoblastic leukemia (ALL), chronic lymphocytic lymphoma (CLL), small lymphocytic lymphoma (SLL), acute myeloid leukemia (AML), chronic myelogenous leukemia (CML), multiple myeloma, acute monocytic leukemia (AMoL), Hodgkin's lymphoma, and non-Hodgkin's lymphoma. The term "B-cell hematological malignancies" refers to hematological malignancies affecting B cells.

[0859] The term "liquid tumor" refers to an abnormal mass of cells that is fluid in nature. Liquid tumor cancers include, but are not limited to, leukemia, myeloma, and lymphoma, as well as other hematological malignancies. TILs obtained from liquid tumors may also be referred to herein as bone marrow-infiltrating lymphocytes (MILs). TILs obtained from liquid tumors, including those circulating in peripheral blood, may also be referred to herein as PBLs. The terms MILs, TILs, and PBLs are used interchangeably herein and differ only based on the tissue type from which the cells are derived.

[0860] The term "microenvironment" as used herein may refer to the solid or hematological tumor microenvironment as a whole, or to individual subsets of cells within the microenvironment. As used herein, the tumor microenvironment refers to a complex mixture of "cells, soluble factors, signaling molecules, extracellular matrix, and mechanical cues that promote neoplastic transformation, support tumor growth and invasion, protect tumors from host immunity, foster therapeutic resistance, and provide a niche for successful and dominant metastasis," as described in Swartz, et al., Cancer Res., 2012, 72, 2473. Tumors express antigens that are recognized by T cells, but tumor clearance by the immune system is rare due to immunosuppression by the microenvironment.

[0861] In one embodiment, the present invention includes a method of treating cancer with a TIL population, wherein the patient is pretreated with non-myeloablative chemotherapy prior to infusion of TILs according to the present invention. In some embodiments, a TIL population can be provided, and the patient is pretreated with non-myeloablative chemotherapy prior to infusion of TILs according to the present invention. In one embodiment, 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 one embodiment, 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.

[0862] Experimental findings indicate that lymphodepletion prior to adoptive transfer of tumor-specific T lymphocytes plays an important role in enhancing therapeutic efficacy by eliminating regulatory T cells and competing elements of the immune system ("cytokine sinks"). Accordingly, some embodiments of the present invention utilize a lymphodepletion step (sometimes referred to as "immunosuppressive conditioning") on patients prior to introducing the TILs of the present invention.

[0863] The term "effective amount" or "therapeutically effective amount" refers to an amount of a compound or combination of compounds described herein sufficient to achieve the intended use, including, but not limited to, disease treatment. A therapeutically effective amount may vary depending on the intended use (in vitro or in vivo), the subject and condition being treated (e.g., the subject's weight, age, and sex), the severity of the condition, or the method of administration. The term also applies to a dose that induces a specific response in target cells (e.g., reduced platelet adhesion and / or cell migration). The specific dose will vary depending on the particular compound selected, the dosing regimen to be followed, whether the compound is administered in combination with other compounds, the timing of administration, the tissue to which it is administered, and the physical delivery system by which the compound is delivered.

[0864] The terms "treatment," "treating," "treating," and the like refer to obtaining a desired pharmacological and / or physiological effect. The effect can be preventative, in terms of completely or partially preventing the disease or condition, and / or therapeutic, in terms of partially or completely curing the disease and / or side effects caused by the disease. "Treatment," as used herein, encompasses any treatment of disease in a mammal, particularly a human, and includes (a) preventing the disease from occurring in a subject who may be susceptible to the disease but has not yet been diagnosed with it; (b) suppressing the disease, i.e., arresting its onset or progression; and (c) palliating the disease, i.e., causing regression of the disease and / or alleviating one or more disease symptoms. "Treatment" is also intended to encompass the delivery of an agent to provide a pharmacological effect even in the absence of a disease or condition. For example, "treatment" encompasses the delivery of a composition capable of eliciting an immune response or conferring immunity in the absence of a pathology, e.g., in the case of a vaccine.

[0865] The term "heterologous," when used with reference to portions of a nucleic acid or protein, indicates that the nucleic acid or protein comprises two or more subsequences that are not found in the same relationship to each other in nature. For example, nucleic acids are typically produced recombinantly, with two or more sequences from unrelated genes arranged to create a new functional nucleic acid, e.g., a promoter from one source and a coding region from another source, or coding regions from different sources. Similarly, a heterologous protein indicates that the protein comprises two or more subsequences that are not found in the same relationship to each other in nature (e.g., a fusion protein).

[0866] The terms "sequence identity," "percent identity," and "percent sequence identity" (or their synonyms, e.g., "99% identical") in the context of two or more nucleic acids or polypeptides refer to two or more sequences or subsequences that are the same or have a specified percentage of the same nucleotide or amino acid residues when compared and aligned for maximum correspondence (introducing gaps, if necessary), without considering any conservative amino acid substitutions as part of the sequence identity. Percent identity can be measured using sequence comparison software or algorithms or by visual inspection. Various algorithms and software that can be used to obtain alignment of amino acid or nucleotide sequences are known in the art. Suitable programs for determining percent sequence identity include, for example, the BLAST suite of programs available from the BLAST website of the U.S. government's National Center for Biotechnology Information. Comparison between two sequences can be performed using either the BLASTN or BLASTP algorithm. BLASTN is used to compare nucleic acid sequences, and BLASTP is used to compare amino acid sequences. ALIGN, ALIGN-2 (Genentech, South San Francisco, California), or MegAlign, available from DNASTAR, are additional publicly available software programs that can be used to align sequences. Those skilled in the art can determine the appropriate parameters for maximum alignment depending on the particular alignment software. In certain embodiments, the default parameters of the alignment software are used.

[0867] As used herein, the term "variant" includes, but is not limited to, an antibody or fusion protein comprising an amino acid sequence that differs from the amino acid sequence of a reference antibody by one or more substitutions, deletions, and / or additions at specific positions within or adjacent to the amino acid sequence of the reference antibody. A variant may contain one or more conservative substitutions in its amino acid sequence compared to the amino acid sequence of the reference antibody. Conservative substitutions may include, for example, substitutions of similarly charged or uncharged amino acids. A variant retains the ability of the reference antibody to specifically bind to an antigen. The term variant also includes pegylated antibodies or proteins.

[0868] As used herein, "tumor infiltrating lymphocytes" or "TILs" refers to a population of cells that are initially acquired as leukocytes that have left a subject's bloodstream and migrated to a tumor. TILs include CD8 + Cytotoxic T cells (lymphocytes), Th1 and Th17 CD4 + These include, but are not limited to, T cells, natural killer cells, dendritic cells, and M1 macrophages. TILs include both primary and secondary TILs. "Primary TILs" are those obtained from a patient tissue sample as outlined herein (sometimes referred to as "freshly harvested"). "Secondary TILs" are any TIL cell populations that have been expanded or propagated as discussed herein, including, but not limited to, bulk TILs, expanded TILs ("REP TILs"), and "reREP TILs" as discussed herein. reREP TILs can include, for example, second expanded TILs or second additional expanded TILs (e.g., those described in step D of FIG. 8, including TILs designated as reREP TILs).

[0869] TILs can generally be defined either biochemically using cell surface markers or functionally by their ability to infiltrate tumors and achieve therapy. TILs can generally be classified by the expression of one or more of the following biomarkers: CD4, CD8, TCRαβ, CD27, CD28, CD56, CCR7, CD45Ra, CD95, PD-1, and CD25. Additionally or alternatively, TILs can be functionally defined by their ability to infiltrate solid tumors upon reintroduction into patients. TILs can be further characterized by efficacy; for example, TILs can be considered potent if their interferon (IFN) release is greater than about 50 pg / mL, greater than about 100 pg / mL, greater than about 150 pg / mL, or greater than about 200 pg / mL.

[0870] 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.

[0871] The term "RNA" defines a molecule containing at least one ribonucleotide residue. The term "ribonucleotide" defines a nucleotide having a hydroxyl group at the 2' position of a bD-ribofuranose moiety. The term RNA includes double-stranded RNA, single-stranded RNA, isolated RNA such as partially purified RNA, essentially pure RNA, synthetic RNA, recombinantly produced RNA, and altered RNA that differs from naturally occurring RNA by the addition, deletion, substitution, and / or alteration of one or more nucleotides. The nucleotides of the RNA molecules described herein can also include non-standard nucleotides, such as non-naturally occurring nucleotides or chemically synthesized nucleotides or deoxynucleotides. These altered RNAs may be referred to as analogs or analogs of naturally occurring RNA.

[0872] The term "pharmaceutically acceptable carrier" or "pharmaceutically acceptable excipient" is intended to include any and all solvents, dispersion media, coatings, antibacterial and antifungal agents, isotonic and absorption delaying agents, and inactive ingredients. The use of such pharmaceutically acceptable carriers or pharmaceutically acceptable excipients for active pharmaceutical ingredients is well known in the art. Except insofar as any conventional pharmaceutically acceptable carrier or pharmaceutically acceptable excipient is incompatible with the active pharmaceutical ingredient, its use in the therapeutic compositions of the invention is contemplated. Additional active pharmaceutical ingredients, such as other drugs, can also be incorporated into the compositions and methods described.

[0873] The terms "about" and "approximately" mean within a statistically significant range of values. Such a range may be within one order of magnitude of a given value or range, preferably within 50%, more preferably within 20%, even more preferably within 10%, and even more preferably within 5%. The acceptable deviation encompassed by the term "about" or "approximately" depends on the particular system under study and can be readily understood by one of ordinary skill in the art. Furthermore, as used herein, the terms "about" and "approximately" mean that dimensions, sizes, formulations, parameters, shapes, and other quantities and characteristics are not, and need not be, exact, but may be approximate and / or larger or smaller, as appropriate, reflecting tolerances, conversion factors, rounding, measurement errors, etc., and other factors known to those of ordinary skill in the art. In general, a dimension, size, formulation, parameter, shape, or other quantity or characteristic is "about" or "approximately" whether or not expressly stated as such. It should be noted that embodiments of widely different sizes, shapes, and dimensions may employ the described configurations.

[0874] When used in the appended claims, the transitional terms "comprising," "consisting essentially of," and "consisting of" define the claim in its original and amended form, in terms of whether additional, unrecited claim elements or steps, if any, are excluded from the 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 element, step, or material other than those specified in the claim, and in the latter case, also excludes impurities normally associated with the specified material(s). The term "consisting essentially of" limits the claim to the specified element, step, or material(s) and does not materially affect the basic and novel feature(s) of the claimed invention. All compositions, methods, and kits described herein embodying the present invention may, in alternative embodiments, be more specifically defined by any of the transitional terms "comprising," "essentially consisting of," and "consisting of."

[0875] 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 VL is composed of three CDRs and four FRs arranged from the amino terminus to the carboxy terminus in the following order: FR1, CDR1, FR2, CDR2, FR3, CDR3, FR4. The variable regions of the heavy and light chains contain binding domains that interact with one or more antigen epitopes. The constant region of the antibody may mediate the binding of the immunoglobulin to host tissues or factors, including various cells of the immune system (e.g., effector cells) and the first component (Clq) of the classical complement system.

[0876] 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.

[0877] The terms "monoclonal antibody," "mAb," "monoclonal antibody composition," or their plurals, refer to a preparation of antibody molecules of single molecular composition. A monoclonal antibody composition exhibits a single binding specificity and affinity for a particular epitope. Monoclonal antibodies specific for a particular receptor can be produced using knowledge and techniques in the art by injecting a test subject with an appropriate antigen and then isolating hybridomas expressing antibodies with the desired sequence or functional characteristics. DNA encoding the monoclonal antibody is readily isolated and sequenced using conventional procedures (e.g., by using oligonucleotide probes that can specifically bind to genes encoding the heavy and light chains of the monoclonal antibody). Hybridoma cells serve as a preferred source of such DNA. Once isolated, the DNA can be placed into an expression vector and then transfected into host cells such as E. coli cells, monkey COS cells, Chinese hamster ovary (CHO) cells, or myeloma cells that do not produce immunoglobulin protein to obtain the synthesis of the monoclonal antibody in the recombinant host cells. Recombinant production of antibodies is described in more detail below.

[0878] As used herein, the term "antigen-binding portion" or "antigen-binding fragment" of an antibody (or simply "antibody portion" or "fragment") refers to one or more fragments of an antibody that retain the ability to specifically bind to an antigen. It has been shown that the antigen-binding function of an antibody can be performed by fragments of a full-length antibody. Examples of binding fragments encompassed by the term "antigen-binding portion" of an antibody include: (i) V L , V H , C L (ii) a Fab fragment, which is a monovalent fragment consisting of two Fab fragments linked by a disulfide bridge at the hinge region; (iii) a V H and an Fd fragment consisting of the CH1 domain, (iv) a V of a single arm of an antibody L and V H Fv fragment consisting of domains, (v) V H or VL and (v) isolated complementarity-determining regions (CDRs). L and V H are encoded by separate genes, which can be synthesized using recombinant methods. L and V H The domains may be joined by a synthetic linker that allows them to pair as a single protein chain to form a monovalent molecule known as a single-chain Fv (scFv) (see, e.g., Bird, et al., Science 1988, 242, 423-426, and Huston, et al., Proc. Natl. Acad. Sci. USA 1988, 85, 5879-5883). Such scFv antibodies are also intended to be encompassed by the term "antigen-binding portion" or "antigen-binding fragment" of an antibody. These antibody fragments are obtained using conventional techniques known to those skilled in the art, and the fragments are screened for utility in the same manner as intact antibodies. In one embodiment, the scFv protein domain comprises a V H Part and V L The scFv molecule contains a V L If the domain is the N-terminal portion of the scFv molecule, V L -LV H , or V H If the domain is the N-terminal portion of the scFv molecule, V H -LV L Methods for producing scFv molecules and designing suitable peptide linkers are described in U.S. Pat. Nos. 4,704,692, 4,946,778, R. Raag and M. Whitlow, "Single Chain Fvs," FASEB Vol. 9:73-80 (1995), and RE Bird and BW Walker, Single Chain Antibody Variable Regions, TIBTECH, Vol. 9:132-137 (1991), the disclosures of which are incorporated herein by reference.

[0879] As used herein, the term "human antibody" is intended to include antibodies having variable regions in which both the framework and CDR regions are derived from human germline immunoglobulin sequences. Furthermore, if the antibody contains a constant region, the constant region also is derived from human germline immunoglobulin sequences. The human antibodies of the invention may include amino acid residues not encoded by human germline immunoglobulin sequences (e.g., mutations introduced by random or site-specific mutagenesis in vitro or by somatic mutation in vivo). As used herein, the term "human antibody" is not intended to include antibodies in which CDR sequences derived from the germline of another mammalian species, such as a mouse, have been grafted onto human framework sequences.

[0880] The term "human monoclonal antibody" refers to antibodies displaying a single binding specificity which have variable regions in which both the framework and CDR regions are derived from human germline immunoglobulin sequences. In 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.

[0881] 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) to thereby modify the V sequences of the recombinant antibodies. H and V L The amino acid sequence of the region is human germline V H and V L These are sequences that are derived from and related to sequences, but may not naturally occur within the human antibody germline repertoire in vivo.

[0882] As used herein, "isotype" refers to the antibody class (e.g., IgM or IgG1) that is encoded by heavy chain constant region genes.

[0883] 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."

[0884] 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.

[0885] The terms "humanized antibody," "humanized antibodies," and "humanization" are intended to refer to antibodies in which CDR sequences derived from the germline of another mammalian species, such as mouse, have been grafted onto human framework sequences. Additional framework region modifications can be made within the human framework sequences. Humanized forms of non-human (e.g., murine) antibodies are chimeric antibodies that contain minimal sequence derived from non-human immunoglobulin. In most cases, humanized antibodies are human immunoglobulins (recipient antibodies) in which residues from a hypervariable region of the recipient are replaced by residues from a hypervariable region of a non-human species (donor antibody) such as mouse, rat, rabbit, or non-human primate having the desired specificity, affinity, and capacity. In some cases, Fv framework (FR) residues of the human immunoglobulin are replaced by corresponding non-human residues. Furthermore, humanized antibodies may contain residues that are not found in the recipient or donor antibody. These modifications are made to further refine antibody performance. Generally, a humanized antibody will comprise substantially all of at least one, and typically two, variable domains, in which all or substantially all of the hypervariable loops correspond to those of a non-human immunoglobulin and all or substantially all of the FR regions are those of a human immunoglobulin sequence. The humanized antibody will also optionally comprise at least a portion of an immunoglobulin constant region (Fc), typically that of a human immunoglobulin. For further details, see Jones, et al., Nature 1986, 321, 522-525; Riechmann, et al., Nature 1988, 332, 323-329; and Presta, Curr. Op. Struct. Biol. 1992, 2, 593-596. The antibodies described herein can also be modified to use any Fc variant known to confer improved (e.g., reduced) effector function and / or FcR binding.Fc variants are described in, for example, International Patent Application Publication Nos. WO1988 / 07089A1, WO1996 / 14339A1, WO1998 / 05787A1, WO1998 / 23289A1, WO1999 / 51642A1, WO99 / 58572A1, WO2000 / 09560A2, WO2000 / 32767A1, WO2000 / 42072A2, and WO2002 / 4 4215A2, WO2002 / 060919A2, WO2003 / 074569A2, WO2004 / 016750A2, WO2004 / 029207A2, WO2004 / 03 5752A2, WO2004 / 063351A2, WO2004 / 074455A2, WO2004 / 099249A2, WO2005 / 040217A2, WO2005 / 07 0963A1, WO2005 / 077981A2, WO2005 / 092925A2, WO2005 / 123780A2, WO2006 / 019447A1, WO2006 / 047350A2, and WO2006 / 085967A2, as well as U.S. Pat. Nos. 5,648,260, 5,739,277, 5,834,250, 5,869,046, 6,096 ,871, 6,121,022, 6,194,551, 6,242,195, 6,277,375, 6,528,624, 6,538,124, 6,737,056, 6,821,505, 6,998,253, and 7,083,784 (the disclosures of which are incorporated herein by reference).

[0886] 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.

[0887] A "diabody" is a small antibody fragment that has two antigen-binding sites. The fragments bind to the same polypeptide chain (VH -V L or V L -V H ) in the light chain variable domain (V L ) connected to the heavy chain variable domain (V H ). When a linker that is too short to pair the two domains on the same chain is used, the domains are forced to pair with complementary domains on another chain and create two antigen-binding sites. Bispecific antibodies are more fully described, 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.

[0888] The term "glycosylation" refers to modified derivatives of antibodies. An aglycosylated antibody lacks glycosylation. Glycosylation can be altered to, for example, increase the affinity of 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 that site. As described in U.S. Pat. Nos. 5,714,350 and 6,350,861, aglycosylation may 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 present invention, thereby producing antibodies with altered glycosylation. For example, the cell lines Ms704, Ms705, and Ms709 lack the fucosyltransferase gene FUT8 (alpha(1,6) fucosyltransferase), such that antibodies expressed in these cell lines lack fucose on their carbohydrates. The Ms704, Ms705, and Ms709 FUT8- / - cell lines were generated by targeted disruption of the FUT8 gene in CHO / DG44 cells using two replacement vectors (see, e.g., U.S. Patent Publication No. 2004 / 0110704 or Yamane-Ohnuki, et al., Biotechnol. Bioeng., 2004, 87, 614-622).As another example, European Patent No. EP 1,176,195 describes cell lines with a functionally disrupted FUT8 gene encoding a fucosyltransferase, thereby resulting in antibodies expressed in such cell lines exhibiting hypofucosylation by reducing or eliminating alpha-1,6 bond-related enzymes. It also describes cell lines with reduced or no enzymatic activity for adding fucose to N-acetylglucosamine linked to the Fc region of antibodies, such as the rat myeloma cell line YB2 / 0 (ATCC CRL 1662). International Patent Publication No. WO 03 / 035835 describes a variant CHO cell line, Lec13 cells, that has a reduced ability to attach fucose to Asn(297)-linked carbohydrates, which also results in hypofucosylation of antibodies expressed in the host cells (see also Shields, et al., J. Biol. Chem. 2002, 277, 26733-26740). International Patent Publication No. WO 99 / 54342 describes cell lines engineered to express glycoprotein-modifying glycosyltransferases (e.g., beta(1,4)-N-acetylglucosaminyltransferase III (GnTIII)), such that antibodies expressed in the engineered cell lines exhibit increased bisecting GlcNac structures, resulting in increased ADCC activity of the antibodies (see also Umana, et al., Nat. Biotech. 1999, 17, 176-180). Alternatively, fucosidase enzymes can be used to cleave fucose residues from antibodies. For example, the fucosidase alpha-L-fucosidase removes fucosyl residues from antibodies, as described in Tarentino, et al., Biochem. 1975, 14, 5516-5523.

[0889] "PEGylation" refers to a modified antibody or fragment thereof that has been reacted with polyethylene glycol (PEG), such as a reactive ester or aldehyde derivative of PEG, under conditions that result in one or more PEG groups being attached to the antibody or antibody fragment. PEGylation can, for example, increase the biological (e.g., serum) half-life of the antibody. Preferably, PEGylation is carried out via an acylation reaction or an alkylation reaction with a reactive PEG molecule (or an analogous reactive water-soluble polymer). As used herein, the term "polyethylene glycol" refers to a mono (C1-C 10 PEG is intended to encompass any of the forms of PEG that have been used to derivatize other proteins, such as alkoxy- or aryloxy-polyethylene glycol, or polyethylene glycol-maleimide. The antibody to be pegylated may be an aglycosylated antibody. Methods for pegylation are known in the art and can be applied to the antibodies of the invention, for example, as described in European Patent Nos. EP 0 154 316 and EP 0 401 384, and U.S. Pat. No. 5,824,778 (the disclosures of each of which are incorporated herein by reference).

[0890] The term "biosimilar" refers to a biological product that is highly similar to a reference biological product approved in the United States, despite minor differences in clinically inactive components, including monoclonal antibodies or proteins, and that has no clinically meaningful differences between the biological product and the reference product in terms of product safety, purity, and potency. Furthermore, a similar biological or "biosimilar" drug is a biological product similar to another biological product already approved for use by the European Medicines Agency. The term "biosimilar" is also used interchangeably by regulatory agencies in other countries and regions. Biological products or biological products are medicines made by or derived from biological sources, such as bacteria or yeast. They can consist of relatively small molecules, such as human insulin or erythropoietin, or complex molecules, such as monoclonal antibodies. For example, if the reference IL-2 protein is aldesleukin (proleukin), a protein approved by a drug regulatory agency for aldesleukin is a "biosimilar" of aldesleukin or a "biosimilar of" aldesleukin. In Europe, a similar biological or "biosimilar" medicinal product is a biological product similar to another biological product already authorized for use by the European Medicines Agency (EMA). The legal basis for similar biological uses in Europe is Article 6 of Regulation (EC) No. 726 / 2004, as amended, and Article 10(4) of Directive 2001 / 83 / EC. Therefore, in Europe, biosimilars may be authorized or approved for authorization or licensing purposes under Article 6 of Regulation (EC) No. 726 / 2004 and Article 10(4) of Directive 2001 / 83 / EC. The original biological product already authorized is sometimes referred to as the "reference medicinal product" in Europe. Some of the requirements for a product to be considered a biosimilar are outlined in the CHMP guideline on biosimilar medicinal products. Additionally, product-specific guidelines, including those related to monoclonal antibody biosimilars, are provided by the EMA on a product-by-product basis and are available on its website.Biosimilars described herein may be similar to the reference medicinal product in terms of quality characteristics, biological activity, mechanism of action, safety profile, and / or efficacy. Furthermore, biosimilars may be used or intended for use to treat the same condition as the reference medicinal product. Thus, biosimilars described herein may be considered to have similar or very similar quality characteristics to the reference medicinal product. Alternatively, or in addition, biosimilars described herein may be considered to have similar or very similar biological activity to the reference medicinal product. Alternatively, or in addition, biosimilars described herein may be considered to have a similar or very similar safety profile to the reference medicinal product. Alternatively, or in addition, biosimilars described herein may be considered to have similar or very similar efficacy to the reference medicinal product. As described herein, biosimilars in Europe are compared to reference medicinal products authorized by the EMA. However, in some cases, biosimilars may be compared in specific studies to biopharmaceuticals authorized outside the European Economic Area (non-EEA-authorized "comparators"). Such studies include, for example, specific clinical studies and in vivo nonclinical studies. As used herein, the term "biosimilar" also refers to a biopharmaceutical that has been or can be compared to a non-EEA-approved comparator. Particular biosimilars are proteins, such as antibodies, antibody fragments (e.g., antigen-binding portions), and fusion proteins. Protein biosimilars may have amino acid sequences with minor modifications to the amino acid structure (e.g., including amino acid deletions, additions, and / or substitutions) that do not significantly affect the function of the polypeptide. A biosimilar may have an amino acid sequence that has 97% or more sequence identity, e.g., 97%, 98%, 99%, or 100%, to the amino acid sequence of its reference pharmaceutical. A biosimilar may include one or more post-translational modifications, such as, but not limited to, glycosylation, oxidation, deamidation, and / or cleavage, that differ from the post-translational modifications of the reference pharmaceutical, provided that the differences do not result in a change in the safety and / or efficacy of the pharmaceutical. A biosimilar may have the same or a different glycosylation pattern as the reference pharmaceutical, provided that the differences do not result in a change in the safety and / or efficacy of the pharmaceutical.In particular, but not exclusively, biosimilars may have different glycosylation patterns if the differences address or are intended to address safety concerns associated with the reference drug. Additionally, biosimilars may deviate from the reference drug, for example, in its strength, dosage form, formulation, excipients, and / or presentation, provided that the drug's safety and efficacy are not compromised. Biosimilars may contain differences, for example, in their pharmacokinetic (PK) and / or pharmacodynamic (PD) profiles, compared to the reference drug, but are still considered sufficiently similar to the reference drug to be approved or deemed suitable for approval. In certain circumstances, biosimilars exhibit different binding characteristics compared to the reference drug, and these different binding characteristics are not considered by regulatory authorities, such as the EMA, to be a barrier to approval as a similar biological product. The term "biosimilar" is also used interchangeably by regulatory agencies in other countries and regions.

[0891] III. Gen2 TIL Manufacturing Process An exemplary family of TIL processes known as Gen2 (also known as Process 2A) that includes some of these features is shown in Figures 1 and 2. An embodiment of Gen2 is shown in Figure 2.

[0892] As discussed herein, the present invention may include steps related to restimulating cryopreserved TILs prior to transplantation into a patient to improve their metabolic activity, and thus their relative health, and methods for testing said metabolic health. As generally outlined herein, TILs are generally harvested from a patient sample and engineered to expand their numbers prior to transplantation into a patient. In some embodiments, TILs may optionally be genetically engineered, as discussed below.

[0893] In some embodiments, TILs can be cryopreserved, and upon thawing, they can be restimulated to enhance their metabolism before infusion into patients.

[0894] In some embodiments, as discussed in detail below and in the Examples and Figures, the first expansion (including the process referred to as pre-REP and the process shown as step A in FIG. 1 ) is shortened to 3-14 days, and the second expansion (including the process referred to as REP and the process shown as step B in 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, as discussed in detail below and in the Examples and Figures, the combined first and second expansion (e.g., the expansion described as steps B and D in FIG. 1 ) is shortened to 22 days.

[0895] 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.

[0896] A. Step A: Obtaining a patient tumor sample Generally, TILs are initially obtained from a patient tumor sample, then expanded into larger populations for further manipulation as described herein, optionally cryopreserved, restimulated as outlined herein, and optionally assessed for phenotypic and metabolic parameters as indicators of TIL health.

[0897] Patient tumor samples can be obtained using methods known in the art, generally via surgical resection, needle biopsy, core biopsy, minibiopsy, 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, minibiopsy, 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. Tumor samples can be liquid tumors, such as tumors obtained from hematological malignancies. Solid tumors can be from lung tissue. In some embodiments, useful TILs are obtained from non-small cell lung cancer (NSCLC).

[0898] Once obtained, tumor samples are generally cut into sections of 1 to approximately 8 mm using sharp dissection. 3 fragmented into small pieces of about 2-3 mm 3are particularly useful. In some embodiments, TILs are cultured from these fragments using an enzymatic tumor digest. Such a tumor digest can be produced by incubation in an enzymatic medium (e.g., Roswell Park Memorial Institute (RPMI) 1640 buffer, 2 mM glutamic acid, 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 the enzymatic medium, mechanically dissociating the tumor for approximately 1 minute, followed by incubation at 37°C in 5% CO2 for 30 minutes, and then repeating cycles of mechanical dissociation and incubation under the aforementioned conditions until only small tissue fragments are present. At the end of this process, if the cell suspension contains a large number of red blood cells or dead cells, density gradient separation using FICOLL branched hydrophilic polysaccharides can be performed to remove these cells. Alternative methods known in the art can be used, such as those described in U.S. Patent Application Publication No. 2012 / 0244133A1, the disclosure of which is incorporated herein by reference. Any of the foregoing methods can be used in any of the embodiments described herein for methods of expanding TILs or methods of treating cancer.

[0899] The tumor dissociation enzyme mixture can include one or more dissociation (digestion) enzymes, such as, but not limited to, collagenase (including any blend or type of collagenase), Accutase™, Accumax™, hyaluronidase, neutral protease (dispase), chymotrypsin, chymopapain, trypsin, caseinase, elastase, papain, type XIV protease (pronase), deoxyribonuclease I (DNase), trypsin inhibitor, any other dissociation enzyme or proteolytic enzyme, and any combination thereof.

[0900] In some embodiments, the dissociation enzyme is reconstituted from a lyophilized enzyme, hi some embodiments, the lyophilized enzyme is reconstituted with an amount of a sterile buffer, such as HBSS.

[0901] In some cases, collagenase (such as animal-free type 1 collagenase) is reconstituted in 10 mL of sterile HBSS or another buffer. Lyophilized stock enzyme may be at a concentration of 2892 PZ U / vial. In some embodiments, collagenase is reconstituted in 5 mL to 15 mL of buffer. In some embodiments, the collagenase stock after reconstitution is in the range of about 100 U / mL to about 400 U / mL, e.g., about 100 U / mL to about 400 U / mL, about 100 U / mL to about 350 U / mL, about 100 U / mL to about 300 U / mL, about 150 U / mL to about 400 U / mL, about 100 U / mL, about 150 U / mL, about 200 U / mL, about 210 U / mL, about 220 U / mL, about 230 U / mL, about 240 U / mL, about 250 U / mL, about 260 U / mL, about 270 U / mL, about 280 U / mL, about 289.2 U / mL, about 300 U / mL, U / mL, about 350 PZ U / mL, or about 400 PZ U / mL.

[0902] In some embodiments, the neutral protease is reconstituted in 1 mL of sterile HBSS or another buffer. The lyophilized stock enzyme may be at a concentration of 175 DMCU / vial. In some embodiments, the neutral protease stock after reconstitution may have a concentration in the range of about 100 DMC / mL to about 400 DMC / mL, e.g., about 100 DMC / mL to about 400 DMC / mL, about 100 DMC / mL to about 350 DMC / mL, about 100 DMC / mL to about 300 DMC / mL, about 150 DMC / mL to about 400 DMC / mL, about 100 DMC / mL, about 110 DMC / mL, or about 120 DMC / mL. C / mL, about 120 DMC / mL, about 130 DMC / mL, about 140 DMC / mL, about 150 DMC / mL, about 160 DMC / mL, about 170 DMC / mL, about 175 DMC / mL, about 180 DMC / mL, about 190 DMC / mL, about 200 DMC / mL, about 250 DMC / mL, about 300 DMC / mL, about 350 DMC / mL, or about 400 DMC / mL.

[0903] In some embodiments, DNAse I is reconstituted in 1 mL of sterile HBSS or another buffer. The lyophilized stock enzyme was at a concentration of 4 KU / vial. In some embodiments, the reconstituted DNase I stock ranges from about 1 KU / mL to 10 KU / mL, e.g., about 1 KU / mL, about 2 KU / mL, about 3 KU / mL, about 4 KU / mL, about 5 KU / mL, about 6 KU / mL, about 7 KU / mL, about 8 KU / mL, about 9 KU / mL, or about 10 KU / mL.

[0904] In some embodiments, enzyme stocks are variable and concentrations may need to be determined. In some embodiments, the concentration of the lyophilized stock can be verified. In some embodiments, the final amount of enzyme added to the digestion cocktail is adjusted based on the determined stock concentration.

[0905] In some embodiments, the enzyme mixture comprises about 10.2 ul of neutral protease (0.36 DMC U / mL), 21.3 μL of collagenase (1.2 PZ / mL), and 250 ul of DNAse I (200 U / mL) in about 4.7 ml of sterile HBSS.

[0906] As noted above, in some embodiments, the TILs are derived from solid tumors. In some embodiments, the solid tumor is not fragmented. In some embodiments, the solid tumor is not fragmented and is subjected to enzymatic digestion as a whole tumor. In some embodiments, the tumor is digested in an enzyme mixture comprising collagenase, DNase, and hyaluronidase. In some embodiments, the tumor is digested in an enzyme mixture comprising collagenase, DNase, and hyaluronidase for 1-2 hours. In some embodiments, the tumor is digested in an enzyme mixture comprising collagenase, DNase, and hyaluronidase for 1-2 hours at 37°C and 5% CO2. In some embodiments, the tumor is digested in an enzyme mixture comprising collagenase, DNase, and hyaluronidase for 1-2 hours at 37°C and 5% CO2 with rotation. In some embodiments, the tumor is digested overnight with constant rotation. In some embodiments, the tumor is digested overnight at 37°C and 5% CO2 with constant rotation. In some embodiments, the whole tumor is combined with the enzymes to form a tumor digestion reaction mixture.

[0907] In some embodiments, the tumor is reconstituted with lyophilized enzyme in a sterile buffer. In some embodiments, the buffer is sterile HBSS.

[0908] In some embodiments, the enzyme mixture includes collagenase. In some embodiments, the collagenase is collagenase IV. In some embodiments, the working stock of collagenase is a 10x working stock of 100 mg / mL.

[0909] In some embodiments, the enzyme mixture comprises DNAse, hi some embodiments, the working stock of DNAse is a 10x working stock of 10,000 IU / mL.

[0910] In some embodiments, the enzyme mixture comprises hyaluronidase. In some embodiments, the working stock of hyaluronidase is a 10x working stock of 10 mg / mL.

[0911] In some embodiments, the enzyme mixture comprises 10 mg / mL collagenase, 1000 IU / mL DNAse, and 1 mg / mL hyaluronidase.

[0912] In some embodiments, the enzyme mixture comprises 10 mg / mL collagenase, 500 IU / mL DNAse, and 1 mg / mL hyaluronidase.

[0913] Generally, the harvested cell suspension is referred to as a "primary cell population" or "freshly harvested" cell population.

[0914] In some embodiments, fragmentation comprises physical fragmentation, including, for example, dissection and digestion. In some embodiments, fragmentation is physical fragmentation. In some embodiments, fragmentation is by dissection. In some embodiments, fragmentation is by digestion. In some embodiments, TILs can be initially cultured from enzymatic tumor digests and tumor fragments obtained from a patient. In some embodiments, TILs can be initially cultured from enzymatic tumor digests and tumor fragments obtained from a patient.

[0915] 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 undergoes physical fragmentation. In some embodiments, fragmentation occurs before cryopreservation. In some embodiments, fragmentation occurs after cryopreservation. In some embodiments, fragmentation occurs after obtaining the tumor in the absence of any cryopreservation. In some embodiments, the tumor is fragmented and 10, 20, 30, 40 or more fragments or pieces are placed in each container for the first expansion. In some embodiments, the tumor is fragmented and 30 or 40 fragments or pieces are placed in each container for the first expansion. In some embodiments, the tumor is fragmented and 40 fragments or pieces are placed in each container for the first expansion. In some embodiments, the plurality of fragments comprises about 4 to about 50 fragments, each fragment being about 27 mm. 3 In some embodiments, the plurality of pieces has a volume of about 1300 mm 3 ~about 1500mm 3 In some embodiments, the plurality of pieces comprises about 30 to about 60 pieces with a total volume of about 1350 mm 3 In some embodiments, the plurality of fragments comprises about 50 fragments with a total mass of about 1 gram to about 1.5 grams. In some embodiments, the plurality of fragments comprises about 4 fragments.

[0916] In some embodiments, the TILs are obtained from tumor fragments. In some embodiments, the tumor fragments are obtained by sharp dissection. In some embodiments, the tumor fragments are approximately 1 mm 3 ~10mm 3 In some embodiments, the tumor fragment is about 1 mm 3 ~8mm 3 In some embodiments, the tumor fragment is about 1 mm 3 In some embodiments, the tumor fragment is about 2 mm 3 In some embodiments, the tumor fragment is about 3 mm 3 In some embodiments, the tumor fragment is about 4 mm 3In some embodiments, the tumor fragment is about 5 mm 3 In some embodiments, the tumor fragment is about 6 mm 3 In some embodiments, the tumor fragment is about 7 mm 3 In some embodiments, the tumor fragment is about 8 mm 3 In some embodiments, the tumor fragment is about 9 mm 3 In some embodiments, the tumor fragment is about 10 mm 3 In some embodiments, the tumor is 1-4 mm x 1-4 mm x 1-4 mm. In some embodiments, the tumor is 1 mm x 1 mm x 1 mm. In some embodiments, the tumor is 2 mm x 2 mm x 2 mm. In some embodiments, the tumor is 3 mm x 3 mm x 3 mm. In some embodiments, the tumor is 4 mm x 4 mm x 4 mm.

[0917] 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.

[0918] In some embodiments, tumor fragmentation is performed to maintain the internal structure of the tumor. In some embodiments, tumor fragmentation is performed without sawing with a scalpel. In some embodiments, TILs are obtained from tumor digests. In some embodiments, tumor digests are generated by incubation in an enzyme medium, such as, but not limited to, RPMI 1640 (2 mM GlutaMAX, 10 mg / mL gentamicin, 30 U / mL DNase, and 1.0 mg / mL collagenase), followed by mechanical dissociation (GentleMACS, Miltenyi Biotec, Auburn, CA). After placing the tumor in the enzyme medium, the tumor may be mechanically dissociated for approximately 1 minute. The solution may then be incubated at 37°C in 5% CO2 for 30 minutes, after which it may be mechanically disrupted again for approximately 1 minute. After again incubating at 37°C in 5% CO2 for 30 minutes, the tumor may be mechanically disrupted a third time for approximately 1 minute. In some embodiments, if large tissue fragments were present, one or two additional rounds of mechanical dissociation were applied to the sample after the third mechanical disruption, with incubation for an additional 30 minutes at 37° C. in 5% CO In some embodiments, if the cell suspension contains a large number of red blood cells or dead cells, density gradient separation using Ficoll can be performed at the end of the final incubation to remove these cells.

[0919] 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.

[0920] In some embodiments, the cells may be optionally frozen after sampling and cryopreserved before undergoing expansion as described in step B, which is described in more detail below and also illustrated in FIG. 1.

[0921] 1. Pleural fluid T cells and TILs In some embodiments, the sample is a pleural fluid sample. In some embodiments, the source of T cell TILs for expansion by the processes described herein is a pleural fluid sample. In some embodiments, the sample is a pleural fluid-derived sample. In some embodiments, the source of TILs for expansion by the processes described herein is a pleural fluid-derived sample. See, e.g., the methods described in U.S. Patent Publication No. 2014 / 0295426, which is incorporated by reference in its entirety for all purposes.

[0922] In some embodiments, any pleural fluid or effusion that appears to be and / or contains TILs can be utilized. Such samples can be derived from primary or metastatic lung cancer, such as NSCLC or SCLC. In some embodiments, the sample can be secondary metastatic cancer cells from another organ, such as the breast, ovary, colon, or prostate. In some embodiments, the sample used in the expansion methods described herein is a pleural effusion. In some embodiments, the sample used in the expansion methods described herein is a pleural transudate. Other biological samples can include other serous fluids containing TILs, including, for example, ascites from the abdomen or pancreatic cyst fluid. Ascites and pleural fluids have very similar chemical systems; both the abdomen and lungs have mesothelial lines and fluid forms in the pleural and abdominal spaces of the same substance in malignant tumors; in some embodiments, such fluids contain TILs. In some embodiments, where the present disclosure exemplifies pleural effusion, the same method can be performed with similar results using pleural fluid or other cyst fluids containing TILs.

[0923] In some embodiments, the pleural fluid is in an unprocessed form removed directly from the patient. In some embodiments, the unprocessed pleural fluid is placed in a standard collection tube, such as an EDTA or heparin tube, before the contacting step. In some embodiments, the unprocessed pleural fluid is placed in a standard CellSave® tube (Veridex) before the contacting step. In some embodiments, the sample is placed in a CellSave tube immediately after collection from the patient to avoid a decrease in the number of viable TILs. The number of viable TILs can decrease significantly within 24 hours if left in unprocessed pleural fluid, even at 4°C. In some embodiments, the sample is placed in an appropriate collection tube within 1 hour, 5 hours, 10 hours, 15 hours, or up to 24 hours after removal from the patient. In some embodiments, the sample is placed in an appropriate collection tube at 4°C within 1 hour, 5 hours, 10 hours, 15 hours, or up to 24 hours after removal from the patient.

[0924] In some embodiments, a pleural fluid sample from a selected subject may be diluted. In one embodiment, the dilution is 1:10 pleural fluid to diluent. In another embodiment, the dilution is 1:9 pleural fluid to diluent. In another embodiment, the dilution is 1:8 pleural fluid to diluent. In another embodiment, the dilution is 1:5 pleural fluid to diluent. In another embodiment, the dilution is 1:2 pleural fluid to diluent. In another embodiment, the dilution is 1:1 pleural fluid to diluent. In some embodiments, the diluent includes saline, phosphate-buffered saline, another buffer, or a physiologically acceptable diluent. In some embodiments, the sample is placed in a CellSave tube immediately after collection and dilution from the patient to avoid a significant loss of viable TILs, which can occur within 24 to 48 hours if left in unprocessed pleural fluid, even at 4°C. In some embodiments, the pleural fluid sample is placed into a suitable collection tube within 1 hour, 5 hours, 10 hours, 15 hours, 24 hours, 36 hours, or up to 48 hours after removal from the patient and dilution. In some embodiments, the pleural fluid sample is placed into a suitable collection tube within 1 hour, 5 hours, 10 hours, 15 hours, 24 hours, 36 hours, or up to 48 hours after removal from the patient and dilution at 4° C.

[0925] In yet another embodiment, the pleural fluid sample is concentrated by conventional means prior to further processing steps. In some embodiments, this pretreatment of the pleural fluid is preferred in situations where the pleural fluid must be cryopreserved (e.g., more than 24-48 hours after collection) for transport to the laboratory where the method will be performed or for subsequent analysis. In some embodiments, the pleural fluid sample is prepared by centrifuging the pleural fluid sample after it is collected from the subject and resuspending the centrate or pellet in a buffer. In some embodiments, the pleural fluid sample is subjected to multiple centrifugations and resuspensions before being cryopreserved for transport or subsequent analysis and / or processing.

[0926] In some embodiments, the pleural fluid sample is concentrated prior to further processing steps by using a filtration method. In some embodiments, the pleural fluid sample used in the contacting step is prepared by filtering the fluid through a filter containing a known, essentially uniform pore size that allows pleural fluid to pass through the membrane but retains tumor cells. In some embodiments, the pore diameter of the membrane can be at least 4 μM. In other embodiments, the pore diameter can be 5 μM or greater, and in other embodiments, 6, 7, 8, 9, or 10 μM. After filtration, the TIL-containing cells retained by the membrane can be rinsed from the membrane into an appropriate physiologically acceptable buffer. The thus-enriched TIL-containing cells can then be used in the contacting step of the method.

[0927] In some embodiments, a pleural fluid sample (e.g., including unprocessed pleural fluid), diluted pleural fluid, or a resuspended cell pellet is contacted with a lysis reagent that specifically lyses nonnucleated red blood cells present in the sample. In some embodiments, this step is performed before further processing steps in situations where the pleural fluid contains a significant number of RBCs. Suitable lysis reagents include a single lysis reagent or a lysis reagent and a quenching reagent, or a lysis agent, a quenching reagent, and a fixation reagent. Suitable lysis systems are commercially available and include the BD Pharm Lyse™ system (Becton Dickenson). Other lysis systems include the Versalyse™ system, the FACSlyse™ system (Becton Dickenson), the Immunoprep™ system, or the Erythrolyse II system (Beckman Coulter, Inc.), or an ammonium chloride system. In some embodiments, the lysis reagent can vary depending on key requirements, such as efficient lysis of red blood cells and preservation of TILs and their phenotypic characteristics in the pleural fluid. In addition to utilizing a single reagent for lysis, lysis systems useful in the methods described herein can include a second reagent, e.g., one that quenches or delays the effect of the lysis reagent during the remaining steps of the method, such as Stabilyse™ reagent (Beckman Coulter, Inc.) Depending on the choice of lysis reagent or the preferred implementation of the method, conventional fixative reagents can also be used.

[0928] In some embodiments, pleural fluid samples that have been unprocessed, diluted, or multiple centrifuged or processed as described herein are stored frozen at a temperature of about -140°C before being further processed and / or expanded as provided herein.

[0929] B. Step B: First Expansion In some embodiments, the methods provide for obtaining young TILs that are capable of increasing their replication cycle upon administration to a subject / patient and may therefore provide additional therapeutic benefit over older TILs (i.e., TILs that have undergone more rounds of replication prior to administration to a subject / patient). The characteristics of young TILs are described in the literature, for example, Donia, et al., Scand. J. Immunol. 2012, 75, 157-167, Dudley, et al., Clin. al.,Clin.Cancer Res.2013,19,OF1-OF9, Besser,et al.,J.Immunother.2009,32,415-423,Robbins,et al.,J.Immunol.2004,173,7125-7130,Shen,et al. al.,J.Immunother.,2007,30,123-129, Zhou,et al., J. Immunother. 2005, 28, 53-62, and Tran, et al., J. Immunother., 2008, 31, 742-751, the disclosures of each of which are incorporated herein by reference.

[0930] The diverse antigen receptors of T and B lymphocytes are produced by somatic recombination of a limited number of gene segments. These gene segments: V (variable), D (diversity), J (joining), and C (constant) determine the binding specificity and downstream applications of immunoglobulins and T cell receptors (TCRs). The present invention provides methods for generating TILs that exhibit and increase T cell repertoire diversity. In some embodiments, TILs obtained by the present methods exhibit increased T cell repertoire diversity. In some embodiments, TILs obtained by the present methods exhibit increased T cell repertoire diversity compared to freshly harvested TILs and / or TILs prepared using methods other than those provided herein, including, for example, methods other than those embodied in FIG. 1. In some embodiments, TILs obtained by the present methods exhibit increased T cell repertoire diversity compared to freshly harvested TILs and / or TILs prepared using a method designated Process 1C as illustrated in FIG. 5 and / or FIG. 6. In some embodiments, the TILs obtained in the first expansion exhibit increased T cell repertoire diversity. In some embodiments, the increased diversity is increased immunoglobulin diversity and / or T cell receptor diversity. In some embodiments, the diversity is in immunoglobulins and in immunoglobulin heavy chains. In some embodiments, the diversity is in immunoglobulins and in immunoglobulin light chains. In some embodiments, the diversity is in T cell receptors. In some embodiments, the diversity is in one of the T cell receptors selected from the group consisting of alpha, beta, gamma, and delta receptors. In some embodiments, expression of T cell receptor (TCR) alpha and / or beta is increased. In some embodiments, expression of T cell receptor (TCR) alpha is increased. In some embodiments, expression of T cell receptor (TCR) beta is increased. In some embodiments, expression of TCRab (i.e., TCRα / β) is increased.

[0931] For example, after dissection or digestion of tumor fragments, such as those described in step A of Figure 1, the resulting cells are cultured in serum containing IL-2 under conditions that favor the growth of TILs over tumor and other cells. In some embodiments, tumor digests are incubated in 2 mL wells in medium containing inactivated human AB serum with 6000 IU / mL of IL-2. This primary cell population is cultured for several days, generally 3-14 days, resulting in a bulk TIL population, generally about 1 x 10 8 In some embodiments, this primary cell population is cultured for 7-14 days, resulting in a bulk TIL population, generally about 1 x 10 8 In some embodiments, this primary cell population is cultured for 10-14 days, resulting in a bulk TIL population, generally about 1 x 10 8 In some embodiments, this primary cell population is cultured for about 11 days, resulting in a bulk TIL population, generally about 1 x 10 8 Generate bulk TIL cells.

[0932] In a preferred embodiment, TIL expansion can be performed using an initial bulk TIL expansion step (such as that described in step B of FIG. 1, which may include a process referred to as pre-REP) as described below and herein, followed by step D below and a second expansion as described herein (step D, which includes a process referred to as the rapid expansion protocol (REP) step), followed by optional cryopreservation, followed by a second step D below and described herein (which includes a process referred to as the restimulation REP step). TILs obtained from this process can optionally be characterized for phenotypic characteristics and metabolic parameters as described herein.

[0933] In embodiments in which TIL cultures are initiated in 24-well plates, e.g., using Costar 24-well cell culture clusters, flat bottom (Corning Incorporated, Corning, NY), 1 x 10 cells are cultured in 2 mL of complete medium (CM) containing IL-2 (6000 IU / mL, Chiron Corp., Emeryville, CA).6 Tumor digested cells or one tumor fragment can be seeded into each well. In some embodiments, the tumor fragment is approximately 1 mm 3 ~10mm 3 is.

[0934] In some embodiments, the first expansion culture medium is referred to as "CM," an abbreviation for culture medium. In some embodiments, the CM in step B consists of RPMI 1640 with GlutaMAX, supplemented with 10% human AB serum, 25 mM Hepes, and 10 mg / mL gentamicin. 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-Rex 10, Wilson Wolf Manufacturing, New Brighton, MN) (Figure 1), each flask contains 10-40 x 10 cells in 10-40 mL of CM containing IL-2. 6 Live tumor cells or 5–30 tumor fragments were loaded. Both G-Rex10 and 24-well plates were incubated in a humidified incubator at 37°C with 5% CO2. Five days after the start of culture, half of the medium was removed and replaced with fresh CM and IL-2. After five days, half of the medium was replaced every 2–3 days.

[0935] 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 with 6000 IU / mL of IL-2 (or in some cases, in the presence of an aAPC cell population, as outlined herein). This primary cell population is cultured for several days, generally 10-14 days, resulting in a bulk TIL population, generally about 1 x 10 8In some embodiments, the growth medium during the first expansion contains IL-2 or a variant thereof. In some embodiments, the IL is recombinant human IL-2 (rhIL-2). In some embodiments, the IL-2 stock solution contains 20-30 x 10 cells per 1 mg vial. 6 IU / mg specific activity. In some embodiments, the IL-2 stock solution contains 20×10 6 IU / mg specific activity. In some embodiments, the IL-2 stock solution contains 25×10 6 IU / mg specific activity. In some embodiments, the IL-2 stock solution contains 30×10 6 IU / mg specific activity. In some embodiments, the IL-2 stock solution contains 4-8 x 10 6 In some embodiments, the IL-2 stock solution has a final concentration of 5-7 x 10 IU / mg IL-2. 6 In some embodiments, the IL-2 stock solution has a final concentration of 6×10 IU / mg of IL-2. 6IU / mg IL-2. In some embodiments, the IL-2 stock solution is prepared as described in Example 5. In some embodiments, the first expansion culture medium contains about 10,000 IU / mL IL-2, about 9,000 IU / mL IL-2, about 8,000 IU / mL IL-2, about 7,000 IU / mL IL-2, about 6,000 IU / mL IL-2, or about 5,000 IU / mL IL-2. In some embodiments, the first expansion culture medium contains about 9,000 IU / mL to about 5,000 IU / mL IL-2. In some embodiments, the first expansion culture medium contains about 8,000 IU / mL to about 6,000 IU / mL IL-2. In some embodiments, the first expansion culture medium comprises about 7,000 IU / mL to about 6,000 IU / mL of IL-2. In some embodiments, the first expansion culture medium comprises about 6,000 IU / mL of IL-2. In certain embodiments, the cell culture medium further comprises IL-2. In some embodiments, the cell culture medium comprises about 3,000 IU / mL of IL-2. In certain embodiments, the cell culture medium further comprises IL-2. In a preferred embodiment, the cell culture medium comprises about 3,000 IU / mL of IL-2. In certain embodiments, the cell culture medium comprises about 1000 IU / mL, about 1500 IU / mL, about 2000 IU / mL, about 2500 IU / mL, about 3000 IU / mL, about 3500 IU / mL, about 4000 IU / mL, about 4500 IU / mL, about 5000 IU / mL, about 5500 IU / mL, about 6000 IU / mL, about 6500 IU / mL, about 7000 IU / mL, about 7500 IU / mL, or about 8000 IU / mL of IL-2. In certain embodiments, the cell culture medium contains 1000-2000 IU / mL, 2000-3000 IU / mL, 3000-4000 IU / mL, 4000-5000 IU / mL, 5000-6000 IU / mL, 6000-7000 IU / mL, 7000-8000 IU / mL, or 8000 IU / mL of IL-2.

[0936] 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 contains about 300 IU / mL to about 100 IU / mL of IL-15. In some embodiments, the first expansion culture medium contains about 200 IU / mL of IL-15. In some embodiments, the cell culture medium contains about 180 IU / mL of IL-15. In certain embodiments, the cell culture medium further contains IL-15. In a preferred embodiment, the cell culture medium contains about 180 IU / mL of IL-15.

[0937] 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 certain 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.

[0938] In certain embodiments, the cell culture medium comprises an OKT-3 antibody. In some embodiments, the cell culture medium comprises about 30 ng / mL of the OKT-3 antibody. In certain embodiments, the cell culture medium comprises about 0.1 ng / mL, about 0.5 ng / mL, about 1 ng / mL, about 2.5 ng / mL, about 5 ng / mL, about 7.5 ng / mL, about 10 ng / mL, about 15 ng / mL, about 20 ng / mL, about 25 ng / mL, about 30 ng / mL, about 35 ng / mL, about 40 ng / mL, about 50 ng / mL, about 60 ng / mL, about 70 ng / mL, about 80 ng / mL, about 90 ng / mL, about 100 ng / mL, about 200 ng / mL, about 500 ng / mL, and about 1 μg / mL of the OKT-3 antibody. In certain embodiments, the cell culture medium comprises an OKT-3 antibody at a concentration of 0.1 ng / mL to 1 ng / mL, 1 ng / mL to 5 ng / mL, 5 ng / mL to 10 ng / mL, 10 ng / mL to 20 ng / mL, 20 ng / mL to 30 ng / mL, 30 ng / mL to 40 ng / mL, 40 ng / mL to 50 ng / mL, or 50 ng / mL to 100 ng / mL. In some embodiments, the cell culture medium does not contain an OKT-3 antibody. In some embodiments, the OKT-3 antibody is muromonab.

[0939] In some embodiments, the cell culture medium comprises one or more TNFRSF agonists in the cell culture medium. In some embodiments, the TNFRSF agonist comprises a 4-1BB agonist. In some embodiments, the TNFRSF agonist is a 4-1BB agonist, and the 4-1BB agonist is selected from the group consisting of urelumab, utomilumab, EU-101, fusion proteins, and fragments, derivatives, variants, biosimilars, and combinations thereof. In some embodiments, the TNFRSF agonist is added at a concentration sufficient to achieve a cell culture medium concentration of 0.1 μg / mL to 100 μg / mL. In some embodiments, the TNFRSF agonist is added at a concentration sufficient to achieve a cell culture medium concentration of 20 μg / mL to 40 μg / mL.

[0940] 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.

[0941] In some embodiments, the first expansion culture medium is referred to as "CM," an abbreviation for culture medium. In some embodiments, it is referred to as CM1 (culture medium 1). In some embodiments, the CM consists of RPMI 1640 with GlutaMAX, supplemented with 10% human AB serum, 25 mM Hepes, and 10 mg / mL gentamicin. The cultures are grown in a 40 mL volume and 10 cm 2 In embodiments initiated in gas-permeable flasks with gas-permeable silicon bottoms (e.g., G-Rex10, Wilson Wolf Manufacturing, New Brighton, MN) (Figure 1), 10-40 x 10 cells in 10-40 mL of CM containing IL-2 are cultured. 6 Viable tumor digested cells or 5-30 tumor fragments were placed in each flask. Both the G-Rex10 and 24-well plates were incubated in a humidified incubator at 37°C with 5% CO2. Five days after the start of culture, half of the medium was removed and replaced with fresh CM and IL-2. After five days, half of the medium was replaced every 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.

[0942] In some embodiments, as discussed in the Examples and Figures, the first expansion (including processes such as those described in step B of FIG. 1, which may include what may be referred to as pre-REP) process is shortened to 3-14 days. In some embodiments, as discussed in the Examples and shown in FIGS. 4 and 5, the first expansion (including processes such as those described in step B of FIG. 1, which may include what may be referred to as pre-REP), including the expansion described in step B of FIG. 1, 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, as discussed in the expansion described in step B of FIG. 1, for example.

[0943] In some embodiments, the first TIL expansion can continue for 1 day, 2 days, 3 days, 4 days, 5 days, 6 days, 7 days, 8 days, 9 days, 10 days, 11 days, 12 days, 13 days, or 14 days. In some embodiments, the first TIL expansion can continue for 1 day to 14 days. In some embodiments, the first TIL expansion can continue for 2 days to 14 days. In some embodiments, the first TIL expansion can continue for 3 days to 14 days. In some embodiments, the first TIL expansion can continue for 4 days to 14 days. In some embodiments, the first TIL expansion can continue for 5 days to 14 days. In some embodiments, the first TIL expansion can continue for 6 days to 14 days. In some embodiments, the first TIL expansion can continue for 7 days to 14 days. In some embodiments, the first TIL expansion can continue for 8 days to 14 days. In some embodiments, the first TIL expansion can continue for 9 days to 14 days. In some embodiments, the first TIL expansion can continue for 10 to 14 days. In some embodiments, the first TIL expansion can continue for 11 to 14 days. In some embodiments, the first TIL expansion can continue for 12 to 14 days. In some embodiments, the first TIL expansion can continue for 13 to 14 days. In some embodiments, the first TIL expansion can continue for 14 days. In some embodiments, the first TIL expansion can continue for 1 to 11 days. In some embodiments, the first TIL expansion can continue for 2 to 11 days. In some embodiments, the first TIL expansion can continue for 3 to 11 days. In some embodiments, the first TIL expansion can continue for 4 to 11 days. In some embodiments, the first TIL expansion can continue for 5 to 11 days. In some embodiments, the first TIL expansion can continue for 6 to 11 days. In some embodiments, the first TIL expansion can continue for 7 to 11 days. In some embodiments, the first TIL expansion can continue for 8 to 11 days.In some embodiments, the first TIL expansion can continue for 9 to 11 days. In some embodiments, the first TIL expansion can continue for 10 to 11 days. In some embodiments, the first TIL expansion can continue for 11 days.

[0944] In some embodiments, a combination of IL-2, IL-7, IL-15, and / or IL-21 is used as a combination during the first expansion. In some embodiments, IL-2, IL-7, IL-15, and / or IL-21, and any combination thereof, may be included during the first expansion, including, for example, during step B of the process according to Figure 1 and described herein. In some embodiments, a combination of IL-2, IL-15, and IL-21 is used as a combination during the first expansion. In some embodiments, IL-2, IL-15, and IL-21, and any combination thereof, may be included during step B of the process according to Figure 1 and described herein.

[0945] In some embodiments, as discussed in the Examples and Figures, the first expansion (including the process referred to as Pre-REP, e.g., step B according to Figure 1) process is shortened to 3-14 days. 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.

[0946] In some embodiments, the first expansion, e.g., step B according to FIG. 1, is performed in a closed system bioreactor. In some embodiments, a closed system is used for TIL expansion as described herein. In some embodiments, a single bioreactor is used. In some embodiments, the single bioreactor used is, for example, a G-REX-10 or G-REX-100. In some embodiments, the closed system bioreactor is a single bioreactor.

[0947] 1. Cytokines and other additives The expansion methods described herein generally use culture media containing high doses of cytokines, particularly IL-2, as known in the art.

[0948] Alternatively, the use of cytokine combinations for rapid and / or secondary expansion of TILs is additionally possible using combinations of two or more of IL-2, IL-15, and IL-21, as described in U.S. Patent Application Publication No. US2017 / 0107490A1 (the disclosure of which is incorporated herein by reference). Thus, possible combinations include IL-2 and IL-15, IL-2 and IL-21, IL-15 and IL-21, and IL-2 or IL-15 and IL-21, the latter finding particular use in many embodiments. The use of cytokine combinations is particularly advantageous for the generation of lymphocytes, particularly T cells as described therein.

[0949] In some embodiments, step B may also include the addition of an OKT-3 antibody or muromonab to the culture medium, as described elsewhere herein. In some embodiments, step B may also include the addition of a 4-1BB agonist to the culture medium, as described elsewhere herein. In some embodiments, step B may also include the addition of an OX-40 agonist to the culture medium, as described elsewhere herein. In other embodiments, additives such as peroxisome proliferator-activated receptor gamma coactivator I-alpha agonists, including proliferator-activated receptor (PPAR)-gamma agonists such as thiazolidinedione compounds, may be used in the culture medium during step B, as described in U.S. Patent Application Publication No. US2019 / 0307796A1 (the disclosure of which is incorporated herein by reference).

[0950] C. Step C: Moving from the first expansion to the second expansion In some cases, the bulk TIL population obtained from the first expansion, including, for example, the TIL population obtained from step B shown in Figure 1, can be immediately cryopreserved using the protocols discussed below. Alternatively, the TIL population obtained from the first expansion, referred to as the second TIL population, can be subjected to a second expansion (which may include an expansion sometimes referred to as REP), as discussed below, and then cryopreserved. Similarly, when genetically modified TILs are used for therapy, the first TIL population (which may be referred to as the bulk TIL population) or the second TIL population (which in some embodiments may include a population referred to as the REP TIL population) can be subjected to genetic modification for the appropriate therapy before expansion or after the first expansion and before the second expansion.

[0951] In some embodiments, TILs obtained from the first expansion (e.g., from step B shown in FIG. 1 ) are stored until phenotyping for selection. In some embodiments, TILs obtained from the first expansion (e.g., from step B shown in FIG. 1 ) are not stored and proceed directly to the second expansion. In some embodiments, TILs obtained from the first expansion are not cryopreserved after the first expansion and before the second expansion. In some embodiments, the transition from the first expansion to the second expansion occurs about 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, or 14 days after fragmentation occurs. In some embodiments, the transition from the first expansion to the second expansion occurs about 3-14 days after fragmentation occurs. In some embodiments, the transition from the first expansion to the second expansion occurs about 4-14 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.

[0952] In some embodiments, the transition from the first expansion to the second expansion occurs 1 day, 2 days, 3 days, 4 days, 5 days, 6 days, 7 days, 8 days, 9 days, 10 days, 11 days, 12 days, 13 days, or 14 days after fragmentation occurs. In some embodiments, the transition from the first expansion to the second expansion occurs 1 to 14 days after fragmentation occurs. In some embodiments, the first TIL expansion can continue for 2 to 14 days. In some embodiments, the transition from the first expansion to the second expansion occurs 3 to 14 days after fragmentation occurs. In some embodiments, the transition from the first expansion to the second expansion occurs 4 to 14 days after fragmentation occurs. In some embodiments, the transition from the first expansion to the second expansion occurs 5 to 14 days after fragmentation occurs. In some embodiments, the transition from the first expansion to the second expansion occurs 6 to 14 days after fragmentation occurs. In some embodiments, the transition from the first expansion to the second expansion occurs 7 to 14 days after fragmentation occurs. In some embodiments, the transition from the first expansion to the second expansion occurs 8 to 14 days after fragmentation occurs. In some embodiments, the transition from the first expansion to the second expansion occurs 9 to 14 days after fragmentation occurs. In some embodiments, the transition from the first expansion to the second expansion occurs 10 to 14 days after fragmentation occurs. In some embodiments, the transition from the first expansion to the second expansion occurs 11 to 14 days after fragmentation occurs. In some embodiments, the transition from the first expansion to the second expansion occurs 12 to 14 days after fragmentation occurs. In some embodiments, the transition from the first expansion to the second expansion occurs 13 to 14 days after fragmentation occurs. In some embodiments, the transition from the first expansion to the second expansion occurs 14 days after fragmentation occurs. In some embodiments, the transition from the first expansion to the second expansion occurs 1 to 11 days after fragmentation occurs. In some embodiments, the transition from the first expansion to the second expansion occurs 2 to 11 days after fragmentation occurs. In some embodiments, the transition from the first expansion to the second expansion occurs 3 to 11 days after fragmentation occurs.In some embodiments, the transition from the first expansion to the second expansion occurs 4 to 11 days after fragmentation occurs. In some embodiments, the transition from the first expansion to the second expansion occurs 5 to 11 days after fragmentation occurs. In some embodiments, the transition from the first expansion to the second expansion occurs 6 to 11 days after fragmentation occurs. In some embodiments, the transition from the first expansion to the second expansion occurs 7 to 11 days after fragmentation occurs. In some embodiments, the transition from the first expansion to the second expansion occurs 8 to 11 days after fragmentation occurs. In some embodiments, the transition from the first expansion to the second expansion occurs 9 to 11 days after fragmentation occurs. In some embodiments, the transition from the first expansion to the second expansion occurs 10 to 11 days after fragmentation occurs. In some embodiments, the transition from the first expansion to the second expansion occurs 11 days after fragmentation occurs.

[0953] In some embodiments, the TILs are not stored after the first expansion and before the second expansion, and the TILs proceed directly to the second expansion (e.g., in some embodiments, they are not stored during the transition from step B to step D shown in FIG. 1). In some embodiments, the transfer occurs in a closed system as described herein. In some embodiments, the second TIL population, the TILs from the first expansion, proceed directly to the second expansion without a transfer period.

[0954] In some embodiments, the transition from the first expansion to the second expansion, e.g., step C according to FIG. 1, is performed in a closed system bioreactor. In some embodiments, a closed system is used for TIL expansion as described herein. In some embodiments, a single bioreactor is used. In some embodiments, the single bioreactor used is, for example, a G-REX-10 or G-REX-100 bioreactor. In some embodiments, the closed system bioreactor is a single biore...

Claims

1. 1. A population of tumor infiltrating lymphocytes (TIL), bone marrow infiltrating lymphocytes (MIL), or peripheral blood lymphocytes (PBL) for use in a method of treating cancer, wherein the population of TIL, MIL, or PBL is genetically modified to express a chimeric costimulatory receptor (CCR), the CCR comprising: i. an extracellular domain; ii. a hinge domain; iii. a transmembrane domain; iv. at least one intracellular domain; The method comprises administering to a patient in need of treatment for cancer the population of TILs, MILs, or PBLs. A population of said TILs, MILs, or PBLs.

2. wherein the cancer is treated by administering a population of TILs, and the method comprises: (a) obtaining and / or receiving a first population of TILs from a tumor excised from the patient by processing a tumor sample obtained from the patient into a plurality of tumor fragments or tumor digests; (b) adding the first population of TILs to a closed system; (c) performing a first expansion by culturing the first TIL population in a first cell culture medium comprising IL-2 and optionally an OKT-3 antibody, and antigen presenting cells (APCs) to produce a second TIL population, wherein the first expansion is performed in a closed container providing a first gas permeable surface area, and the first expansion is performed for about 3-14 days to obtain the second TIL population, and the transition from step (b) to step (c) occurs without opening the system. (d) genetically modifying the second TIL population to express the CCR; and (e) performing a second expansion of the second TIL population in a second cell culture medium comprising IL-2, OKT-3 antibody, and APCs to produce a third TIL population, wherein the second expansion is performed for about 3-14 days to obtain the third TIL population, the third TIL population being a therapeutic TIL population, and wherein the second expansion is performed in a sealed container providing a second gas permeable surface area; (f) harvesting the therapeutic TIL population obtained from step (e); (g) transferring the harvested TIL population from step (f) to an infusion bag, wherein the transition from step (e) to (f) occurs without opening the system; (h) cryopreserving the infusion bag containing the harvested TIL population from step (f) using a cryopreservation process; 2. The population of TILs, MILs, or PBLs of claim 1, comprising: (i) administering a therapeutically effective dose of the third population of TILs to the patient from the infusion bag of step (g). (i) the extracellular domain comprises an scFv binding domain, and optionally the scFv binding domain binds to a protein selected from the group consisting of CD19, CD20, CD22, CD24, CD33, CD38, CD39, CD73, CD123, CD138, CD228, LRRC15, CEA, FRα, EPCAM, PD-L1, PSMA, gp100, MUC1, MCSP, EGFR, GD2, TROP-2, GPC3, MICA, MICB, VISTA, ULBP, HER2, MCM5, FAP, 5T4, LFA-1, B7-H3, IL-13Rα2, FAS, TGFβ, TGFβRII, and MUC16; (ii) the extracellular domain is selected from the group consisting of a PD-1 domain, a FAS domain, and a TGFβRII domain; (iii) the intracellular domain is selected from the group consisting of CD28, CD134 (OX40), CD278 (ICOS), CD137 (4-1BB), CD27, CD40L, STAT3, IL-2Rβ, IL-2Rγ, IL-18R1, IL-18RAP, IL-7Rα, IL-12R1, IL-12R2, IL-15Rα, IL-21R, LTBR, ​​and combinations thereof; or (iv) the population of TILs, MILs, or PBLs of claim 1 or 2, wherein the transmembrane domain is selected from the group consisting of the transmembrane regions of CD3α, CD3β, CDζ, CD3ε, CD4, CD5, CD8α, CD9, CD16, CD22, CD27, CD28, CD33, CD37, CD45, CD64, CD80, CD86, CD134, CD137, CD154, IgG1, IgG4, IgD, IL-2Rα, IL-2Rβ, IL-2Rγ, and CD40L.

4. The population of TILs, MILs, or PBLs of claim 2 or 3, wherein step (d) further comprises genetically modifying the TILs using a lentivirus to express said CCR.

5. 5. The population of TILs, MILs, or PBLs of any one of claims 1 to 4, wherein the TILs, MILs, or PBLs are further genetically modified to stably or transiently reduce expression of a gene selected from the group consisting of PD-1, LAG-3, TIM-3, CTLA-4, TIGIT, CISH, TGFβR2, PKA, CBL-B, BAFF (BR3), SOCS1, ANKRD11, BCOR, and combinations thereof.

6. the cancer is a solid tumor cancer that is treated by administration of TILs; Optionally, the cancer is selected from the group consisting of sarcoma, pancreatic cancer, liver cancer, glioblastoma, gastrointestinal cancer, melanoma, ovarian cancer, endometrial cancer, thyroid cancer, colorectal cancer, cervical cancer, lung cancer, non-small cell lung cancer, small cell lung cancer, mesothelioma, bladder cancer, breast cancer, cancer caused by human papillomavirus, head and neck cancer, renal cancer, and renal cell carcinoma, and the patient is a human; Further optionally, the cancer is non-small cell lung cancer and the patient is:

1. A predefined tumor proportion score (TPS) of PD-L1 < 1%; 2. A Tumor Proportion Score (TPS) of 1% to 49% PD-L1; or 3. Having at least one of the predetermined absence of one or more driver mutations; Further optionally, the patient has a TIL, MIL or PBL population of any one of claims 1 to 5, wherein the patient has a TPS of PD-L1 of <1%.

7. (a) the patient has a cancer that is not indicated for treatment with an EGFR inhibitor, a BRAF inhibitor, an ALK inhibitor, a c-Ros inhibitor, a RET inhibitor, an ERBB2 inhibitor, a BRCA inhibitor, a MAP2K1 inhibitor, a PIK3CA inhibitor, a CDKN2A inhibitor, a PTEN inhibitor, a UMD inhibitor, an NRAS inhibitor, a KRAS inhibitor, a NF1 inhibitor, a MET inhibitor, a TP53 inhibitor, a CREBBP inhibitor, a KMT2C inhibitor, a KMT2D mutation, an ARID1A mutation, a RB1 inhibitor, an ATM inhibitor, a SETD2 inhibitor, a FLT3 inhibitor, a PTPN11 inhibitor, an FGFR1 inhibitor, an EP300 inhibitor, a MYC inhibitor, an EZH2 inhibitor, a JAK2 inhibitor, a FBXW7 inhibitor, a CCND3 inhibitor, and a GNA11 inhibitor; (b) the patient has the absence of one or more driver mutations, and optionally the one or more driver mutations are EGFR mutation, EGFR insertion, EGFR exon 20, KRAS mutation, BRAF mutation, BRAF V600 mutation, ALK mutation, c-ROS mutation (ROS1 mutation), ROS1 fusion, RET mutation, RET fusion, ERBB2 mutation, ERBB2 amplification, BRCA mutation, MAP2K1 mutation, PIK3CA, CDKN2A, PTEN mutation, UMD mutation, NRAS mutation, KRAS mutation, NF1 mutation, MET mutation, MET splice and / or altered MET sequence. signal transmission, TP53 mutation, CREBBP mutation, KMT2C mutation, KMT2D mutation, ARID1A mutation, RB1 mutation, ATM mutation, SETD2 mutation, FLT3 mutation, PTPN11 mutation, FGFR1 mutation, EP300 mutation, MYC mutation, EZH2 mutation, JAK2 mutation, FBXW7 mutation, CCND3 mutation, and GNA11 mutation; (c) the cancer is refractory or resistant to treatment with a chemotherapeutic agent or chemotherapy regimen; (d) the cancer is refractory or resistant to treatment with a VEGF-A inhibitor, and optionally the VEGF-A inhibitor is selected from the group consisting of bevacizumab, ranibizumab, icrucumab, and fragments, variants, and biosimilars thereof; (e) the cancer is refractory or resistant to treatment with a PD-1 inhibitor or a PD-L1 inhibitor, and optionally the PD-1 or PD-L1 inhibitor is selected from the group consisting of nivolumab, pembrolizumab, cemiplimab, tislelizumab, sintilimab, toripalimab, dostarimab, durvalumab, avelumab, atezolizumab, retifanlimab, and fragments, variants, and biosimilars thereof; (f) the cancer is refractory or resistant to treatment with a CTLA-4 inhibitor, and the CTLA-4 inhibitor is selected from the group consisting of ipilumumab, tremelimumab, zalifrelimab, and fragments, variants, and biosimilars thereof; (g) the IL-2 is initially present in the first cell culture medium and in the second cell culture medium at an initial concentration of 1000 IU / mL to 6000 IU / mL; (h) the OKT-3 antibody is initially present in the second cell culture medium at an initial concentration of about 30 ng / mL; (i) the first or second cell culture medium further comprises a cytokine selected from the group consisting of IL-4, IL-7, IL-15, IL-21, a 4-1BB agonist, an OX-40 agonist, an AKT inhibitor, and combinations thereof; or (k) 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.

8. The population of TILs, MILs, or PBLs of any one of claims 1 to 7, wherein the method further comprises treating the patient with a non-myeloablative lymphodepletion regimen prior to administering the third TIL population to the patient, optionally wherein the non-myeloablative lymphodepletion regimen comprises administering cyclophosphamide at a dose of 60 mg / m2 / day for two days, followed by fludarabine at a dose of 25 mg / m2 / day for five days, and further optionally wherein the non-myeloablative lymphodepletion regimen comprises administering cyclophosphamide at a dose of 60 mg / m2 / day and fludarabine at a dose of 25 mg / m2 / day for two days, followed by fludarabine at a dose of 25 mg / m2 / day for three days.

9. The method according to claim 1, (a) further comprising the step of treating said patient with an IL-2 regimen beginning the day after administration of said third population of TILs to said patient; or (b) treating the patient with an IL-2 regimen beginning on the same day as administering the third population of TILs to the patient; Optionally, (i) the IL-2 regimen is a high-dose IL-2 regimen comprising 600,000 or 720,000 IU / kg aldesleukin, or a fragment, variant, or biosimilar thereof, administered as a 15-minute bolus intravenous infusion every 8 hours until tolerated; (ii) the IL-2 regimen comprises administration of bempegaldesleukin, or a fragment, variant, or biosimilar thereof; (iii) the IL-2 regimen comprises administration of THOR-707, or a fragment, variant, or biosimilar thereof; (iv) the IL-2 regimen comprises administration of nembareukin alfa, or a fragment, variant, or biosimilar thereof; or (v) The population of TILs, MILs, or PBLs of any one of claims 1 to 8, wherein the IL-2 regimen comprises administration of an antibody, or a fragment, variant, or biosimilar thereof, comprising a heavy chain selected from the group consisting of SEQ ID NO:29 and SEQ ID NO:38, and a light chain selected from the group consisting of SEQ ID NO:37 and SEQ ID NO:

39.

10. A therapeutically effective population of TILs is administered, approximately 2×10 ９ ~Approx. 15×10 １０ TIL, and optionally said first expansion is performed for a period of 11 days or less; Further optionally, the second expansion is performed for a period of up to 11 days.

11. 1. A composition comprising tumor infiltrating lymphocytes (TIL), bone marrow infiltrating lymphocytes (MIL), or peripheral blood lymphocytes (PBL) genetically modified to express a chimeric costimulatory receptor (CCR), wherein the CCR is i. an extracellular domain; ii. a hinge domain; iii. a transmembrane domain; iv. at least one intracellular domain.

12. (a) the extracellular domain comprises an scFv binding domain, and optionally the scFv binding domain is selected from the group consisting of an anti-CD19 domain, an anti-CD20 domain, an anti-CD22 domain, an anti-CD24 domain, an anti-CD33 domain, an anti-CD38 domain, an anti-CD39 domain, an anti-CD73 domain, an anti-CD123 domain, an anti-CD138 domain, an anti-CD228 domain, an anti-LRRC15 domain, an anti-CEA domain, an anti-FRα domain, an anti-EPCAM domain, an anti-PD-L1 domain, in, an anti-PSMA domain, an anti-gp100 domain, an anti-MUC1 domain, an anti-MCSP domain, an anti-EGFR domain, an anti-GD2 domain, an anti-TROP-2 domain, an anti-GPC3 domain, an anti-MICA domain, an anti-MICB domain, an anti-VISTA domain, an anti-ULBP domain, an anti-HER2 domain, an anti-MCM5 domain, an anti-FAP domain, an anti-5T4 domain, an anti-LFA-1 domain, an anti-B7-H3 domain, and an anti-MUC16 domain; (b) the extracellular domain is a PD-1 domain, a FAS domain, or a TGFβRII domain; (c) the intracellular domain is selected from the group consisting of a CD28 domain, a CD134 (OX40) domain, a CD278 (ICOS) domain, a CD137 (4-1BB) domain, a CD27 domain, a STAT3 domain, an IL-2Rβ domain, an IL-2Rγ domain, an IL-18R1 domain, an IL-18RAP domain, an IL-7Rα domain, an IL-12R1 domain, an IL-12R2 domain, an IL-15Rα domain, an IL-21R domain, and combinations thereof; or (d) the transmembrane domain is selected from the group consisting of a CD3α domain, a CD3β domain, a CDζ domain, a CD3ε domain, a CD4 domain, a CD5 domain, a CD8α domain, a CD9 domain, a CD16 domain, a CD22 domain, a CD27 domain, a CD28 domain, a CD33 domain, a CD37 domain, a CD45 domain, a CD64 domain, a CD80 domain, a CD86 domain, a CD134 domain, a CD137 domain, a CD154 domain, an IgG1 domain, an IgG4 domain, 12. The composition of claim 11, wherein the TIL, MIL, or PBL is selected from the group consisting of: main, IgD domain, IL-2Rα domain, IL-2Rβ domain, and IL-2Rγ domain, and optionally the TIL, MIL, or PBL is further genetically modified to stably or transiently reduce expression of a gene selected from the group consisting of PD-1, LAG-3, TIM-3, CTLA-4, TIGIT, CISH, TGFβR2, PKA, CBL-B, BAFF (BR3), and combinations thereof.

13. 1. A composition comprising a chimeric costimulatory receptor (CCR), the CCR comprising: i. an extracellular protein domain; ii. a hinge protein domain; iii. a transmembrane protein domain; iv. at least one intracellular protein domain. (a) the extracellular protein domain comprises an scFv binding domain, and optionally the scFv binding domain is selected from the group consisting of an anti-CD19 domain, an anti-CD20 domain, an anti-CD22 domain, an anti-CD24 domain, an anti-CD33 domain, an anti-CD38 domain, an anti-CD39 domain, an anti-CD73 domain, an anti-CD123 domain, an anti-CD138 domain, an anti-CD228 domain, an anti-LRRC15 domain, an anti-CEA domain, an anti-FRα domain, an anti-EPCAM domain, an anti-PD-L1 domain, an anti-PSMA domain, an anti-g selected from the group consisting of a p100 domain, an anti-MUC1 domain, an anti-MCSP domain, an anti-EGFR domain, an anti-GD2 domain, an anti-TROP-2 domain, an anti-GPC3 domain, an anti-MICA domain, an anti-MICB domain, an anti-VISTA domain, an anti-ULBP domain, an anti-HER2 domain, an anti-MCM5 domain, an anti-FAP domain, an anti-5T4 domain, an anti-LFA-1 domain, an anti-B7-H3 domain, an anti-IL-13Rα2 domain, an anti-FAS domain, an anti-TGFβRII domain, and an anti-MUC16 domain; (b) the extracellular protein domain is a PD-1 domain, a FAS domain, or a TGFβRII domain; (c) the intracellular domain is selected from the group consisting of a CD28 domain, a CD134 (OX40) domain, a CD278 (ICOS) domain, a CD137 (4-1BB) domain, a CD27 domain, an IL-2Rβ domain, an IL-2Rγ domain, an IL-18R1 domain, an IL-18RAP domain, an IL-7Rα domain, an IL-12R1 domain, an IL-12R2 domain, an IL-15Rα domain, an IL-21R domain, and combinations thereof; (d) the transmembrane protein domain is selected from the group consisting of a CD3α domain, a CD3β domain, a CDζ domain, a CD3ε domain, a CD4 domain, a CD5 domain, a CD8α domain, a CD9 domain, a CD16 domain, a CD22 domain, a CD27 domain, a CD28 domain, a CD33 domain, a CD37 domain, a CD45 domain, a CD64 domain, a CD80 domain, a CD86 domain, a CD134 domain, a CD137 domain, a CD154 domain, an IgG1 domain, an IgG4 domain, an IgD domain, an IL-2Rα domain, an IL-2Rβ domain, and an IL-2Rγ domain; or (e) the hinge protein domain is selected from the group consisting of a CD3α domain, a CD3β domain, a CDζ domain, a CD3ε domain, a CD4 domain, a CD5 domain, a CD8α domain, a CD9 domain, a CD16 domain, a CD22 domain, a CD27 domain, a CD28 domain, a CD33 domain, a CD37 domain, a CD45 domain, a CD64 domain, a CD80 domain, a CD86 domain, a CD134 domain, a CD137 domain, a CD154 domain, an IgG1 domain, an IgG4 domain, an IgD domain, an IL-2Rα domain, an IL-2Rβ domain, and an IL-2Rγ domain.

15. (i) tumor-infiltrating lymphocytes; (ii) bone marrow infiltrating lymphocytes; or (iii) peripheral blood lymphocytes; 15. The composition of claim 13 or 14, further comprising: