Methods for tumor infiltrating lymphocyte (TIL) expansion related to cd39 / cd103 selection

Abstract

Provided herein are TILs that are in CD39, CD103 and / or both. In some embodiments, the subject TILs are produced by genetically manipulating a population of TILs that have been selected for expression of (i) PD-1, (ii) CD39, (iii) CD103, (iv) the combination of (i) and (ii), (v) the combination of (i) and (iii), or (vi) the combination of (ii) and (iii). Also provided herein are expansion methods for producing such populations of TILs and methods of treatment using such TILs.

Description

METHODS FOR TUMOR INFILTRATING LYMPHOCYTE (TIL) EXPANSION RELATED TO CD39 / CD103 SELECTION CROSS-REFERENCE TO RELATED APPLICATION

[0001] The present application claims priority to U.S. provisional application No.63 / 382,460 filed November 4, 2022, the entire disclosure of which is incorporated herein by reference in its entirety. BACKGROUND OF THE INVENTION

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

[0003] Furthermore, current TIL manufacturing and treatment processes are limited by length, cost, sterility concerns, and other factors described herein such that the potential to treat patients which are refractory other checkpoint inhibitor therapies have been severely limited. There is an urgent need to provide TIL manufacturing processes and therapies based on such processes that are appropriate for use in treating patients for whom very few or no viable treatment options remain. The present invention meets this need by providing a manufacturing process for use in generating more robust TILs.

[0004] The present invention provides improved and / or shortened processes and methods for preparing TILs with particular combinations of cytokines and other cell culture medium constitutents in order to prepare therapeutic populations of TILs with increased therapeutic efficacy for the treatment of cancer with TILs.BRIEF SUMMARY OF THE INVENTION

[0005] Provided herein are TILs that are genetically modified to silence or reduce expression of endogenous PD-1, CD39 and / or CD103. In some embodiments, the subject TILs are produced by genetically manipulating a population of TILs that have been selected for CD39 and / or CD103 expression (i.e., a CD39 and / or CD103 expressing TIL population). Expression of CD39 and CD103 are believed to correlate with PD-1 expression in TILs. PD-1 expressing TILs are believed to have enhanced anti-tumor activity. However, PD-1 is known to be immunosuppressive. Also provided herein are expansion methods for producing such genetically modified TILs and methods of treatment using such TILs.

[0006] In one aspect, provided herein is a method of treating a cancer in a patient or subject in need thereof comprising administering a population of modified tumor infiltrating lymphocytes (TILs), the method comprising the steps of: (a) obtaining and / or receiving a first population of TILs in a plurality of tumor fragments obtained from a tumor sample resected from a tumor in the subject or patient; (b) selecting CD39 and / or CD103 positive TILs from the first population of TILs in (a) to obtain a population of CD39 and / or CD103 expressing TILs; (c) adding the population of CD39 and / or CD103 expressing TILs into a closed system; (d) performing a first expansion by culturing the population of CD39 and / or CD103 expressing TILs in a first cell culture medium supplemented with IL-2 to produce a second population of TILs, wherein the first expansion is performed in a closed container providing a first gas-permeable surface area, wherein the first expansion is performed for about 3-14 days to obtain the second population of TILs, and wherein the transition from step (c) to step (d) occurs without opening the system; (e) performing a second expansion by culturing the second population of TILs in a second cell culture medium supplemented with IL-2, OKT-3, and antigen presenting cells (APCs), to produce a third population of TILs, wherein the second expansion is performed for about 7-14 days to obtain the third population of TILs, wherein the third population of TILs is a therapeutic population of TILs, wherein the second expansion is performed in a closed container providing a second gas-permeable surface area, and wherein the transition from step (d) to step (e) occurs without opening the system; (f) harvesting the therapeutic population of TILs obtained from step (e), wherein the transition from step (e) to step (f) occurs without opening the system; (g) transferring the harvested therapeutic population of TILs from step (f) to an infusion bag, wherein the transfer from step (f) to (g) occurs without opening the system; (h) cryopreserving the infusion bag using a cryopreservation process; (i) administering a therapeutically effective dosage of the therapeutic population of TILs from the infusion bag in step (h) to the subject; and (j) genetically modifying the population of CD39 and / or CD103 expressing TILs, the second population of TILsand / or the third population of TILs at any time after the selecting CD39 and / or CD103 positive TILs (b) and prior to the administering (i) such that the administered therapeutic population of TILs comprises genetically modified TILs comprising a genetic modification that reduces expression of PD- 1, CD39 and / or CD103.

[0007] In another aspect, provided herein is a method of treating a cancer in a patient or subject in need thereof comprising administering a population of modified tumor infiltrating lymphocytes (TILs), the method comprising the steps of: (a) selecting CD39 and / or CD103 positive TILs from a first population of TILs in a tumor digest produced by digesting in an enzymatic digest medium a tumor sample resected from a tumor in the patient or subject to obtain a population of PD-1 expressing TILs; (b) performing a first expansion by culturing the population of CD39 and / or CD103 expressing TILs in a first cell culture medium supplemented with IL-2 to produce a second population of TILs, wherein the first expansion is performed in a closed container providing a first gas-permeable surface area, wherein the first expansion is performed for about 3-14 days to obtain the second population of TILs; (c) performing a second expansion by culturing the second population of TILs in a second cell culture medium supplemented with IL-2, OKT-3, and antigen presenting cells (APCs), to produce a third population of TILs, wherein the second expansion is performed for about 7-14 days to obtain the third population of TILs, wherein the third population of TILs is a therapeutic population of TILs, wherein the second expansion is performed in a closed container providing a second gas-permeable surface area, and wherein the transition from step (b) to step (c) occurs without opening the system; (d) harvesting the therapeutic population of TILs obtained from step (c), wherein the transition from step (c) to step (d) occurs without opening the system; (e) transferring the harvested therapeutic population of TILs from step (d) to an infusion bag, wherein the transfer from step (d) to (e) occurs without opening the system; (f) cryopreserving the infusion bag using a cryopreservation process; (g) administering a therapeutically effective dosage of the therapeutic population of TILs from the infusion bag in step (f) to the subject; and (h) genetically modifying the population of CD39 and / or CD103 expressing TILs, the second population of TILs and / or the third population of TILs at any time after the selecting CD39 and / or CD103 positive TILs (a) and prior to the administering (g) such that the administered therapeutic population of TILs comprises genetically modified TILs comprising a genetic modification that reduces expression of PD- 1, CD39 and / or CD103. In some embodiments, step (a) comprises selecting CD39 and / or CD103 positive TILs from a first population of TILs in a tumor digest produced by digesting in an enzymatic digest medium a plurality of tumor fragments prepared from a tumor sample resected from a tumor in the patient or subject to obtain a population of CD39 and / or CD103 expressing TILs.

[0008] In one aspect, provided herein is a method of treating a cancer in a patient or subject in need thereof comprising administering a population of tumor infiltrating lymphocytes (TILs), the method comprising the steps of: (a) obtaining a first population of TILs in a plurality of tumor fragments prepared from a tumor sample resected from a tumor in the patient or subject; (b) selecting CD39 and / or CD103 positive TILs from the first population of TILs in (a) to obtain a population of CD39 and / or CD103 expressing TILs; (c) adding the population of CD39 and / or CD103 expressing TILs into a closed system; (d) performing a first expansion by culturing the population of CD39 and / or CD103 expressing TILs in a first cell culture medium supplemented with IL-2 to produce a second population of TILs, wherein the first expansion is performed in a closed container providing a first gas-permeable surface area, wherein the first expansion is performed for about 3-11 days to obtain the second population of TILs, and wherein the transition from step (c) to step (d) occurs without opening the system; (e) performing a second expansion by culturing the second population of TILs in a second cell culture medium supplemented with IL-2, OKT-3, and antigen presenting cells (APCs), to produce a third population of TILs, wherein the second expansion is performed for about 7-11 days to obtain the third population of TILs, wherein the second expansion is performed in a closed container providing a second gas-permeable surface area, and wherein the transition from step (d) to step (e) occurs without opening the system; (f) harvesting the third population of TILs obtained from step (e), wherein the transition from step (e) to step (f) occurs without opening the system; (g) transferring the harvested third population of TILs from step (f) to an infusion bag, wherein the transfer from step (f) to (g) occurs without opening the system; (h) cryopreserving the infusion bag using a cryopreservation process; (i) administering a therapeutically effective dosage of the third population of TILs from the infusion bag in step (h) to the subject; and (j) genetically modifying the population of CD39 and / or CD103 expressing TILs, the second population of TILs and / or the third population of TILs at any time after the selecting CD39 and / or CD103 positive TILs (b) and prior to the administering (i) such that the administered third population of TILs comprising a genetic modification that reduces expression of PD-1, CD39 and / or CD103.

[0009] In one aspect, provided herein is a method of treating a cancer in a patient or subject in need thereof comprising administering a population of tumor infiltrating lymphocytes (TILs), the method comprising the steps of: (a) selecting CD39 and / or CD103 positive TILs from a first population of TILs in a tumor digest produced by digesting in an enzymatic digest medium a tumor sample resected from a tumor in the patient or subject to obtain a population of CD39 and / or CD103 expressing TILs; (b) performing a first expansion by culturing the population of CD39 and / or CD103 expressing TILs in a first cell culture medium supplemented with IL-2 to produce a second population of TILs, wherein the first expansion is performed in a closed container providing a first gas-permeable surface area, wherein the first expansion is performed for about 3-11 days to obtain the second population of TILs; (c) performing a second expansion by culturing the second population of TILs in a second cell culture medium supplemented with IL-2, OKT-3, and antigen presenting cells (APCs), to produce a third population of TILs, wherein the second expansion is performed for about 7-11 days to obtain the third population of TILs, wherein the second expansion is performed in a closed container providing a second gas-permeable surface area, and wherein the transition from step (b) to step (c) occurs without opening the system; (d) harvesting the third population of TILs obtained from step (c), wherein the transition from step (c) to step (d) occurs without opening the system; (e) transferring the harvested third TIL population from step (d) to an infusion bag, wherein the transfer from step (d) to (e) occurs without opening the system; (f) cryopreserving the infusion bag using a cryopreservation process; (g) administering a therapeutically effective dosage of the third population of TILs from the infusion bag in step (f) to the subject; and (h) genetically modifying the population of CD39 and / or CD103 expressing TILs, the second population of TILs and / or the third population of TILs at any time after the selecting CD39 and / or CD103 positive TILs (a) and prior to the administering (g) such that the administered third population of TILs comprising a genetic modification that reduces expression of PD-1, CD39 and / or CD103.

[0010] In some embodiments, step (a) comprises selecting CD39 and / or CD103 positive TILs from a first population of TILs in a tumor digest produced by digesting in an enzymatic digest medium a plurality of tumor fragments prepared from a tumor sample resected from a tumor in the patient or subject to obtain a population of CD39 and / or CD103 expressing TILs.

[0011] In another aspect, provided herein is a method of treating a cancer in a patient or subject in need thereof comprising administering a population of tumor infiltrating lymphocytes (TILs), the method comprising the steps of: (a) obtaining and / or receiving a first population of TILs in a tumor sample obtained from surgical resection, needle biopsy, core biopsy, small biopsy, or other means for obtaining a sample that contains a mixture of tumor and TIL cells from the cancer in the patient or subject, (b) selecting CD39 and / or CD103 positive TILs from the first population of TILs in (a) to obtain a population of CD39 and / or CD103 expressing TILs; (c) adding the population of CD39 and / or CD103 expressing TILs into a closed system; (d) performing a first expansion by culturing the population of CD39 and / or CD103 expressing TILs in a first cell culture medium supplemented with IL-2 to produce a second population of TILs, wherein the first expansion is performed in a closed container providing a first gas-permeable surface area, wherein the first expansion is performed for about 3-11 days to obtain the second population of TILs, and wherein the transition from step (c) to step (d) occurs without opening the system; (e) performing a second expansion by culturing thesecond population of TILs in a second cell culture medium supplemented with IL-2, OKT-3, and antigen presenting cells (APCs), to produce a third population of TILs, wherein the second expansion is performed for about 7-11 days to obtain the third population of TILs, wherein the second expansion is performed in a closed container providing a second gas-permeable surface area, and wherein the transition from step (d) to step (e) occurs without opening the system; (f) harvesting the third population of TILs obtained from step (e), wherein the transition from step (e) to step (f) occurs without opening the system; (g) transferring the harvested third population of TILs from step (f) to an infusion bag, wherein the transfer from step (e) to (f) occurs without opening the system; (h) cryopreserving the infusion bag using a cryopreservation process; i) administering a therapeutically effective dosage of the third population of TILs from the infusion bag in step (h) to the subject; and (j) genetically modifying the population of CD39 and / or CD103 expressing TILs, the second population of TILs and / or the third population of TILs at any time after the selecting CD39 and / or CD103 positive TILs (b) and prior to the administering (i) such that the administered third population of TILs comprises genetically modified TILs comprising a genetic modification that reduces expression of PD-1, CD39 and / or CD103.

[0012] In one aspect, provided herein is of treating a cancer in a patient or subject in need thereof comprising administering a population of modified tumor infiltrating lymphocytes (TILs), the method comprising the steps of: (a) resecting a tumor sample from a tumor in the subject or patient, the tumor comprising a first population of TILs, optionally from surgical resection, needle biopsy, core biopsy, small biopsy, or other means for obtaining a sample that contains a mixture of tumor and TIL cells from the cancer; (b) processing the tumor sample into a plurality of tumor fragments; (c) enzymatically digesting in an enzymatic digest medium the plurality of tumor fragments to obtain the first population of TILs; (d) selecting CD39 and / or CD103 positive TILs from the first population of TILs in (c) to obtain a population of CD39 and / or CD103 expressing TILs; (e) adding the population of CD39 and / or CD103 expressing TILs into a closed system; (f) performing a first expansion by culturing the population of CD39 and / or CD103 expressing TILs in a first cell culture medium supplemented with IL-2 to produce a second population of TILs, wherein the first expansion is performed in a closed container providing a first gas-permeable surface area, wherein the first expansion is performed for about 3-11 days to obtain the second population of TILs, and wherein the transition from step (e) to step (f) occurs without opening the system; (g) performing a second expansion by culturing the second population of TILs in a second cell culture medium supplemented with IL-2, OKT-3, and antigen presenting cells (APCs), to produce a third population of TILs, wherein the second expansion is performed for about 7-11 days to obtain the third population of TILs, wherein the second expansion is performed in a closed container providing a second gas-permeable surface area,and wherein the transition from step (f) to step (g) occurs without opening the system; (h) harvesting the third population of TILs obtained from step (g), wherein the transition from step (g) to step (h) occurs without opening the system; (i) transferring the harvested third TIL population from step (h) to an infusion bag, wherein the transfer from step (h) to (i) occurs without opening the system; (j) cryopreserving the infusion bag using a cryopreservation process; (k) administering a therapeutically effective dosage of the third population of TILs from the infusion bag in step (j) to the subject or patient with the cancer; and (k) genetically modifying the population of CD39 and / or CD103 expressing TILs, the second population of TILs and / or the third population of TILs at any time after the selecting CD39 and / or CD103 positive TILs (d) and prior to the administering (i) such that the administered third population of TILs comprises genetically modified TILs comprising a genetic modification that reduces expression of PD-1, CD39 and / or CD103.

[0013] In one aspect, provided herein is a method of treating a cancer in a patient or subject in need thereof comprising administering a population of tumor infiltrating lymphocytes (TILs), the method comprising the steps of: (a) selecting CD39 and / or CD103 positive TILs from a first population of TILs in a tumor digest prepared by digesting in an enzymatic digest medium a tumor sample obtained or received from surgical resection, needle biopsy, core biopsy, small biopsy, or other means for obtaining a sample that contains a mixture of tumor and TIL cells from the cancer in the patient or subject, to produce a population of CD39 and / or CD103 expressing TILs; (b) performing a first expansion by culturing the population of CD39 and / or CD103 expressing TILs in a first cell culture medium supplemented with IL-2 to produce a second population of TILs, wherein the first expansion is performed in a closed container providing a first gas-permeable surface area, wherein the first expansion is performed for about 3-11 days to obtain the second population of TILs; (c) performing a second expansion by culturing the second population of TILs in a second cell culture medium supplemented with IL-2, OKT-3, and antigen presenting cells (APCs), to produce a third population of TILs, wherein the second expansion is performed for about 7-11 days to obtain the third population of TILs, wherein the second expansion is performed in a closed container providing a second gas-permeable surface area, and wherein the transition from step (b) to step (c) occurs without opening the system; (d) harvesting the third population of TILs obtained from step (c), wherein the transition from step (c) to step (d) occurs without opening the system; (e) transferring the harvested third population of TILs from step (d) to an infusion bag, wherein the transfer from step (d) to (e) occurs without opening the system; (f) cryopreserving the infusion bag using a cryopreservation process; (g) administering a therapeutically effective dosage of the third population of TILs from the infusion bag in step (f) to the subject; and (h) genetically modifying the population of CD39 and / or CD103 expressing TILs, the second population of TILs and / or the thirdpopulation of TILs at any time after the selecting CD39 and / or CD103 positive TILs and prior to the administering (g) such that the administered third population of TILs comprises genetically modified TILs comprising a genetic modification that reduces expression of PD-1, CD39 and / or CD103.

[0014] In some embodiments, step (a) comprises selecting CD39 and / or CD103 positive TILs from a first population of TILs in a tumor digest prepared by digesting in an enzymatic digest medium a plurality of tumor fragments prepared from a tumor sample obtained or received from surgical resection, needle biopsy, core biopsy, small biopsy, or other means for obtaining a sample that contains a mixture of tumor and TIL cells from the cancer in the patient or subject, to produce a population of CD39 and / or CD103 expressing TILs.

[0015] In one aspect, provided herein is a method of treating a cancer in a patient or subject in need thereof comprising administering a population of tumor infiltrating lymphocytes (TILs), the method comprising the steps of: (a) obtaining and / or receiving a first population of TILs in a tumor sample obtained from surgical resection, needle biopsy, core biopsy, small biopsy, or other means for obtaining a sample that contains a mixture of tumor and TIL cells from the subject or patient; (b) selecting CD39 and / or CD103 positive TILs from the first population of TILs in (a) to obtain a population of CD39 and / or CD103 expressing TILs; (c) performing an initial expansion (or priming first expansion) of the population of CD39 and / or CD103 expressing TILs in a first cell culture medium to obtain a second population of TILs, wherein the first cell culture medium is supplemented with s IL-2, optionally OKT-3 (anti-CD3 antibody), and optionally antigen presenting cells (APCs), where the priming first expansion occurs for a period of 1 to 8 days; (d) performing a rapid second expansion of the second population of TILs in a second cell culture medium to obtain a third population of TILs, wherein the second cell culture medium is supplemented with IL-2, OKT-3 (anti-CD3 antibody), and APCs; and wherein the rapid expansion is performed over a period of 14 days or less, optionally the rapid second expansion can proceed for 1 day, 2 days, 3 days, 4, days, 5 days, 6 days, 7 days, 8 days, 9 days or 10 days after initiation of the rapid second expansion; (e) harvesting the third population of TILs; (f) administering a therapeutically effective dosage of the third population of TILs to the subject or patient with the cancer; and (g) genetically modifying the population of CD39 and / or CD103 expressing TILs, the second population of TILs and / or the third population of TILs at any time after the selecting CD39 and / or CD103 positive TILs (b) and prior to the administering (f) such that the administered third population of TILs comprises genetically modified TILs comprising a genetic modification that reduces expression of PD-1, CD39 and / or CD103.

[0016] In another aspect, provided herein is a method of treating a cancer in a patient or subject in need thereof comprising administering a population of tumor infiltrating lymphocytes (TILs), the method comprising the steps of: (a) obtaining a tumor sample from the cancer in the subject or patient, the tumor sample comprising a first population of TILs, optionally from surgical resection, needle biopsy, core biopsy, small biopsy, or other means for obtaining a sample that contains a mixture of tumor and TIL cells from the cancer;(b) fragmenting the tumor into a plurality of tumor fragments; (c) selecting CD39 and / or CD103 positive TILs from the first population of TILs of the plurality of tumor fragments to obtain a population of CD39 and / or CD103 expressing TILs; (d) performing an initial expansion (or priming first expansion) of the population of CD39 and / or CD103 expressing TILs in a first cell culture medium to obtain a second population of TILs, wherein the first cell culture medium is supplemented with IL-2, optionally OKT-3 (anti-CD3 antibody), and optionally antigen presenting cells (APCs), where the priming first expansion occurs for a period of 1 to 8 days; (e) performing a rapid second expansion of the second population of TILs in a second cell culture medium to obtain a third population of TILs, wherein the second cell culture medium is supplemented with IL-2, OKT-3 (anti-CD3 antibody), and APCs, and wherein the rapid expansion is performed over a period of 14 days or less, optionally the rapid second expansion can proceed for 1 day, 2 days, 3 days, 4, days, 5 days, 6 days, 7 days, 8 days, 9 days or 10 days after initiation of the rapid second expansion; (f) harvesting the third population of TILs; (g) administering a therapeutically effective dosage of the third population of TILs to the subject or patient with the cancer; and (h) genetically modifying the population of CD39 and / or CD103 expressing TILs, the second population of TILs and / or the third population of TILs at any time after the selecting CD39 and / or CD103 positive TILs (c) and prior to the administering (g) such that the administered third population of TILs comprises genetically modified TILs comprising a genetic modification that reduces expression of PD-1, CD39 and / or CD103.

[0017] In another aspect, provided herein is a method of treating a cancer in a patient or subject in need thereof comprising administering a population of tumor infiltrating lymphocytes (TILs), the method comprising the steps of: (a) selecting CD39 and / or CD103 positive TILs from a first population of TILs in a tumor digest prepared by digesting in an enzymatic digest medium a tumor sample obtained or received from surgical resection, needle biopsy, core biopsy, small biopsy, or other means for obtaining a sample that contains a mixture of tumor and TIL cells from the cancer in the patient or subject, to produce a population of CD39 and / or CD103 expressing TILs; (b) performing an initial expansion (or priming first expansion) of the population of CD39 and / or CD103 expressing TILs in a first cell culture medium to obtain a second population of TILs, wherein the first cell culture medium is supplemented with IL-2, optionally OKT-3 (anti-CD3 antibody), and optionallyantigen presenting cells (APCs), where the priming first expansion occurs for a period of 1 to 8 days; (c) performing a rapid second expansion of the second population of TILs in a second cell culture medium to obtain a third population of TILs, wherein the second cell culture medium is supplemented with IL-2, OKT-3 (anti-CD3 antibody), and APCs; and wherein the rapid expansion is performed over a period of 14 days or less, optionally the rapid second expansion can proceed for 1 day, 2 days, 3 days, 4, days, 5 days, 6 days, 7 days, 8 days, 9 days or 10 days after initiation of the rapid second expansion; (d) harvesting the third population of TILs; (e) administering a therapeutically effective dosage of the third population of TILs to the subject or patient with the cancer; and (f) genetically modifying the population of CD39 and / or CD103 expressing TILs, the second population of TILs and / or the third population of TILs at any time after the selecting CD39 and / or CD103 positive TILs (a) and prior to the administering (e) such that the administered third population of TILs comprises genetically modified TILs comprising a genetic modification that reduces expression of PD-1, CD39 and / or CD103. In some embodiments, step (a) comprises selecting CD39 and / or CD103 positive TILs from a first population of TILs in a tumor digest prepared by digesting in an enzymatic digest medium a plurality of tumor fragments prepared from a tumor sample obtained or received from surgical resection, needle biopsy, core biopsy, small biopsy, or other means for obtaining a sample that contains a mixture of tumor and TIL cells from the cancer in the patient or subject, to produce a population of CD39 and / or CD103 expressing TILs.

[0018] In one aspect, provided herein is a method of treating a cancer in a patient or subject in need thereof comprising administering a population of tumor infiltrating lymphocytes (TILs), the method comprising the steps of: (a) obtaining and / or receiving a first population of TILs in a tumor sample obtained from surgical resection, needle biopsy, core biopsy, small biopsy, or other means for obtaining a sample that contains a mixture of tumor and TIL cells from the cancer in the patient or subject, (b) selecting CD39 and / or CD103 positive TILs from the first population of TILs in (a) to obtain a population of CD39 and / or CD103 expressing TILs; (c) performing a priming first expansion by culturing the CD39 and / or CD103 expressing TIL population in a first cell culture medium supplemented with IL-2, OKT-3, and antigen presenting cells (APCs) to produce a second population of TILs, wherein the priming first expansion is performed in a container comprising a first gas- permeable surface area, wherein the priming first expansion is performed for first period of about 3- 14 days to obtain the second population of TILs, wherein the second population of TILs is greater in number than the first population of TILs; (d) restimulating the second population of TILs with OKT-3; (e) genetically modifying the second population of TILs to produce a modified second population of TILs, wherein the modified second population of TILs comprises a genetic modification that reduces expression of PD-1, CD39 and / or CD103; (f) performing a rapid second expansion by culturing themodified second population of TILs in a second culture medium supplemented with IL-2, OKT-3, and APCs, to produce a third population of TILs, wherein the rapid second expansion is performed for a second period of about 14 days or less to obtain the third population of TILs, wherein the third population of TILs is a therapeutic population of TILs comprising the genetic modification that reduces expression of PD-1, CD39 and / or CD103; (g) harvesting the therapeutic population of TILs; and (h) administering a therapeutically effective portion of the therapeutic population of TILs to the subject or patient with the cancer.

[0019] In one aspect, provided herein is a method of treating a cancer in a patient or subject in need thereof comprising administering a population of tumor infiltrating lymphocytes (TILs), the method comprising the steps of: (a) selecting CD39 and / or CD103 positive TILs from a first population of TILs in a tumor digest prepared by digesting in an enzymatic digest medium a tumor sample obtained or received from surgical resection, needle biopsy, core biopsy, small biopsy, or other means for obtaining a sample that contains a mixture of tumor and TIL cells from the cancer in the patient or subject, to produce a population of CD39 and / or CD103 expressing TILs; (b) performing a priming first expansion by culturing the CD39 and / or CD103 expressing TIL population in a first cell culture medium supplemented with IL-2, OKT-3, and antigen presenting cells (APCs) to produce a second population of TILs, wherein the priming first expansion is performed in a container comprising a first gas-permeable surface area, wherein the priming first expansion is performed for first period of about 3-14 days to obtain the second population of TILs, wherein the second population of TILs is greater in number than the first population of TILs; (c) restimulating the second population of TILs with OKT-3; (d) genetically modifying the second population of TILs to produce a modified second population of TILs, wherein the modified second population of TILs comprises a genetic modification that reduces expression of PD-1, CD39 and / or CD103; (e) performing a rapid second expansion by culturing the modified second population of TILs in a second culture medium supplemented with IL-2, OKT-3, and APCs, to produce a third population of TILs, wherein the rapid second expansion is performed for a second period of about 14 days or less to obtain the third population of TILs, wherein the third population of TILs is a therapeutic population of TILs comprising the genetic modification that reduces expression of PD-1, CD39 and / or CD103; (f) harvesting the therapeutic population of TILs; and (g) administering a therapeutically effective portion of the therapeutic population of TILs to the subject or patient with the cancer. In some embodiments, step (a) comprises selecting CD39 and / or CD103 positive TILs from a first population of TILs in a tumor digest prepared by digesting in an enzymatic digest medium a plurality of tumor fragments prepared from a tumor sample obtained or received from surgical resection, needle biopsy, core biopsy, smallbiopsy, or other means for obtaining a sample that contains a mixture of tumor and TIL cells from the cancer in the patient or subject, to produce a population of CD39 and / or CD103 expressing TILs.

[0020] In one aspect, provided herein is a method of expanding tumor infiltrating lymphocytes (TILs) into a therapeutic population of TILs comprising: (a) obtaining and / or receiving a first population of TILs in a plurality of tumor fragments prepared from a tumor sample resected from a cancer in a subject; (b) selecting CD39 and / or CD103 positive TILs from the first population of TILs in step (a) to obtain a population of CD39 and / or CD103 expressing TILs; (c) performing a priming first expansion by culturing the PD-l expressing TIL population in a first cell culture medium supplemented with IL-2, OKT-3, and antigen presenting cells (APCs) to produce a second population of TILs, wherein the priming first expansion is performed in a container comprising a first gas- permeable surface area, wherein the priming first expansion is performed for first period of about 1 to 7 / 8 days to obtain the second population of TILs, wherein the second population of TILs is greater in number than the first population of TILs; (d) performing a rapid second expansion by culturing the second population of TILs in a second culture medium supplemented with IL-2, OKT-3, and APCs, to produce a third population of TILs, wherein the number of APCs added in the rapid second expansion is at least twice the number of APCs added in step (b), wherein the rapid second expansion is performed for a second period of about 1 to 11 days to obtain the third population of TILs, wherein the third population of TILs is a therapeutic population of TILs, wherein the rapid second expansion is performed in a container comprising a second gas-permeable surface area; (e) harvesting the therapeutic population of TILs obtained from step (d); (f) transferring the harvested therapeutic population of TILs from step (e) to an infusion bag, and (g) genetically modifying the population of CD39 and / or CD103 expressing TILs, the second population of TILs and / or the third population of TILs at any time after the selecting CD39 and / or CD103 positive TILs (b) and prior to the transfer to the infusion bag (f) such that the transferred therapeutic population of TILs comprises genetically modified TILs comprising a genetic modification that reduces expression of PD-1, CD39 and / or CD103.

[0021] In one aspect, provided herein is a method for expanding tumor infiltrating lymphocytes (TILs) into a therapeutic population of TILs comprising: (a) selecting CD39 and / or CD103 positive TILs from a first population of TILs in a tumor digest obtained from digesting in an enzymatic digest medium a plurality of tumor fragments prepared from a tumor sample resected from a cancer in a subject to obtain a population of CD39 and / or CD103 expressing TILs; (b) performing a priming first expansion by culturing the CD39 and / or CD103 expressing TIL population in a first cell culture medium supplemented with IL-2, OKT-3, and antigen presenting cells (APCs) to produce a secondpopulation of TILs, wherein the priming first expansion is performed in a container comprising a first gas-permeable surface area, wherein the priming first expansion is performed for first period of about 1 to 7 / 8 days to obtain the second population of TILs, wherein the second population of TILs is greater in number than the first population of TILs; (c) performing a rapid second expansion by culturing the second population of TILs in a second culture medium supplemented with IL-2, OKT-3, and APCs, to produce a third population of TILs, wherein the number of APCs added in the rapid second expansion is at least twice the number of APCs added in step (a), wherein the rapid second expansion is performed for a second period of about 1 to 11 days to obtain the third population of TILs, wherein the third population of TILs is a therapeutic population of TILs, wherein the rapid second expansion is performed in a container comprising a second gas-permeable surface area; (d) harvesting the therapeutic population of TILs obtained from step (c); (e) transferring the harvested therapeutic population of TILs from step (d) to an infusion bag, and (f) genetically modifying the population of CD39 and / or CD103 expressing TILs, the second population of TILs and / or the third population of TILs at any time after the selecting CD39 and / or CD103 positive TILs (a) and prior to the transfer to the infusion bag (e) such that the transferred therapeutic population of TILs comprises genetically modified TILs comprising a genetic modification that reduces expression of PD- 1, CD39 and / or CD103.

[0022] In another aspect, provided herein is a method of expanding tumor infiltrating lymphocytes (TILs) into a therapeutic population of TILs, the method comprising the steps of: (a) obtaining and / or receiving a first population of TILs in a plurality of tumor fragments prepared from a tumor sample resected from a cancer in a subject or patient; (b) selecting CD39 and / or CD103 positive TILs from the first population of TILs in (a) to obtain a population of CD39 and / or CD103 expressing TILs; (c) adding the population of CD39 and / or CD103 expressing TILs into a closed system; (d) performing a first expansion by culturing the population of CD39 and / or CD103 expressing TILs in a first cell culture medium supplemented with IL-2 to produce a second population of TILs, wherein the first expansion is performed in a closed container providing a first gas-permeable surface area, wherein the first expansion is performed for about 3-14 days to obtain the second population of TILs, and wherein the transition from step (c) to step (d) occurs without opening the system; (e) performing a second expansion by culturing the second population of TILs in a second cell culture medium supplemented with IL-2, OKT-3, and antigen presenting cells (APCs), to produce a third population of TILs, wherein the second expansion is performed for about 7-14 days to obtain the third population of TILs, wherein the third population of TILs is a therapeutic population of TILs, wherein the second expansion is performed in a closed container providing a second gas-permeable surface area, and wherein the transition from step (d) to step (e) occurs without opening the system; (f) harvesting thetherapeutic population of TILs obtained from step (e), wherein the transition from step (e) to step (f) occurs without opening the system; (g) transferring the harvested therapeutic population of TILs from step (f) to an infusion bag, wherein the transfer from step (f) to (g) occurs without opening the system; and (h) genetically modifying the population of CD39 and / or CD103 expressing TILs, the second population of TILs and / or the third population of TILs at any time after the selecting CD39 and / or CD103 positive TILs (b) and prior to the transfer to the infusion bag (g) such that the transferred third population of TILs comprises genetically modified TILs comprising a genetic modification that reduces expression of PD-1, CD39 and / or CD103.

[0023] In another aspect, provided herein is a method of expanding tumor infiltrating lymphocytes (TILs) into a therapeutic population of TILs, the method comprising the steps of: (a) selecting CD39 and / or CD103 positive TILs from a first population of TILs in a tumor digest prepared by digesting in an enzymatic digest medium a plurality of tumor fragments prepared from a tumor sample obtained or received from surgical resection, needle biopsy, core biopsy, small biopsy, or other means for obtaining a sample that contains a mixture of tumor and TIL cells from the cancer in the patient or subject, to produce a population of CD39 and / or CD103 expressing TILs; (b) performing a first expansion by culturing the population of CD39 and / or CD103 expressing TILs in a first cell culture medium supplemented with IL-2 to produce a second population of TILs, wherein the first expansion is performed in a closed container providing a first gas-permeable surface area, wherein the first expansion is performed for about 3-14 days to obtain the second population of TILs; (c) performing a second expansion by culturing the second population of TILs in a second cell culture medium supplemented with IL-2, OKT-3, and antigen presenting cells (APCs), to produce a third population of TILs, wherein the second expansion is performed for about 7-14 days to obtain the third population of TILs, wherein the third population of TILs is a therapeutic population of TILs, wherein the second expansion is performed in a closed container providing a second gas-permeable surface area, and wherein the transition from step (b) to step (c) occurs without opening the system; (d) harvesting the therapeutic population of TILs obtained from step (c), wherein the transition from step (c) to step (d) occurs without opening the system; (e) transferring the harvested therapeutic population of TILs from step (d) to an infusion bag, wherein the transfer from step (d) to (e) occurs without opening the system; and (f) genetically modifying the population of CD39 and / or CD103 expressing TILs, the second population of TILs and / or the third population of TILs at any time after the selecting CD39 and / or CD103 positive TILs (a) and prior to the transfer to the infusion bag (e) such that the transferred third population of TILs comprises genetically modified TILs comprising a genetic modification that reduces expression of PD-1, CD39 and / or CD103.

[0024] In another aspect, provided herein is a method of expanding tumor infiltrating lymphocytes (TILs) into a therapeutic population of TILs, the method comprising the steps of: (a) obtaining a first population of TILs in a plurality of tumor fragments prepared from a tumor sample resected from a cancer in a subject; (b) selecting CD39 and / or CD103 positive TILs from the first population of TILs in (a) to obtain a population of CD39 and / or CD103 expressing TILs; (c) adding the population of CD39 and / or CD103 expressing TILs into a closed system; (d) performing a first expansion by culturing population of CD39 and / or CD103 expressing TILs in a first cell culture medium supplemented with IL-2 to produce a second population of TILs, wherein the first expansion is performed in a closed container providing a first gas-permeable surface area, wherein the first expansion is performed for about 3-11 days to obtain the second population of TILs, and wherein the transition from step (c) to step (d) occurs without opening the system; (e) performing a second expansion by culturing the second population of TILs in a second cell culture medium supplemented with IL-2, OKT-3, and antigen presenting cells (APCs), to produce a third population of TILs, wherein the second expansion is performed for about 7-11 days to obtain the third population of TILs, wherein the second expansion is performed in a closed container providing a second gas-permeable surface area, and wherein the transition from step (d) to step (e) occurs without opening the system; (f) harvesting the third population of TILs obtained from step (e), wherein the transition from step (e) to step (f) occurs without opening the system; (g) transferring the harvested third population of TILs from step (f) to an infusion bag, wherein the transfer from step (f) to (g) occurs without opening the system; and (h) genetically modifying the population of CD39 and / or CD103 expressing TILs, the second population of TILs and / or the third population of TILs at any time after the selecting CD39 and / or CD103 positive TILs (b) and prior to the transfer to the infusion bag (g) such that the transferred third population of TILs comprises genetically modified TILs comprising a genetic modification that reduces expression of PD-1, CD39 and / or CD103.

[0025] In one aspect, provided herein is a method of expanding tumor infiltrating lymphocytes (TILs) into a therapeutic population of TILs, the method comprising the steps of: (a) selecting CD39 and / or CD103 positive TILs from a first population of TILs in a tumor digest produced by digesting in an enzymatic digest medium a tumor sample resected from a cancer in a patient or subject to obtain a population of CD39 and / or CD103 expressing TILs; (b) performing a first expansion by culturing population of CD39 and / or CD103 expressing TILs in a first cell culture medium supplemented with IL-2 to produce a second population of TILs, wherein the first expansion is performed in a closed container providing a first gas-permeable surface area, wherein the first expansion is performed for about 3-11 days to obtain the second population of TILs; (c) performing a second expansion by culturing the second population of TILs in a second cell culture medium supplemented with IL-2,OKT-3, and antigen presenting cells (APCs), to produce a third population of TILs, wherein the second expansion is performed for about 7-11 days to obtain the third population of TILs, wherein the second expansion is performed in a closed container providing a second gas-permeable surface area, and wherein the transition from step (b) to step (c) occurs without opening the system; (d) harvesting the third population of TILs obtained from step (c), wherein the transition from step (c) to step (d) occurs without opening the system; (e) transferring the harvested third population of TILs from step (d) to an infusion bag, wherein the transfer from step (d) to (e) occurs without opening the system; and (f) genetically modifying the population of CD39 and / or CD103 expressing TILs, the second population of TILs and / or the third population of TILs at any time after the selecting CD39 and / or CD103 positive TILs (a) and prior to the transfer to the infusion bag (e) such that the transferred third population of TILs comprises genetically modified TILs comprising a genetic modification that reduces expression of PD-1, CD39 and / or CD103. In some embodiments, step (a) comprises selecting CD39 and / or CD103 positive TILs from a first population of TILs in a tumor digest produced by digesting in an enzymatic digest medium a plurality of tumor fragments prepared from a tumor sample resected from a cancer in a patient or subject to obtain a population of CD39 and / or CD103 expressing TILs.

[0026] In another aspect, provided herein is a method of expanding tumor infiltrating lymphocytes (TILs) into a therapeutic population of TILs, the method comprising the steps of: (a) obtaining and / or receiving a first population of TILs in a tumor sample obtained from surgical resection, needle biopsy, core biopsy, small biopsy, or other means for obtaining a sample that contains a mixture of tumor and TIL cells from a cancer in a patient or subject; (b) selecting CD39 and / or CD103 positive TILs from the first population of TILs in (a) to obtain a population of CD39 and / or CD103 expressing TILs; (c) adding the population of CD39 and / or CD103 expressing TILs into a closed system; (d) performing a first expansion by culturing the population of CD39 and / or CD103 expressing TILs in a first cell culture medium supplemented with IL-2 to produce a second population of TILs, wherein the first expansion is performed in a closed container providing a first gas-permeable surface area, wherein the first expansion is performed for about 3-11 days to obtain the second population of TILs, and wherein the transition from step (c) to step (d) occurs without opening the system; (e) performing a second expansion by culturing the second population of TILs in a second cell culture medium supplemented with IL-2, OKT-3, and antigen presenting cells (APCs), to produce a third population of TILs, wherein the second expansion is performed for about 7-11 days to obtain the third population of TILs, wherein the second expansion is performed in a closed container providing a second gas-permeable surface area, and wherein the transition from step (d) to step (e) occurs without opening the system; (f) harvesting the third population of TILs obtained from step (e),wherein the transition from step (e) to step (f) occurs without opening the system; (g) transferring the harvested third population of TILs from step (f) to an infusion bag, wherein the transfer from step (e) to (f) occurs without opening the system; and (h) genetically modifying the population of CD39 and / or CD103 expressing TILs, the second population of TILs and / or the third population of TILs at any time after the selecting CD39 and / or CD103 positive TILs (b) and prior to the transfer to the infusion bag (g) such that the transferred third population of TILs comprises genetically modified TILs comprising a genetic modification that reduces expression of PD-1, CD39 and / or CD103.

[0027] In another aspect, provided herein is a method of expanding tumor infiltrating lymphocytes (TILs) to a therapeutic population of TILs, the method comprising the steps of: (a) resecting a tumor sample from a cancer in subject or patient, the tumor sample comprising a first population of TILs, optionally from surgical resection, needle biopsy, core biopsy, small biopsy, or other means for obtaining a sample that contains a mixture of tumor and TIL cells from the cancer; (b) processing the tumor sample into a plurality of tumor fragments; (c) enzymatically digesting in an enzymatic digest medium the plurality of tumor fragments to obtain the first population of TILs; (d) selecting CD39 and / or CD103 positive TILs from the first population of TILs in (c) to obtain a population of CD39 and / or CD103 expressing TILs; (e) adding the population of CD39 and / or CD103 expressing TILs into a closed system; (f) performing a first expansion by culturing the population of CD39 and / or CD103 expressing TILs in a first cell culture medium supplemented with IL-2 to produce a second population of TILs, wherein the first expansion is performed in a closed container providing a first gas- permeable surface area, wherein the first expansion is performed for about 3-11 days to obtain the second population of TILs, and wherein the transition from step (e) to step (f) occurs without opening the system; (g) performing a second expansion by culturing the second population of TILs in a second cell culture medium supplemented with IL-2, OKT-3, and antigen presenting cells (APCs), to produce a third population of TILs, wherein the second expansion is performed for about 7-11 days to obtain the third population of TILs, wherein the second expansion is performed in a closed container providing a second gas-permeable surface area, and wherein the transition from step (f) to step (g) occurs without opening the system; (h) harvesting the third population of TILs obtained from step (g), wherein the transition from step (g) to step (h) occurs without opening the system; (i) transferring the harvested third TIL population from step (h) to an infusion bag, wherein the transfer from step (h) to (i) occurs without opening the system; and (j) genetically modifying the population of CD39 and / or CD103 expressing TILs, the second population of TILs and / or the third population of TILs at any time after the selecting CD39 and / or CD103 positive TILs (d) and prior to the transfer to the infusion bag (h) such that the transferred third population of TILs comprises genetically modified TILs comprising a genetic modification that reduces expression of PD-1, CD39 and / or CD103.

[0028] In another aspect, provided herein is a method of expanding tumor infiltrating lymphocytes (TILs) into a therapeutic population of TILs, the method comprising the steps of: (a) selecting CD39 and / or CD103 positive TILs from a first population of TILs in a tumor digest prepared by digesting in an enzymatic digest medium a tumor sample obtained or received from surgical resection, needle biopsy, core biopsy, small biopsy, or other means for obtaining a sample that contains a mixture of tumor and TIL cells from a cancer in a patient or subject, to produce a population of CD39 and / or CD103 expressing TILs; (b) performing a first expansion by culturing the population of CD39 and / or CD103 expressing TILs in a first cell culture medium supplemented with IL-2 to produce a second population of TILs, wherein the first expansion is performed in a closed container providing a first gas-permeable surface area, wherein the first expansion is performed for about 3-11 days to obtain the second population of TILs; (c) performing a second expansion by culturing the second population of TILs in a second cell culture medium supplemented with IL-2, OKT-3, and antigen presenting cells (APCs), to produce a third population of TILs, wherein the second expansion is performed for about 7-11 days to obtain the third population of TILs, wherein the second expansion is performed in a closed container providing a second gas-permeable surface area, and wherein the transition from step (b) to step (c) occurs without opening the system; (d) harvesting the third population of TILs obtained from step (c), wherein the transition from step (c) to step (d) occurs without opening the system; (e) transferring the harvested third population of TILs from step (d) to an infusion bag, wherein the transfer from step (d) to (e) occurs without opening the system; and (f) genetically modifying the population of CD39 and / or CD103 expressing TILs, the second population of TILs and / or the third population of TILs at any time after the selecting CD39 and / or CD103 positive TILs (a) and prior to the transfer to the infusion bag (e) such that the transferred third population of TILs comprises genetically modified TILs comprising a genetic modification that reduces expression of PD- 1, CD39 and / or CD103. In some embodiments, step (a) comprises selecting CD39 and / or CD103 positive TILs from a first population of TILs in a tumor digest prepared by digesting in an enzymatic digest medium a plurality of tumor fragments prepared from a tumor sample obtained or received from surgical resection, needle biopsy, core biopsy, small biopsy, or other means for obtaining a sample that contains a mixture of tumor and TIL cells from a cancer in a patient or subject, to produce a population of CD39 and / or CD103 expressing TILs.

[0029] In another aspect, provided herein is a method of expanding tumor infiltrating lymphocytes (TILs) into a therapeutic population of TILs, the method comprising the steps of: (a) obtaining and / or receiving a first population of TILs in a tumor sample obtained from surgical resection, needle biopsy, core biopsy, small biopsy, or other means for obtaining a sample that contains a mixture of tumor and TIL cells from a cancer in the subject or patient; (b) selecting CD39 and / or CD103 positiveTILs from the first population of TILs in (a) to obtain a population of CD39 and / or CD103 expressing TILs; (c) performing an initial expansion (or priming first expansion) of the population of CD39 and / or CD103 expressing TILs in a first cell culture medium to obtain a second population of TILs, wherein the first cell culture medium is supplemented with IL-2, optionally OKT-3 (anti-CD3 antibody), and optionally antigen presenting cells (APCs), where the priming first expansion occurs for a period of 1 to 8 days; (d) performing a rapid second expansion of the second population of TILs in a second cell culture medium to obtain a third population of TILs, wherein the second cell culture medium is supplemented with IL-2, OKT-3 (anti-CD3 antibody), and APCs, and wherein the rapid expansion is performed over a period of 14 days or less, optionally the rapid second expansion can proceed for 1 day, 2 days, 3 days, 4, days, 5 days, 6 days, 7 days, 8 days, 9 days or 10 days after initiation of the rapid second expansion; (e) harvesting the third population of TILs; and (f) genetically modifying the population of CD39 and / or CD103 expressing TILs, the second population of TILs and / or the third population of TILs at any time after the selecting CD39 and / or CD103 positive TILs (b) and prior to the harvesting (f) such that the harvested third population of TILs comprises genetically modified TILs comprising a genetic modification that reduces expression of PD-1, CD39 and / or CD103.

[0030] In one aspect, provided herein is a method of expanding tumor infiltrating lymphocytes (TILs) into a therapeutic population of TILs, the method comprising the steps of: (a) obtaining a tumor sample from the cancer in the subject or patient, the tumor sample comprising a first population of TILs, optionally from surgical resection, needle biopsy, core biopsy, small biopsy, or other means for obtaining a sample that contains a mixture of tumor and TIL cells from the cancer; (b) fragmenting the tumor sample into a plurality of tumor fragments; (c) selecting CD39 and / or CD103 positive TILs from the first population of TILs of the tumor fragments to obtain a population of CD39 and / or CD103 expressing TILs; (d) performing an initial expansion (or priming first expansion) of the population of CD39 and / or CD103 expressing TILs in a first cell culture medium to obtain a second population of TILs, wherein the first cell culture medium is supplemented with IL-2, optionally OKT-3 (anti-CD3 antibody), and optionally antigen presenting cells (APCs), where the priming first expansion occurs for a period of 1 to 8 days; (e) performing a rapid second expansion of the second population of TILs in a second cell culture medium to obtain a third population of TILs, wherein the second cell culture medium is supplemented with IL-2, OKT-3 (anti-CD3 antibody), and APCs, and wherein the rapid expansion is performed over a period of 14 days or less, optionally the rapid second expansion can proceed for 1 day, 2 days, 3 days, 4, days, 5 days, 6 days, 7 days, 8 days, 9 days or 10 days after initiation of the rapid second expansion; (f) harvesting the third population of TILs; and (g) genetically modifying the population of CD39 and / or CD103 expressing TILs, the second population of TILs and / or the third population of TILs at any time after the selecting CD39 and / orCD103 positive TILs (c) and prior to the harvesting (f) such that the harvested third population of TILs comprises genetically modified TILs comprising a genetic modification that reduces expression of PD- 1, CD39 and / or CD103.

[0031] In another aspect, provided herein is a method of expanding tumor infiltrating lymphocytes (TILs) into a therapeutic population of TILs, the method comprising the steps of: (a) selecting CD39 and / or CD103 positive TILs from a first population of TILs in a tumor digest prepared by digesting in an enzymatic digest medium a tumor sample obtained or received from surgical resection, needle biopsy, core biopsy, small biopsy, or other means for obtaining a sample that contains a mixture of tumor and TIL cells from a cancer in a patient or subject, to produce a population of CD39 and / or CD103 expressing TILs; (b) performing an initial expansion (or priming first expansion) of the population of CD39 and / or CD103 expressing TILs in a first cell culture medium to obtain a second population of TILs, wherein the first cell culture medium is supplemented with IL-2, optionally OKT-3 (anti-CD3 antibody), and optionally antigen presenting cells (APCs), where the priming first expansion occurs for a period of 1 to 8 days; (c) performing a rapid second expansion of the second population of TILs in a second cell culture medium to obtain a third population of TILs, wherein the second cell culture medium is supplemented with IL-2, OKT-3 (anti-CD3 antibody), and APCs, and wherein the rapid expansion is performed over a period of 14 days or less, optionally the rapid second expansion can proceed for 1 day, 2 days, 3 days, 4, days, 5 days, 6 days, 7 days, 8 days, 9 days or 10 days after initiation of the rapid second expansion; (d) harvesting the third population of TILs; and (e) genetically modifying the population of CD39 and / or CD103 expressing TILs, the second population of TILs and / or the third population of TILs at any time after the selecting CD39 and / or CD103 positive TILs (a) and prior to the harvesting (d) such that the harvested third population of TILs comprises genetically modified TILs comprising a genetic modification that reduces expression of PD-1, CD39 and / or CD103.

[0032] In some embodiments, step (a) comprises selecting CD39 and / or CD103 positive TILs from a first population of TILs in a tumor digest prepared by digesting in an enzymatic digest medium a plurality of tumor fragments prepared from a tumor sample obtained or received from surgical resection, needle biopsy, core biopsy, small biopsy, or other means for obtaining a sample that contains a mixture of tumor and TIL cells from the cancer in the patient or subject, to produce a population of CD39 and / or CD103 expressing TILs.

[0033] In one aspect, provided herein is a method for expanding tumor infiltrating lymphocytes (TILs) into a therapeutic population of TILs comprising: (a) obtaining and / or receiving a first population of TILs in a plurality of tumor fragments prepared from a tumor sample resected from acancer in a subject; (b) enzymatically digesting in an enzymatic digest medium the plurality of tumor fragments to obtain the first population of TILs; (c) selecting PD-l positive TILs from the first population of TILs in step (b) to obtain a population of CD39 and / or CD103 expressing TILs; (d) performing a priming first expansion by culturing the population of CD39 and / or CD103 expressing TILs in a first cell culture medium supplemented with IL-2, anti-CD3 agonist antibody, and antigen presenting cells (APCs), to produce a second population of TILs, wherein the priming first expansion is performed for a first period of about 1 to 11 days to obtain the second population of TILs, wherein the second population of TILs is greater in number than the first population of TILs; (e) restimulating the second population of TILs with anti-CD3 agonist antibody; (f) genetically modifying the second population of TILs to produce a modified second population of TILs, wherein the modified second population of TILs comprises a genetic modification that reduces expression of PD-1, CD39 and / or CD103; (g) performing a rapid second expansion by culturing the modified second population of TILs in a second cell culture medium supplemented with IL-2, anti-CD3 agonist antibody, and APCs, to produce a third population of TILs, wherein the rapid second expansion is performed for a second period of about 1 to 11 days to obtain the third population of TILs, wherein the third population of TILs is a therapeutic population of TILs; and (h) harvesting the therapeutic population of TILs obtained from step (g).

[0034] In certain embodiments, provided herein is a method for expanding tumor infiltrating lymphocytes (TILs) into a therapeutic population of TILs comprising: (a) selecting CD39 and / or CD103 positive TILs from a first population of TILs in a tumor digest prepared by enzymatically digesting in an enzymatic digest medium a plurality of tumor fragments prepared from a tumor sample obtained or received from surgical resection, needle biopsy, core biopsy, small biopsy, or other means for obtaining a sample that contains a mixture of tumor and TIL cells from a cancer in a patient or subject, to produce a population of CD39 and / or CD103 expressing TILs; (b) performing a priming first expansion by culturing the population of CD39 and / or CD103 expressing TILs in a first cell culture medium supplemented with IL-2, anti-CD3 agonist antibody, and antigen presenting cells (APCs), to produce a second population of TILs, wherein the priming first expansion is performed for a first period of about 1 to 11 days to obtain the second population of TILs, wherein the second population of TILs is greater in number than the first population of TILs; (c) restimulating the second population of TILs with anti-CD3 agonist antibody; (d) genetically modifying the second population of TILs to produce a modified second population of TILs, wherein the modified second population of TILs comprises a genetic modification that reduces expression of PD-1, CD39 and / or CD103; (e) performing a rapid second expansion by culturing the modified second population of TILs in a second cell culture medium supplemented with IL-2, anti-CD3 agonist antibody, and APCs, to produce a thirdpopulation of TILs, wherein the rapid second expansion is performed for a second period of about 1 to 11 days to obtain the third population of TILs, wherein the third population of TILs is a therapeutic population of TILs; and (f) harvesting the therapeutic population of TILs obtained from step (e). In some embodiments, wherein in step (d) the cell culture medium further comprises antigen- presenting cells (APCs), and wherein the number of APCs in the culture medium in step (e) is greater than the number of APCs in the culture medium in step (d).

[0035] In another aspect, provided herein is a method for expanding tumor infiltrating lymphocytes (TILs) into a therapeutic population of TILs comprising: (a) obtaining and / or receiving a first population of TILs in a tumor sample obtained from surgical resection, needle biopsy, core biopsy, small biopsy, or other means for obtaining a sample that contains a mixture of tumor and TIL cells from a cancer in a patient or subject, (b) enzymatically digesting in an enzymatic digest medium the tumor sample to obtain the first population of TILs; (c) selecting CD39 and / or CD103 positive TILs from the first population of TILs in (b) to obtain a population of CD39 and / or CD103 expressing TILs; (d) performing a priming first expansion by culturing the PD-l expressing TIL population in a first cell culture medium supplemented with IL-2, anti-CD3 agonist antibody, and antigen presenting cells (APCs) to produce a second population of TILs, wherein the priming first expansion is performed in a container comprising a first gas-permeable surface area, wherein the priming first expansion is performed for first period of about 3-14 days to obtain the second population of TILs, wherein the second population of TILs is greater in number than the first population of TILs; (e) restimulating the second population of TILs with anti-CD3 agonist antibody; (f) genetically modifying the second population of TILs to produce a modified second population of TILs, wherein the modified second population of TILs comprises a genetic modification that reduces expression of PD-1, CD39 and / or CD103; (g) performing a rapid second expansion by culturing the modified second population of TILs in a second culture medium supplemented with IL-2, anti-CD3 agonist antibody, and APCs, to produce a third population of TILs, wherein the rapid second expansion is performed for a second period of about 14 days or less to obtain the third population of TILs, wherein the third population of TILs comprises the genetic modification that reduces expression of PD-1, CD39 and / or CD103; and (h) harvesting the third population of TILs.

[0036] In one aspect, provided herein is a method for expanding tumor infiltrating lymphocytes (TILs) into a therapeutic population of TILs comprising: (a) selecting CD39 and / or CD103 positive TILs from a first population of TILs in a tumor digest prepared by enzymatically digesting in an enzymatic digest medium a tumor sample obtained or received from surgical resection, needle biopsy, core biopsy, small biopsy, or other means for obtaining a sample that contains a mixture of tumor and TILcells from a cancer in a patient or subject, to produce a population of CD39 and / or CD103 expressing TILs; (b) performing a priming first expansion by culturing the PD-l expressing TIL population in a first cell culture medium supplemented with IL-2, anti-CD3 agonist antibody, and antigen presenting cells (APCs) to produce a second population of TILs, wherein the priming first expansion is performed in a container comprising a first gas-permeable surface area, wherein the priming first expansion is performed for first period of about 3-14 days to obtain the second population of TILs, wherein the second population of TILs is greater in number than the first population of TILs; (c) restimulating the second population of TILs with anti-CD3 agonist antibody; (d) genetically modifying the second population of TILs to produce a modified second population of TILs, wherein the modified second population of TILs comprises a genetic modification that reduces expression of PD-1, CD39 and / or CD103; (e) performing a rapid second expansion by culturing the modified second population of TILs in a second culture medium supplemented with IL-2, anti-CD3 agonist antibody, and APCs, to produce a third population of TILs, wherein the rapid second expansion is performed for a second period of about 14 days or less to obtain the third population of TILs, wherein the third population of TILs comprises the genetic modification that reduces expression of PD-1, CD39 and / or CD103; and (f) harvesting the third population of TILs. In some embodiments, step (a) comprises selecting CD39 and / or CD103 positive TILs from a first population of TILs in a tumor digest prepared by digesting in an enzymatic digest medium a plurality of tumor fragments prepared from a tumor sample obtained or received from surgical resection, needle biopsy, core biopsy, small biopsy, or other means for obtaining a sample that contains a mixture of tumor and TIL cells from a cancer in a patient or subject, to produce a population of CD39 and / or CD103 expressing TILs.

[0037] In some embodiments, the anti-CD3 agonist antibody is OKT-3.

[0038] In some embodiments of the subject method, the cancer is selected from the group consisting of melanoma, ovarian cancer, cervical cancer, non-small-cell lung cancer (NSCLC), lung cancer, bladder cancer, breast cancer, triple negative breast cancer, cancer caused by human papilloma virus, head and neck cancer (including head and neck squamous cell carcinoma (HNSCC)), renal cancer, and renal cell carcinoma.

[0039] In one aspect, provided herein is a method for expanding tumor infiltrating lymphocytes (TILs) into a therapeutic population of TILs comprising: (a) performing a priming first expansion by culturing a first population of CD39 and / or CD103 expressing TILs in a first cell culture medium supplemented with IL-2, optionally OKT-3, and optionally comprising antigen presenting cells (APCs), to produce a second population of TILs, wherein the priming first expansion is performed for a first period of about 1 to 11 days to obtain the second population of TILs, whereinthe second population of TILs is greater in number than the first population of TILs; (b) performing a rapid second expansion by culturing the second population of TILs in a second cell culture medium supplemented with IL-2, OKT-3, and APCs, to produce a third population of TILs, wherein the rapid second expansion is performed for a second period of about 1 to 11 days to obtain the third population of TILs, wherein the third population of TILs is a therapeutic population of TILs; (c) harvesting the third population of TILs obtained from step (b); and (d) genetically modifying the population of TILs, the second population of TILs and / or the third population of TILs at any time prior to the harvesting step (c) such that the harvested third population of TILs comprises genetically modified TILs comprising a genetic modification that reduces or increases the expression of one or more T cell exhaustion markers and / or one or more factors phenotypically associated with said T cell exhaustion markers. In some embodiments, in step (a) the cell culture medium further comprises antigen-presenting cells (APCs), and wherein the number of APCs in the culture medium in step (c) is greater than the number of APCs in the culture medium in step (b).

[0040] In another aspect, provided herein is a method of expanding T cells comprising: (a) performing a priming first expansion of a first population of TILs obtained from a donor by culturing the first population of TILs to effect growth and to prime an activation of the first population of T cells, wherein the first population of TILs is a population of TILs that expresses at least CD39 and CD103; (b) after the activation of the first population of T cells primed in step (a) begins to decay, performing a rapid second expansion of the first population of T cells by culturing the first population of T cells to effect growth and to boost the activation of the first population of T cells to obtain a second population of T cells; (c) harvesting the second population of T cells; and (d) genetically modifying the first population of TILs and / or the second population of TILs such that the harvested second population of TILs comprises genetically modified TILs comprising a genetic modification that reduces or increases the expression of one or more T cell exhaustion markers and / or one or more factors phenotypically associated with said T cell exhaustion markers.

[0041] In one aspect, provided herein is a method of expanding T cells comprising: (a) performing a priming first expansion of a first population of T cells from a tumor sample obtained from one or more small biopsies, core biopsies, or needle biopsies of a tumor in a donor by culturing the first population of T cells to effect growth and to prime an activation of the first population of T cells, wherein the first population of T cells is a population of T cells that expresses at least CD39 and CD103; (b) after the activation of the first population of T cells primed in step (a) begins to decay, performing a rapid second expansion of the first population of T cells by culturing the first population of T cells to effect growth and to boost the activation of the first population of T cells toobtain a second population of T cells; (c) harvesting the second population of T cells; and (d) genetically modifying the first population of T cells and / or the second population of TILs such that the harvested second population of T cells comprises genetically modified TILs comprising a genetic modification that reduces or increases the expression of one or more T cell exhaustion markers and / or one or more factors phenotypically associated with said T cell exhaustion markers.

[0042] In some embodiments, the one or more T cell exhaustion markers is selected from PD-1 and CD39, and a combination thereof. In some embodiments, the one or more factors phenotypically associated with said T cell exhaustion markers is CD103.

[0043] In another aspect, the methods herein described comprise an additional selection step. In some embodiments, the method further comprises a second selecting, from the population of TILs, a population of TILs that expresses at least one protein selected from the group consisting of: OX40, 4- 1BB, and combinations thereof. In some embodiments, the second selecting occurs prior to the start of the first expansion. In some embodiments, the second selecting occurs within five days of the start of the first expansion, wherein no OKT-3 has yet been added. In some embodiments, the method further comprises a second selecting, from the second population of TILs, a population of TILs that expresses at least one protein selected from the group consisting of: OX40, 4-1BB and combinations thereof.

[0044] In some embodiments, the modifying is carried out on the second population of TILs from the first expansion, or the third population of TILs from the second expansion, or both. In some embodiments, the modifying is carried out on the second population of TILs from the priming first expansion, or the third population of TILs from the rapid second expansion, or both. In some embodiments, the modifying is carried out on the second population of TILs from the first expansion and before the second expansion. In some embodiments, the modifying is carried out the second population of TILs from the priming first expansion and before the rapid second expansion. In some embodiments, the modifying is carried out on the third population of TILs from the second expansion. In some embodiments, the modifying is carried out on the third population of TILs from the rapid second expansion. In some embodiments, the modifying is carried out after the harvesting.

[0045] In some embodiments, the first expansion is performed over a period of about 11 days. In some embodiments, the priming first expansion is performed over a period of about 11 days.

[0046] In some embodiments, the IL-2 is present at an initial concentration of between 1000 IU / mL and 6000 IU / mL in the cell culture medium in the first expansion. The In some embodiments, the IL-2 is present at an initial concentration of between 1000 IU / mL and 6000 IU / mL in the cell culture medium in the priming first expansion.

[0047] In some embodiments, in the second expansion step, the IL-2 is present at an initial concentration of between 1000 IU / mL and 6000 IU / mL and the OKT-3 antibody is present at an initial concentration of about 30 ng / mL. In some embodiments, in the rapid second expansion step, the IL-2 is present at an initial concentration of between 1000 IU / mL and 6000 IU / mL and the OKT-3 antibody is present at an initial concentration of about 30 ng / mL.

[0048] In some embodiments, the first expansion is performed using a gas permeable container. In some embodiments, the priming first expansion is performed using a gas permeable container. In some embodiments, the second expansion is performed using a gas permeable container. In some embodiments, the rapid second expansion is performed using a gas permeable container.

[0049] In some embodiments, the cell culture medium of the first expansion further comprises a cytokine selected from the group consisting of IL-4, IL-7, IL-15, IL-21, and combinations thereof.

[0050] In some embodiments, the cell culture medium of the priming first expansion further comprises a cytokine selected from the group consisting of IL-4, IL-7, IL-15, IL-21, and combinations thereof.

[0051] In some embodiments, the cell culture medium of the second expansion further comprises a cytokine selected from the group consisting of IL-4, IL-7, IL-15, IL-21, and combinations thereof.

[0052] In some embodiments, the cell culture medium of the rapid second expansion further comprises a cytokine selected from the group consisting of IL-4, IL-7, IL-15, IL-21, and combinations thereof.

[0053] In some embodiments, the method further comprises the step of treating the patient with a non-myeloablative lymphodepletion regimen prior to administering the therapeutic population of TILs to the patient.

[0054] In some embodiments, the non-myeloablative lymphodepletion regimen comprises the steps of administration of cyclophosphamide at a dose of 60 mg / m2 / day for two days followed by administration of fludarabine at a dose of 25 mg / m2 / day for three days. In some embodiments, the non-myeloablative lymphodepletion regimen comprises the steps of administration of cyclophosphamide at a dose of 60 mg / m2 / day and fludarabine at a dose of 25 mg / m2 / day for two days followed by administration of fludarabine at a dose of 25 mg / m2 / day for three days. In some embodiments, the non-myeloablative lymphodepletion regimen comprises the steps ofadministration of cyclophosphamide at a dose of 60 mg / m2 / day and fludarabine at a dose of 25 mg / m2 / day for two days followed by administration of fludarabine at a dose of 25 mg / m2 / day for one day.

[0055] In some embodiments, the non-myeloablative lymphodepletion regimen comprises the steps of administration of cyclophosphamide at a dose of 60 mg / m2 / day for two days followed by administration of fludarabine at a dose of 25 mg / m2 / day for five days.

[0056] In some embodiments, the method further comprises the step cyclophosphamide is administered with mesna.

[0057] In some embodiments, the method further comprises the step of treating the patient with an IL-2 regimen starting on the day after the administration of TILs to the patient.

[0058] In some embodiments, the method further comprises the step of treating the patient with an IL-2 regimen starting on the same day as administration of TILs to the patient.

[0059] In some embodiments, the IL-2 regimen is a high-dose IL-2 regimen comprising 600,000 or 720,000 IU / kg of aldesleukin, or a biosimilar or variant thereof, administered as a 15-minute bolus intravenous infusion every eight hours until tolerance.

[0060] In some embodiments, the therapeutically effective population of TILs comprises from about 2.3×1010to about 13.7×1010TILs.

[0061] In some embodiments, the priming first expansion and rapid second expansion are performed over a period of 21 days or less. In certain embodiments, the priming first expansion and rapid second expansion are performed over a period of 16 or 17 days or less. In certain embodiments, the priming first expansion is performed over a period of 7 or 8 days or less. In certain embodiments, the rapid second expansion is performed over a period of 11 days or less. In some embodiments, the priming first expansion and the rapid second expansion are each individually performed within a period of 11 days.

[0062] In some embodiments of the method, all steps are performed within about 26 days. In certain embodiments, the first cell culture medium and the second cell culture medium are different. In some embodiments, the first cell culture medium and the second cell culture medium are the same.

[0063] In some embodiments, at about 4 or 5 days after initiation of the rapid second expansion the culture is divided into a plurality of subcultures and cultured in a third culture medium supplemented with IL-2 for a period of about 6 or 7 days to produce the third population of TILs.

[0064] In certain embodiments, the priming first expansion is performed in a closed container comprising a first gas permeable surface area, the rapid second expansion is initiated in a closed container comprising a second gas permeable surface area, and the plurality of subcultures are cultured in a plurality of closed containers comprising a third gas permeable surface area.

[0065] In some embodiments, the transfer of the second population of TILs from the closed container comprising the first gas permeable surface area to the closed container comprising the second gas permeable surface area is effected without opening the system, wherein the transfer of the second population of TILs from the closed container comprising the second gas permeable surface area to the plurality of closed containers comprising the third gas permeable surface area is effected without opening the system, and wherein the third population of TILs is harvested from the plurality of closed containers comprising the third gas permeable surface area without opening the system.

[0066] In some embodiments, at about 4 or 5 days after initiation of the second expansion, the culture is divided into a plurality of closed subculture containers each comprising a third gas permeable surface area and cultured in a third cell culture medium supplemented with IL-2 for a period of about 6 or 7 days to produce the third population of TILs.

[0067] In certain embodiments, the division of the culture into the plurality of closed subculture containers effects a transfer of the culture from the closed container comprising the second gas permeable surface to the plurality of subculture containers without opening the system.

[0068] In certain embodiments, the genetically modified TILs further comprises an additional genetic modification that reduces expression of one or more of the following immune checkpoint genes selected from the group comprising CTLA-4, LAG-3, HAVCR2 (TIM-3), Cish, TGFβ, PKA, CBL-B, PPP2CA, PPP2CB, PTPN6, PTPN22, BTLA, CD160, TIGIT, CD96, CRTAM, LAIR1, SIGLEC7, SIGLEC9, CD244, TNFRSF10B, TNFRSF10A, CASP8, CASP10, CASP3, CASP6, CASP7, FADD, FAS, SMAD2, SMAD3, SMAD4, SMAD10, SKI, SKIL, TGIF1, IL10RA, IL10RB, HMOX2, IL6R, IL6ST, EIF2AK4, CSK, PAG1, SIT1, FOXP3, PRDM1, BATF, GUCY1A2, GUCY1A3, GUCY1B2, GUCY1B3, TOX, SOCS1, ANKRD11, and BCOR. In exemplary embodiments, the one or more immune checkpoint genes is / are selected from the group comprising PD-1, CTLA-4, LAG-3, HAVCR2 (TIM-3), Cish, TGFβ, and PKA.

[0069] In some embodiments, the genetically modified TILs further comprises an additional genetic modification that causes expression of one or more immune checkpoint genes to be enhanced in at least a portion of the therapeutic population of TILs, the immune checkpoint gene(s) being selected from the group comprising CCR2, CCR4, CCR5, CXCR2, CXCR3, CX3CR1, IL-2, IL-4, IL-7, IL-10, IL-15, IL- 21, the NOTCH 1 / 2 intracellular domain (ICD), and / or the NOTCH ligand mDLL1.

[0070] In certain embodiments, the genetic modification step is performed on the second population of TILs before initiation of the second expansion or rapid second expansion, and wherein the method comprises restimulating the second population of TILs with OKT-3 for about 2 days before performing the genetic modification step.

[0071] In some embodiments, the modified second population of TILs is rested for about 1 day after the genetic modification step and before initiation of the second expansion or rapid second expansion.

[0072] In some embodiments, the genetically modifying step is performed using a programmable nuclease that mediates the generation of a double-strand or single-strand break at said one or more immune checkpoint genes.

[0073] In some embodiments, the genetically modifying step is performed using one or more methods selected from a CRISPR method, a TALE method, a zinc finger method, and a combination thereof. In some embodiments, the genetically modifying step is performed using a CRISPR method. In some embodiments, the CRISPR method is a CRISPR / Cas9 method. In some embodiments, the genetically modifying step is performed using a TALE method. In some embodiments, the genetically modifying step is performed using a zinc finger method.

[0074] In some embodiments, processing a tumor sample obtained from the subject into a tumor digest comprises incubating the tumor sample in an enzymatic media. In some embodiments, processing a tumor sample obtained from the subject into a tumor digest further comprises disrupting the tumor sample mechanically so as to dissociate the tumor sample. In some embodiments, processing a tumor sample obtained from the subject into a tumor digest further comprises purifying the disassociated tumor sample using a density gradient separation.

[0075] In some embodiments, the tumor sample or plurality of tumor fragments are digested in an enzymatic digest medium before the selecting step to produce a tumor digest comprising the first population of TILs. In some embodiments, the enzymatic digest medium comprises a mixture of enzymes. In some embodiments, the enzymatic digest medium comprises a collagenase, a neutralprotease, and a DNase. In some embodiments, the enzymatic media comprises 30 units / mL of DNase. In some embodiments, the enzymatic digest medium comprises a collagenase. In some embodiments, the enzymatic media comprises 1.0 mg / mL of collagenase. In some embodiments, the enzymatic digest medium comprises a DNase. In some embodiments, the enzymatic digest medium comprises a neutral protease. In some embodiments, the enzymatic digest medium comprises a hyaluronidase.

[0076] In some embodiments, the tumor sample or plurality of tumor fragments are subjected to mechanical dissociation before, during and / or after the digestion of the tumor sample or plurality of tumor fragments.

[0077] In one aspect, the methods and compositions herein disclosed describe therapeutic populations of TILs. In some embodiments, the therapeutic population of TILs harvested comprises sufficient TILs for use in administering a therapeutically effective dosage to a subject. In some embodiments, the therapeutically effective dosage comprises from about 1×109to about 9×1010TILs. In some embodiments, the therapeutic population of TILs harvested exhibits an increased subpopulation of CD8+ cells relative to the first and / or second population of TILs.

[0078] In some embodiments, the APCs comprise peripheral blood mononuclear cells (PBMCs). In some embodiments, the PBMCs are supplemented at a ratio of about 1:25 TIL:PBMCs.

[0079] In some embodiments, the first expansion and the second expansion are each individually performed within a period of 11-12 days. In some embodiments, steps (a) through (e), (f), or (g) of the methods described above are performed in about 10 days to about 24 days. In some embodiments, steps (a) through (e), (f), or (g) of the methods described above are performed in about 15 days to about 24 days. In some embodiments, steps (a) through (e), (f), or (g) of the methods described above are performed in about 20 days to about 24 days. In some embodiments, steps (a) through (e), (f), or (g) of the methods described above are performed in about 20 days to about 22 days.

[0080] In some embodiments, the second population of TILs is at least 50-fold greater in number than the first population of TILs.

[0081] In one aspect, the instant disclosure comprises a population of TILs according to any of the methods described above.BRIEF DESCRIPTION OF THE DRAWINGS

[0082] Figure 1: Exemplary Process 2A chart providing an overview of Steps A through F.

[0083] Figures 2A-2C: Process Flow Chart of Process 2A.

[0084] Figure 3: Shows a diagram of an embodiment of a cryopreserved TIL exemplary manufacturing process (~22 days).

[0085] Figure 4: Shows a diagram of an embodiment of process 2A, a 22-day process for TIL manufacturing.

[0086] Figure 5: Comparison table of Steps A through F from exemplary embodiments of process 1C and process 2A.

[0087] Figure 6: Detailed comparison of an embodiment of process 1C and an embodiment of process 2A.

[0088] Figure 7: Exemplary GEN 3 type process for tumors.

[0089] Figure 8A-8G: A) Shows a comparison between the 2A process (approximately 22-day process) and an embodiment of the Gen 3 process for TIL manufacturing (approximately 14-days to 16-days process). B) Exemplary Process Gen3 chart providing an overview of Steps A through F (approximately 14-days to 16-days process). C) Chart providing three exemplary Gen 3 processes with an overview of Steps A through F (approximately 14-days to 16-days process) for each of the three process variations. D) Exemplary Modified Gen 2-like process providing an overview of Steps A through F (approximately 22-days process). E) Shows a comparison between the 2A process (approximately 22-day process) and an embodiment of the Gen 3 process for TIL manufacturing (approximately 14-days to 22-days process). F) Exemplary Process CD39-103 Gen3 chart providing an overview of Steps A through F (approximately 14-days to 22-days process). G) Exemplary Process OX4-1 Gen3 chart providing an overview of Steps A through F (approximately 14-days to 22-days process).

[0090] Figure 9: Provides an experimental flow chart for comparability between GEN 2 (process 2A) versus GEN 3.

[0091] Figure 10: Shows a comparison between various Gen 2 (2A process) and the Gen 3.1 process embodiment.

[0092] Figure 11: Table describing various features of embodiments of the Gen 2, Gen 2.1 and Gen 3.0 process.

[0093] Figure 12: Overview of the media conditions for an embodiment of the Gen 3 process, referred to as Gen 3.1.

[0094] Figure 13: Table describing various features of embodiments of the Gen 2, Gen 2.1 and Gen 3.0 process.

[0095] Figure 14: Table comparing various features of embodiments of the Gen 2 and Gen 3.0 processes.

[0096] Figure 15: Table providing media uses in the various embodiments of the described expansion processes.

[0097] Figure 16: Schematic of an exemplary embodiment of the Gen 3 process (a 16-day process).

[0098] Figure 17: Schematic of an exemplary embodiment of a method for expanding T cells from hematopoietic malignancies using Gen 3 expansion platform.

[0099] Figure 18: Provides the structures I-A and I-B, the cylinders refer to individual polypeptide binding domains. Structures I-A and I-B comprise three linearly-linked TNFRSF binding domains derived from e.g., 4-1BBL or an antibody that binds 4-1BB, which fold to form a trivalent protein, which is then linked to a second trivalent protein through IgG1-Fc (including CH3 and CH2 domains) is then used to link two of the trivalent proteins together through disulfide bonds (small elongated ovals), stabilizing the structure and providing an agonists capable of bringing together the intracellular signaling domains of the six receptors and signaling proteins to form a signaling complex. The TNFRSF binding domains denoted as cylinders may be scFv domains comprising, e.g., a VH and a VL chain connected by a linker that may comprise hydrophilic residues and Gly and Ser sequences for flexibility, as well as Glu and Lys for solubility.

[0100] Figure 19: Schematic of an exemplary embodiment of the Gen 3 process (a 16-day process).

[0101] Figure 20: Provides a process overview for an exemplary embodiment (Gen 3.1 Test) of the Gen 3.1 process (a 16 day process).

[0102] Figure 21: Schematic of an exemplary embodiment of the Gen 3.1 Test (Gen 3.1 optimized) process (a 16-17 day process).

[0103] Figure 22: Schematic of an exemplary embodiment of the Gen 3 process (a 16-day process).

[0104] Figure 23A-23B: Comparison tables for exemplary Gen 2 and exemplary Gen 3 processes with exemplary differences highlighted.

[0105] Figure 24: Schematic of an exemplary embodiment of the Gen 3 process (a 16 / 17 day process) preparation timeline.

[0106] Figure 25: Schematic of an exemplary embodiment of the Gen 3 process (a 14-16 day process).

[0107] Figure 26A-26B: Schematic of an exemplary embodiment of the Gen 3 process (a 16 day process).

[0108] Figure 27: Schematic of an exemplary embodiment of the Gen 3 process (a 16 day process).

[0109] Figure 28: Comparison of Gen 2, Gen 2.1 and an embodiment of the Gen 3 process (a 16 day process).

[0110] Figure 29: Comparison of Gen 2, Gen 2.1 and an embodiment of the Gen 3 process (a 16 day process).

[0111] Figure 30: Gen 3 embodiment components.

[0112] Figure 31: Gen 3 embodiment flow chart comparison (Gen 3.0, Gen 3.1 control, Gen 3.1 Test).

[0113] Figure 32: Shown are the components of an exemplary embodiment of the Gen 3 process (Gen 3-Optimized, a 16-17 day process).

[0114] Figure 33: Schematic of exemplary workflow in TIL generation process, generally highlighting selection (e.g., of CD39- and / or CD103-expressing cells) prior to expansion.

[0115] Figure 34A-34B: (A) Summary of two independent experiments, in which 22 tumor samples were analyzed for markers associated with activation and exhaustion (Experiment 1: 12 tumor digests from 5 melanoma, 3 HNSCC, 1 cervical, 1 ovarian, 1 prostate, 1 NSCLC; Experiment 2: 10 tumor digests from 1 melanoma, 3 HNSCC, 1 cervical, 2 ovarian and 3 NSCLC). (B) Illustration of biased approach for identifying TIL populations for selection based upon unsupervised clusteringwith PD-1. In this instance, PD-1-selected TILs are the baseline for selection alonge with other factors clustering with it.

[0116] Figure 35A-35E: Assessment of 21 tumor digests (3 cervical, 6 HNSCC, 3 NSCLC, 5 Melanoma, 3 Ovarian and 1 Prostate) for phenotypic expression of markers associated with activation, exhaustion, and tumor specificity. Tumor digests were gated on PD-1high, PD-1int, PD-1negTIL and compared to the CD3+ population.

[0117] Figure 36A-36I: Exemplary cluster analysis of 12 tumor digests (5 Melanoma, 3 HNSCC, 1 Cervical, 1 Ovarian, 1 Prostate, 1 NSCLC) showing the phenotypic profile of the various tumors (A-D), and clustering of specific markers (E-I).

[0118] Figure 37: Summary of various phenotypic markers in either Ki67- or Ki67+ CD4 and CD8 TILs.

[0119] Figures 38A-38D: In vitro expandion of PD-1highCD39+ selected TILs.

[0120] Figure 39: Expression of CD3 and phenotypic markers that define T cell lineage, memory, and differentiation by PD-1highCD39+ selected TILs.

[0121] Figures 40A-40C: Expression of CD3 and phenotypic markers that define T cell activation and exhaustion by PD-1highCD39+ selected TILs.

[0122] Figure 41: IFNγ secretion by PD-1highCD39+ selected TILs. (Note: H3099, H3117 and CC10052 are not included.)

[0123] Figure 42: Tumor reactivity of PD-1highCD39+ selected TILs. BRIEF DESCRIPTION OF THE SEQUENCE LISTING

[0124] SEQ ID NO:1 is the amino acid sequence of the heavy chain of muromonab.

[0125] SEQ ID NO:2 is the amino acid sequence of the light chain of muromonab.

[0126] SEQ ID NO:3 is the amino acid sequence of a recombinant human IL-2 protein.

[0127] SEQ ID NO:4 is the amino acid sequence of aldesleukin.

[0128] SEQ ID NO:5 is an IL-2 form.

[0129] SEQ ID NO:6 is an IL-2 form.

[0130] SEQ ID NO:7 is an IL-2 form.

[0131] SEQ ID NO:8 is a mucin domain polypeptide.

[0132] SEQ ID NO:9 is the amino acid sequence of a recombinant human IL-4 protein.

[0133] SEQ ID NO:10 is the amino acid sequence of a recombinant human IL-7 protein.

[0134] SEQ ID NO:11 is the amino acid sequence of a recombinant human IL-15 protein.

[0135] SEQ ID NO:12 is the amino acid sequence of a recombinant human IL-21 protein.

[0136] SEQ ID NO:13 is an IL-2 sequence.

[0137] SEQ ID NO:14 is an IL-2 mutein sequence.

[0138] SEQ ID NO:15 is an IL-2 mutein sequence.

[0139] SEQ ID NO:16 is the HCDR1_IL-2 for IgG.IL2R67A.H1.

[0140] SEQ ID NO:17 is the HCDR2 for IgG.IL2R67A.H1.

[0141] SEQ ID NO:18 is the HCDR3 for IgG.IL2R67A.H1.

[0142] SEQ ID NO:19 is the HCDR1_IL-2 kabat for IgG.IL2R67A.H1.

[0143] SEQ ID NO:20 is the HCDR2 kabat for IgG.IL2R67A.H1.

[0144] SEQ ID NO:21 is the HCDR3 kabat for IgG.IL2R67A.H1.

[0145] SEQ ID NO:22 is the HCDR1_IL-2 clothia for IgG.IL2R67A.H1.

[0146] SEQ ID NO:23 is the HCDR2 clothia for IgG.IL2R67A.H1.

[0147] SEQ ID NO:24 is the HCDR3 clothia for IgG.IL2R67A.H1.

[0148] SEQ ID NO:25 is the HCDR1_IL-2 IMGT for IgG.IL2R67A.H1.

[0149] SEQ ID NO:26 is the HCDR2 IMGT for IgG.IL2R67A.H1.

[0150] SEQ ID NO:27 is the HCDR3 IMGT for IgG.IL2R67A.H1.

[0151] SEQ ID NO:28 is the VH chain for IgG.IL2R67A.H1.

[0152] SEQ ID NO:29 is the heavy chain for IgG.IL2R67A.H1.

[0153] SEQ ID NO:30 is the LCDR1 kabat for IgG.IL2R67A.H1.

[0154] SEQ ID NO:31 is the LCDR2 kabat for IgG.IL2R67A.H1.

[0155] SEQ ID NO:32 is the LCDR3 kabat for IgG.IL2R67A.H1.

[0156] SEQ ID NO:33 is the LCDR1 chothia for IgG.IL2R67A.H1.

[0157] SEQ ID NO:34 is the LCDR2 chothia for IgG.IL2R67A.H1.

[0158] SEQ ID NO:35 is the LCDR3 chothia for IgG.IL2R67A.H1.

[0159] SEQ ID NO:36 is a VL chain.

[0160] SEQ ID NO:37 is a light chain.

[0161] SEQ ID NO:38 is a light chain.

[0162] SEQ ID NO:39 is a light chain.

[0163] SEQ ID NO:40 is the amino acid sequence of human 4-1BB.

[0164] SEQ ID NO:41 is the amino acid sequence of murine 4-1BB.

[0165] SEQ ID NO:42 is the heavy chain for the 4-1BB agonist monoclonal antibody utomilumab (PF-05082566).

[0166] SEQ ID NO:43 is the light chain for the 4-1BB agonist monoclonal antibody utomilumab (PF-05082566).

[0167] SEQ ID NO:44 is the heavy chain variable region (VH) for the 4-1BB agonist monoclonal antibody utomilumab (PF-05082566).

[0168] SEQ ID NO:45 is the light chain variable region (VL) for the 4-1BB agonist monoclonal antibody utomilumab (PF-05082566).

[0169] SEQ ID NO:46 is the heavy chain CDR1 for the 4-1BB agonist monoclonal antibody utomilumab (PF-05082566).

[0170] SEQ ID NO:47 is the heavy chain CDR2 for the 4-1BB agonist monoclonal antibody utomilumab (PF-05082566).

[0171] SEQ ID NO:48 is the heavy chain CDR3 for the 4-1BB agonist monoclonal antibody utomilumab (PF-05082566).

[0172] SEQ ID NO:49 is the light chain CDR1 for the 4-1BB agonist monoclonal antibody utomilumab (PF-05082566).

[0173] SEQ ID NO:50 is the light chain CDR2 for the 4-1BB agonist monoclonal antibody utomilumab (PF-05082566).

[0174] SEQ ID NO:51 is the light chain CDR3 for the 4-1BB agonist monoclonal antibody utomilumab (PF-05082566).

[0175] SEQ ID NO:52 is the heavy chain for the 4-1BB agonist monoclonal antibody urelumab (BMS-663513).

[0176] SEQ ID NO:53 is the light chain for the 4-1BB agonist monoclonal antibody urelumab (BMS- 663513).

[0177] SEQ ID NO:54 is the heavy chain variable region (VH) for the 4-1BB agonist monoclonal antibody urelumab (BMS-663513).

[0178] SEQ ID NO:55 is the light chain variable region (VL) for the 4-1BB agonist monoclonal antibody urelumab (BMS-663513).

[0179] SEQ ID NO:56 is the heavy chain CDR1 for the 4-1BB agonist monoclonal antibody urelumab (BMS-663513).

[0180] SEQ ID NO:57 is the heavy chain CDR2 for the 4-1BB agonist monoclonal antibody urelumab (BMS-663513).

[0181] SEQ ID NO:58 is the heavy chain CDR3 for the 4-1BB agonist monoclonal antibody urelumab (BMS-663513).

[0182] SEQ ID NO:59 is the light chain CDR1 for the 4-1BB agonist monoclonal antibody urelumab (BMS-663513).

[0183] SEQ ID NO:60 is the light chain CDR2 for the 4-1BB agonist monoclonal antibody urelumab (BMS-663513).

[0184] SEQ ID NO:61 is the light chain CDR3 for the 4-1BB agonist monoclonal antibody urelumab (BMS-663513).

[0185] SEQ ID NO:62 is an Fc domain for a TNFRSF agonist fusion protein.

[0186] SEQ ID NO:63 is a linker for a TNFRSF agonist fusion protein.

[0187] SEQ ID NO:64 is a linker for a TNFRSF agonist fusion protein.

[0188] SEQ ID NO:65 is a linker for a TNFRSF agonist fusion protein.

[0189] SEQ ID NO:66 is a linker for a TNFRSF agonist fusion protein.

[0190] SEQ ID NO:67 is a linker for a TNFRSF agonist fusion protein.

[0191] SEQ ID NO:68 is a linker for a TNFRSF agonist fusion protein.

[0192] SEQ ID NO:69 is a linker for a TNFRSF agonist fusion protein.

[0193] SEQ ID NO:70 is a linker for a TNFRSF agonist fusion protein.

[0194] SEQ ID NO:71 is a linker for a TNFRSF agonist fusion protein.

[0195] SEQ ID NO:72 is a linker for a TNFRSF agonist fusion protein.

[0196] SEQ ID NO:73 is an Fc domain for a TNFRSF agonist fusion protein.

[0197] SEQ ID NO:74 is a linker for a TNFRSF agonist fusion protein.

[0198] SEQ ID NO:75 is a linker for a TNFRSF agonist fusion protein.

[0199] SEQ ID NO:76 is a linker for a TNFRSF agonist fusion protein.

[0200] SEQ ID NO:77 is a 4-1BB ligand (4-1BBL) amino acid sequence.

[0201] SEQ ID NO:78 is a soluble portion of 4-1BBL polypeptide.

[0202] SEQ ID NO:79 is a heavy chain variable region (VH) for the 4-1BB agonist antibody 4B4-1-1 version 1.

[0203] SEQ ID NO:80 is a light chain variable region (VL) for the 4-1BB agonist antibody 4B4-1-1 version 1.

[0204] SEQ ID NO:81 is a heavy chain variable region (VH) for the 4-1BB agonist antibody 4B4-1-1 version 2.

[0205] SEQ ID NO:82 is a light chain variable region (VL) for the 4-1BB agonist antibody 4B4-1-1 version 2.

[0206] SEQ ID NO:83 is a heavy chain variable region (VH) for the 4-1BB agonist antibody H39E3- 2.

[0207] SEQ ID NO:84 is a light chain variable region (VL) for the 4-1BB agonist antibody H39E3-2.

[0208] SEQ ID NO:85 is the amino acid sequence of human OX40.

[0209] SEQ ID NO:86 is the amino acid sequence of murine OX40.

[0210] SEQ ID NO:87 is the heavy chain for the OX40 agonist monoclonal antibody tavolixizumab (MEDI-0562).

[0211] SEQ ID NO:88 is the light chain for the OX40 agonist monoclonal antibody tavolixizumab (MEDI-0562).

[0212] SEQ ID NO:89 is the heavy chain variable region (VH) for the OX40 agonist monoclonal antibody tavolixizumab (MEDI-0562).

[0213] SEQ ID NO:90 is the light chain variable region (VL) for the OX40 agonist monoclonal antibody tavolixizumab (MEDI-0562).

[0214] SEQ ID NO:91 is the heavy chain CDR1 for the OX40 agonist monoclonal antibody tavolixizumab (MEDI-0562).

[0215] SEQ ID NO:92 is the heavy chain CDR2 for the OX40 agonist monoclonal antibody tavolixizumab (MEDI-0562).

[0216] SEQ ID NO:93 is the heavy chain CDR3 for the OX40 agonist monoclonal antibody tavolixizumab (MEDI-0562).

[0217] SEQ ID NO:94 is the light chain CDR1 for the OX40 agonist monoclonal antibody tavolixizumab (MEDI-0562).

[0218] SEQ ID NO:95 is the light chain CDR2 for the OX40 agonist monoclonal antibody tavolixizumab (MEDI-0562).

[0219] SEQ ID NO:96 is the light chain CDR3 for the OX40 agonist monoclonal antibody tavolixizumab (MEDI-0562).

[0220] SEQ ID NO:97 is the heavy chain for the OX40 agonist monoclonal antibody 11D4.

[0221] SEQ ID NO:98 is the light chain for the OX40 agonist monoclonal antibody 11D4.

[0222] SEQ ID NO:99 is the heavy chain variable region (VH) for the OX40 agonist monoclonal antibody 11D4.

[0223] SEQ ID NO:100 is the light chain variable region (VL) for the OX40 agonist monoclonal antibody 11D4.

[0224] SEQ ID NO:101 is the heavy chain CDR1 for the OX40 agonist monoclonal antibody 11D4.

[0225] SEQ ID NO:102 is the heavy chain CDR2 for the OX40 agonist monoclonal antibody 11D4.

[0226] SEQ ID NO:103 is the heavy chain CDR3 for the OX40 agonist monoclonal antibody 11D4.

[0227] SEQ ID NO:104 is the light chain CDR1 for the OX40 agonist monoclonal antibody 11D4.

[0228] SEQ ID NO:105 is the light chain CDR2 for the OX40 agonist monoclonal antibody 11D4.

[0229] SEQ ID NO:106 is the light chain CDR3 for the OX40 agonist monoclonal antibody 11D4.

[0230] SEQ ID NO:107 is the heavy chain for the OX40 agonist monoclonal antibody 18D8.

[0231] SEQ ID NO:108 is the light chain for the OX40 agonist monoclonal antibody 18D8.

[0232] SEQ ID NO:109 is the heavy chain variable region (VH) for the OX40 agonist monoclonal antibody 18D8.

[0233] SEQ ID NO:110 is the light chain variable region (VL) for the OX40 agonist monoclonal antibody 18D8.

[0234] SEQ ID NO:111 is the heavy chain CDR1 for the OX40 agonist monoclonal antibody 18D8.

[0235] SEQ ID NO:112 is the heavy chain CDR2 for the OX40 agonist monoclonal antibody 18D8.

[0236] SEQ ID NO:113 is the heavy chain CDR3 for the OX40 agonist monoclonal antibody 18D8.

[0237] SEQ ID NO:114 is the light chain CDR1 for the OX40 agonist monoclonal antibody 18D8.

[0238] SEQ ID NO:115 is the light chain CDR2 for the OX40 agonist monoclonal antibody 18D8.

[0239] SEQ ID NO:116 is the light chain CDR3 for the OX40 agonist monoclonal antibody 18D8.

[0240] SEQ ID NO:117 is the heavy chain variable region (VH) for the OX40 agonist monoclonal antibody Hu119-122.

[0241] SEQ ID NO:118 is the light chain variable region (VL) for the OX40 agonist monoclonal antibody Hu119-122.

[0242] SEQ ID NO:119 is the heavy chain CDR1 for the OX40 agonist monoclonal antibody Hu119- 122.

[0243] SEQ ID NO:120 is the heavy chain CDR2 for the OX40 agonist monoclonal antibody Hu119- 122.

[0244] SEQ ID NO:121 is the heavy chain CDR3 for the OX40 agonist monoclonal antibody Hu119- 122.

[0245] SEQ ID NO:122 is the light chain CDR1 for the OX40 agonist monoclonal antibody Hu119- 122.

[0246] SEQ ID NO:123 is the light chain CDR2 for the OX40 agonist monoclonal antibody Hu119- 122.

[0247] SEQ ID NO:124 is the light chain CDR3 for the OX40 agonist monoclonal antibody Hu119- 122.

[0248] SEQ ID NO:125 is the heavy chain variable region (VH) for the OX40 agonist monoclonal antibody Hu106-222.

[0249] SEQ ID NO:126 is the light chain variable region (VL) for the OX40 agonist monoclonal antibody Hu106-222.

[0250] SEQ ID NO:127 is the heavy chain CDR1 for the OX40 agonist monoclonal antibody Hu106- 222.

[0251] SEQ ID NO:128 is the heavy chain CDR2 for the OX40 agonist monoclonal antibody Hu106- 222.

[0252] SEQ ID NO:129 is the heavy chain CDR3 for the OX40 agonist monoclonal antibody Hu106- 222.

[0253] SEQ ID NO:130 is the light chain CDR1 for the OX40 agonist monoclonal antibody Hu106- 222.

[0254] SEQ ID NO:131 is the light chain CDR2 for the OX40 agonist monoclonal antibody Hu106- 222.

[0255] SEQ ID NO:132 is the light chain CDR3 for the OX40 agonist monoclonal antibody Hu106- 222.

[0256] SEQ ID NO:133 is an OX40 ligand (OX40L) amino acid sequence.

[0257] SEQ ID NO:134 is a soluble portion of OX40L polypeptide.

[0258] SEQ ID NO:135 is an alternative soluble portion of OX40L polypeptide.

[0259] SEQ ID NO:136 is the heavy chain variable region (VH) for the OX40 agonist monoclonal antibody 008.

[0260] SEQ ID NO:137 is the light chain variable region (VL) for the OX40 agonist monoclonal antibody 008.

[0261] SEQ ID NO:138 is the heavy chain variable region (VH) for the OX40 agonist monoclonal antibody 011.

[0262] SEQ ID NO:139 is the light chain variable region (VL) for the OX40 agonist monoclonal antibody 011.

[0263] SEQ ID NO:140 is the heavy chain variable region (VH) for the OX40 agonist monoclonal antibody 021.

[0264] SEQ ID NO:141 is the light chain variable region (VL) for the OX40 agonist monoclonal antibody 021.

[0265] SEQ ID NO:142 is the heavy chain variable region (VH) for the OX40 agonist monoclonal antibody 023.

[0266] SEQ ID NO:143 is the light chain variable region (VL) for the OX40 agonist monoclonal antibody 023.

[0267] SEQ ID NO:144 is the heavy chain variable region (VH) for an OX40 agonist monoclonal antibody.

[0268] SEQ ID NO:145 is the light chain variable region (VL) for an OX40 agonist monoclonal antibody.

[0269] SEQ ID NO:146 is the heavy chain variable region (VH) for an OX40 agonist monoclonal antibody.

[0270] SEQ ID NO:147 is the light chain variable region (VL) for an OX40 agonist monoclonal antibody.

[0271] SEQ ID NO:148 is the heavy chain variable region (VH) for a humanized OX40 agonist monoclonal antibody.

[0272] SEQ ID NO:149 is the heavy chain variable region (VH) for a humanized OX40 agonist monoclonal antibody.

[0273] SEQ ID NO:150 is the light chain variable region (VL) for a humanized OX40 agonist monoclonal antibody.

[0274] SEQ ID NO:151 is the light chain variable region (VL) for a humanized OX40 agonist monoclonal antibody.

[0275] SEQ ID NO:152 is the heavy chain variable region (VH) for a humanized OX40 agonist monoclonal antibody.

[0276] SEQ ID NO:153 is the heavy chain variable region (VH) for a humanized OX40 agonist monoclonal antibody.

[0277] SEQ ID NO:154 is the light chain variable region (VL) for a humanized OX40 agonist monoclonal antibody.

[0278] SEQ ID NO:155 is the light chain variable region (VL) for a humanized OX40 agonist monoclonal antibody.

[0279] SEQ ID NO:156 is the heavy chain variable region (VH) for an OX40 agonist monoclonal antibody.

[0280] SEQ ID NO:157 is the light chain variable region (VL) for an OX40 agonist monoclonal antibody.

[0281] SEQ ID NO:158 is the heavy chain amino acid sequence of the PD-1 inhibitor nivolumab.

[0282] SEQ ID NO:159 is the light chain amino acid sequence of the PD-1 inhibitor nivolumab.

[0283] SEQ ID NO:160 is the heavy chain variable region (VH) amino acid sequence of the PD-1 inhibitor nivolumab.

[0284] SEQ ID NO:161 is the light chain variable region (VL) amino acid sequence of the PD-1 inhibitor nivolumab.

[0285] SEQ ID NO:162 is the heavy chain CDR1 amino acid sequence of the PD-1 inhibitor nivolumab.

[0286] SEQ ID NO:163 is the heavy chain CDR2 amino acid sequence of the PD-1 inhibitor nivolumab.

[0287] SEQ ID NO:164 is the heavy chain CDR3 amino acid sequence of the PD-1 inhibitor nivolumab.

[0288] SEQ ID NO:165 is the light chain CDR1 amino acid sequence of the PD-1 inhibitor nivolumab.

[0289] SEQ ID NO:166 is the light chain CDR2 amino acid sequence of the PD-1 inhibitor nivolumab.

[0290] SEQ ID NO:167 is the light chain CDR3 amino acid sequence of the PD-1 inhibitor nivolumab.

[0291] SEQ ID NO:168 is the heavy chain amino acid sequence of the PD-1 inhibitor pembrolizumab.

[0292] SEQ ID NO:169 is the light chain amino acid sequence of the PD-1 inhibitor pembrolizumab.

[0293] SEQ ID NO:170 is the heavy chain variable region (VH) amino acid sequence of the PD-1 inhibitor pembrolizumab.

[0294] SEQ ID NO:171 is the light chain variable region (VL) amino acid sequence of the PD-1 inhibitor pembrolizumab.

[0295] SEQ ID NO:172 is the heavy chain CDR1 amino acid sequence of the PD-1 inhibitor pembrolizumab.

[0296] SEQ ID NO:173 is the heavy chain CDR2 amino acid sequence of the PD-1 inhibitor pembrolizumab.

[0297] SEQ ID NO:174 is the heavy chain CDR3 amino acid sequence of the PD-1 inhibitor pembrolizumab.

[0298] SEQ ID NO:175 is the light chain CDR1 amino acid sequence of the PD-1 inhibitor pembrolizumab.

[0299] SEQ ID NO:176 is the light chain CDR2 amino acid sequence of the PD-1 inhibitor pembrolizumab.

[0300] SEQ ID NO:177 is the light chain CDR3 amino acid sequence of the PD-1 inhibitor pembrolizumab.

[0301] SEQ ID NO:178 is the heavy chain amino acid sequence of the PD-L1 inhibitor durvalumab.

[0302] SEQ ID NO:179 is the light chain amino acid sequence of the PD-L1 inhibitor durvalumab.

[0303] SEQ ID NO:180 is the heavy chain variable region (VH) amino acid sequence of the PD-L1 inhibitor durvalumab.

[0304] SEQ ID NO:181 is the light chain variable region (VL) amino acid sequence of the PD-L1 inhibitor durvalumab.

[0305] SEQ ID NO:182 is the heavy chain CDR1 amino acid sequence of the PD-L1 inhibitor durvalumab.

[0306] SEQ ID NO:183 is the heavy chain CDR2 amino acid sequence of the PD-L1 inhibitor durvalumab.

[0307] SEQ ID NO:184 is the heavy chain CDR3 amino acid sequence of the PD-L1 inhibitor durvalumab.

[0308] SEQ ID NO:185 is the light chain CDR1 amino acid sequence of the PD-L1 inhibitor durvalumab.

[0309] SEQ ID NO:186 is the light chain CDR2 amino acid sequence of the PD-L1 inhibitor durvalumab.

[0310] SEQ ID NO:187 is the light chain CDR3 amino acid sequence of the PD-L1 inhibitor durvalumab.

[0311] SEQ ID NO:188 is the heavy chain amino acid sequence of the PD-L1 inhibitor avelumab.

[0312] SEQ ID NO:189 is the light chain amino acid sequence of the PD-L1 inhibitor avelumab.

[0313] SEQ ID NO:190 is the heavy chain variable region (VH) amino acid sequence of the PD-L1 inhibitor avelumab.

[0314] SEQ ID NO:191 is the light chain variable region (VL) amino acid sequence of the PD-L1 inhibitor avelumab.

[0315] SEQ ID NO:192 is the heavy chain CDR1 amino acid sequence of the PD-L1 inhibitor avelumab.

[0316] SEQ ID NO:193 is the heavy chain CDR2 amino acid sequence of the PD-L1 inhibitor avelumab.

[0317] SEQ ID NO:194 is the heavy chain CDR3 amino acid sequence of the PD-L1 inhibitor avelumab.

[0318] SEQ ID NO:195 is the light chain CDR1 amino acid sequence of the PD-L1 inhibitor avelumab.

[0319] SEQ ID NO:196 is the light chain CDR2 amino acid sequence of the PD-L1 inhibitor avelumab.

[0320] SEQ ID NO:197 is the light chain CDR3 amino acid sequence of the PD-L1 inhibitor avelumab.

[0321] SEQ ID NO:198 is the heavy chain amino acid sequence of the PD-L1 inhibitor atezolizumab.

[0322] SEQ ID NO:199 is the light chain amino acid sequence of the PD-L1 inhibitor atezolizumab.

[0323] SEQ ID NO:200 is the heavy chain variable region (VH) amino acid sequence of the PD-L1 inhibitor atezolizumab.

[0324] SEQ ID NO:201 is the light chain variable region (VL) amino acid sequence of the PD-L1 inhibitor atezolizumab.

[0325] SEQ ID NO:202 is the heavy chain CDR1 amino acid sequence of the PD-L1 inhibitor atezolizumab.

[0326] SEQ ID NO:203 is the heavy chain CDR2 amino acid sequence of the PD-L1 inhibitor atezolizumab.

[0327] SEQ ID NO:204 is the heavy chain CDR3 amino acid sequence of the PD-L1 inhibitor atezolizumab.

[0328] SEQ ID NO:205 is the light chain CDR1 amino acid sequence of the PD-L1 inhibitor atezolizumab.

[0329] SEQ ID NO:206 is the light chain CDR2 amino acid sequence of the PD-L1 inhibitor atezolizumab.

[0330] SEQ ID NO:207 is the light chain CDR3 amino acid sequence of the PD-L1 inhibitor atezolizumab.

[0331] SEQ ID NO:208 is the heavy chain amino acid sequence of the CTLA-4 inhibitor ipilimumab.

[0332] SEQ ID NO:209 is the light chain amino acid sequence of the CTLA-4 inhibitor ipilimumab.

[0333] SEQ ID NO:210 is the heavy chain variable region (VH) amino acid sequence of the CTLA-4 inhibitor ipilimumab.

[0334] SEQ ID NO:211 is the light chain variable region (VL) amino acid sequence of the CTLA-4 inhibitor ipilimumab.

[0335] SEQ ID NO:212 is the heavy chain CDR1 amino acid sequence of the CTLA-4 inhibitor ipilimumab.

[0336] SEQ ID NO:213 is the heavy chain CDR2 amino acid sequence of the CTLA-4 inhibitor ipilimumab.

[0337] SEQ ID NO:214 is the heavy chain CDR3 amino acid sequence of the CTLA-4 inhibitor ipilimumab.

[0338] SEQ ID NO:215 is the light chain CDR1 amino acid sequence of the CTLA-4 inhibitor ipilimumab.

[0339] SEQ ID NO:216 is the light chain CDR2 amino acid sequence of the CTLA-4 inhibitor ipilimumab.

[0340] SEQ ID NO:217 is the light chain CDR3 amino acid sequence of the CTLA-4 inhibitor ipilimumab.

[0341] SEQ ID NO:218 is the heavy chain amino acid sequence of the CTLA-4 inhibitor tremelimumab.

[0342] SEQ ID NO:219 is the light chain amino acid sequence of the CTLA-4 inhibitor tremelimumab.

[0343] SEQ ID NO:220 is the heavy chain variable region (VH) amino acid sequence of the CTLA-4 inhibitor tremelimumab.

[0344] SEQ ID NO:221 is the light chain variable region (VL) amino acid sequence of the CTLA-4 inhibitor tremelimumab.

[0345] SEQ ID NO:222 is the heavy chain CDR1 amino acid sequence of the CTLA-4 inhibitor tremelimumab.

[0346] SEQ ID NO:223 is the heavy chain CDR2 amino acid sequence of the CTLA-4 inhibitor tremelimumab.

[0347] SEQ ID NO:224 is the heavy chain CDR3 amino acid sequence of the CTLA-4 inhibitor tremelimumab.

[0348] SEQ ID NO:225 is the light chain CDR1 amino acid sequence of the CTLA-4 inhibitor tremelimumab.

[0349] SEQ ID NO:226 is the light chain CDR2 amino acid sequence of the CTLA-4 inhibitor tremelimumab.

[0350] SEQ ID NO:227 is the light chain CDR3 amino acid sequence of the CTLA-4 inhibitor tremelimumab.

[0351] SEQ ID NO:228 is the heavy chain amino acid sequence of the CTLA-4 inhibitor zalifrelimab.

[0352] SEQ ID NO:229 is the light chain amino acid sequence of the CTLA-4 inhibitor zalifrelimab.

[0353] SEQ ID NO:230 is the heavy chain variable region (VH) amino acid sequence of the CTLA-4 inhibitor zalifrelimab.

[0354] SEQ ID NO:231 is the light chain variable region (VL) amino acid sequence of the CTLA-4 inhibitor zalifrelimab.

[0355] SEQ ID NO:232 is the heavy chain CDR1 amino acid sequence of the CTLA-4 inhibitor zalifrelimab.

[0356] SEQ ID NO:233 is the heavy chain CDR2 amino acid sequence of the CTLA-4 inhibitor zalifrelimab.

[0357] SEQ ID NO:234 is the heavy chain CDR3 amino acid sequence of the CTLA-4 inhibitor zalifrelimab.

[0358] SEQ ID NO:235 is the light chain CDR1 amino acid sequence of the CTLA-4 inhibitor zalifrelimab.

[0359] SEQ ID NO:236 is the light chain CDR2 amino acid sequence of the CTLA-4 inhibitor zalifrelimab.

[0360] SEQ ID NO:237 is the light chain CDR3 amino acid sequence of the CTLA-4 inhibitor zalifrelimab.

[0361] SEQ ID NO:238 is a target PD-1 sequence.

[0362] SEQ ID NO:239 is a target PD-1 sequence.

[0363] SEQ ID NO:240 is a repeat PD-1 left repeat sequence.

[0364] SEQ ID NO:241 is a repeat PD-1 right repeat sequence.

[0365] SEQ ID NO:242 is a repeat PD-1 left repeat sequence.

[0366] SEQ ID NO:243 is a repeat PD-1 right repeat sequence.

[0367] SEQ ID NO:244 is a PD-1 left TALEN nuclease sequence.

[0368] SEQ ID NO:245 is a PD-1 right TALEN nuclease sequence.

[0369] SEQ ID NO:246 is a PD-1 left TALEN nuclease sequence.

[0370] SEQ ID NO:247 is a PD-1 right TALEN nuclease sequence.

[0371] Unless defined otherwise, all technical and scientific terms used herein have the same meaning as is commonly understood by one of skill in the art to which this invention belongs. All patents and publications referred to herein are incorporated by reference in their entireties. DETAILED DESCRIPTION OF THE INVENTION I. Introduction

[0372] Provided herein are TILs that express CD39, CD103 and / or both. In some embodiments, the subject TILs are produced by genetically manipulating a population of TILs that have been selected for expression of (i) PD-1, (ii) CD39, (iii) CD103, (iv) the combination of (i) and (ii), (v) the combination of (i) and (iii), or (vi) the combination of (ii) and (iii). Also provided herein are expansion methods for producing such populations of TILs and methods of treatment using such TILs.

[0373] Definitions

[0374] Unless defined otherwise, all technical and scientific terms used herein have the same meaning as is commonly understood by one of skill in the art to which this invention belongs. All patents and publications referred to herein are incorporated by reference in their entireties.

[0375] The terms “co-administration,” “co-administering,” “administered in combination with,” “administering in combination with,” “simultaneous,” and “concurrent,” as used herein, encompass administration of two or more active pharmaceutical ingredients (in some embodiments of the present invention, for example, a plurality of TILs) to a subject so that both active pharmaceutical ingredients and / or their metabolites are present in the subject at the same time. Co- administration includes simultaneous administration in separate compositions, administration at different times in separate compositions, or administration in a composition in which two or more active pharmaceutical ingredients are present. Simultaneous administration in separate compositions and administration in a composition in which both agents are present are preferred.

[0376] The term “in vivo” refers to an event that takes place in a subject's body.

[0377] The term “in vitro” refers to an event that takes places outside of a subject's body. In vitro assays encompass cell-based assays in which cells alive or dead are employed and may also encompass a cell-free assay in which no intact cells are employed.

[0378] The term “ex vivo” refers to an event which involves treating or performing a procedure on a cell, tissue and / or organ which has been removed from a subject’s body. Aptly, the cell, tissue and / or organ may be returned to the subject’s body in a method of surgery or treatment.

[0379] The term “rapid expansion” means an increase in the number of antigen-specific TILs of at least about 3-fold (or 4-, 5-, 6-, 7-, 8-, or 9-fold) over a period of a week, more preferably at least about 10-fold (or 20-, 30-, 40-, 50-, 60-, 70-, 80-, or 90-fold) over a period of a week, or most preferably at least about 100-fold over a period of a week. A number of rapid expansion protocols are described herein.

[0380] By “tumor infiltrating lymphocytes” or “TILs” herein is meant a population of cells originally obtained as white blood cells that have left the bloodstream of a subject and migrated into a tumor. TILs include, but are not limited to, CD8+cytotoxic T cells (lymphocytes), Th1 and Th17 CD4+T cells, natural killer cells, dendritic cells and M1 macrophages. TILs include both primary and secondary TILs. “Primary TILs” are those that are obtained from patient tissue samples as outlined herein (sometimes referred to as “freshly harvested”), and “secondary TILs” are any TIL cell populations that have been expanded or proliferated as discussed herein, including, but not limited to bulk TILs and expanded TILs (“REP TILs” or “post-REP TILs”). TIL cell populations can include genetically modified TILs.

[0381] TILs can generally be defined either biochemically, using cell surface markers, or functionally, by their ability to infiltrate tumors and effect treatment. TILs can be generally categorized by expressing one or more of the following biomarkers: CD4, CD8, 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 a patient. TILS may further be characterized by potency – for example, TILS may be considered potent if, for example, interferon (IFN) release is greater than about 50 pg / mL, greater than about 100 pg / mL, greater than about 150 pg / mL, or greater than about 200 pg / mL. TILs may be considered potent if, for example, interferon (IFNγ) release is greater than about 50 pg / mL, greater than about 100 pg / mL, greater than about 150 pg / mL, or greater than about 200 pg / mL, greater than about 300 pg / mL, greater than about 400 pg / mL, greater than about 500 pg / mL, greater than about 600 pg / mL, greater than about 700 pg / mL, greater than about 800 pg / mL, greater than about 900 pg / mL, greater than about 1000 pg / mL.

[0382] By “population of cells” (including TILs) herein is meant a number of cells that share common traits. In general, populations generally range from 1 X 106to 1 X 1010in number, with different TIL populations comprising different numbers. For example, initial growth of primary TILs in the presence of IL-2 results in a population of bulk TILs of roughly 1 × 108cells. REP expansion is generally done to provide populations of 1.5 × 109to 1.5 × 1010cells for infusion.

[0383] By “cryopreserved TILs” herein is meant that TILs, either primary, bulk, or expanded (REP TILs), are treated and stored in the range of about -150°C to -60°C. General methods for cryopreservation are also described elsewhere herein, including in the Examples. For clarity, “cryopreserved TILs” are distinguishable from frozen tissue samples which may be used as a source of primary TILs.

[0384] By “thawed cryopreserved TILs” herein is meant a population of TILs that was previously cryopreserved and then treated to return to room temperature or higher, including but not limited to cell culture temperatures or temperatures wherein TILs may be administered to a patient.

[0385] TILs can generally be defined either biochemically, using cell surface markers, or functionally, by their ability to infiltrate tumors and effect treatment. TILs can be generally categorized by expressing one or more of the following biomarkers: CD4, CD8, 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 a patient.

[0386] The term “cryopreservation media” or “cryopreservation medium” refers to any medium that can be used for cryopreservation of cells. Such media can include media comprising 7% to 10% DMSO. Exemplary media include CryoStor CS10, Hyperthermasol, as well as combinations thereof. The term “CS10” refers to a cryopreservation medium which is obtained from Stemcell Technologies or from Biolife Solutions. The CS10 medium may be referred to by the trade name “CryoStor® CS10”. The CS10 medium is a serum-free, animal component-free medium which comprises DMSO.

[0387] The term “central memory T cell” refers to a subset of T cells that in the human are CD45R0+ and constitutively express CCR7 (CCR7hi) and CD62L (CD62hi). The surface phenotype of central memory T cells also includes TCR, CD3, CD127 (IL-7R), and IL-15R. Transcription factors for central memory T cells include BCL-6, BCL-6B, MBD2, and BMI1. Central memory T cells primarily secret IL-2 and CD40L as effector molecules after TCR triggering. Central memory T cells are predominant in the CD4 compartment in blood, and in the human are proportionally enriched in lymph nodes and tonsils.

[0388] The term “effector memory T cell” refers to a subset of human or mammalian T cells that, like central memory T cells, are CD45R0+, but have lost the constitutive expression of CCR7 (CCR7lo) and are heterogeneous or low for CD62L expression (CD62Llo). The surface phenotype of central memory T cells also includes TCR, CD3, CD127 (IL-7R), and IL-15R. Transcription factors for central memory T cells include BLIMP1. Effector memory T cells rapidly secret high levels of inflammatory cytokines following antigenic stimulation, including interferon-γ, IL-4, and IL-5. Effector memory T cells are predominant in the CD8 compartment in blood, and in the human are proportionally enriched in the lung, liver, and gut. CD8+ effector memory T cells carry large amounts of perforin.

[0389] The term “closed system” refers to a system that is closed to the outside environment. Any closed system appropriate for cell culture methods can be employed with the methods of the present invention. Closed systems include, for example, but are not limited to, closed G-containers. Once a tumor segment is added to the closed system, the system is no opened to the outside environment until the TILs are ready to be administered to the patient.

[0390] The terms “fragmenting,” “fragment,” and “fragmented,” as used herein to describe processes for disrupting a tumor, includes mechanical fragmentation methods such as crushing, slicing, dividing, and morcellating tumor tissue as well as any other method for disrupting the physical structure of tumor tissue.

[0391] The terms “peripheral blood mononuclear cells” and “PBMCs” refers to a peripheral blood cell having a round nucleus, including lymphocytes (T cells, B cells, NK cells) and monocytes. When used as an antigen presenting cell (PBMCs are a type of antigen-presenting cell), the peripheral blood mononuclear cells are preferably irradiated allogeneic peripheral blood mononuclear cells.

[0392] The terms “peripheral blood lymphocytes” and “PBLs” refer to T cells expanded from peripheral blood. In some embodiments, PBLs are separated from whole blood or apheresis product from a donor. In some embodiments, PBLs are separated from whole blood or apheresis product from a donor by positive or negative selection of a T cell phenotype, such as the T cell phenotype of CD3+ CD45+.

[0393] The term “anti-CD3 antibody” refers to an antibody or variant thereof, e.g., a monoclonal antibody and including human, humanized, chimeric or murine antibodies which are 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.

[0394] The term “OKT-3” (also referred to herein as “OKT3”) refers to a monoclonal antibody or biosimilar or variant thereof, including human, humanized, chimeric, or murine antibodies, directed against the CD3 receptor in the T cell antigen receptor of mature T cells, and includes commercially-available forms such as OKT-3 (30 ng / mL, MACS GMP CD3 pure, Miltenyi Biotech, Inc., San Diego, CA, USA) and muromonab or variants, conservative amino acid substitutions, glycoforms, or biosimilars thereof. The amino acid sequences of the heavy and light chains of muromonab are given in Table 1 (SEQ ID NO:1 and SEQ ID NO:2). A hybridoma capable of producing OKT-3 is deposited with the American Type Culture Collection and assigned the ATCC accession number CRL 8001. A hybridoma capable of producing OKT-3 is also deposited with European Collection of Authenticated Cell Cultures (ECACC) and assigned Catalogue No.86022706. TABLE 1. Amino acid sequences of muromonab (exemplary OKT-3 antibody).

[0395] The term “IL-2” (also referred to herein as “IL2”) refers to the T cell growth factor known as interleukin-2, and includes all forms of IL-2 including human and mammalian forms, conservative amino acid substitutions, glycoforms, biosimilars, and variants thereof. IL-2 is described, e.g., in Nelson, J. Immunol.2004, 172, 3983-88 and Malek, Annu. Rev. Immunol.2008, 26, 453-79, the disclosures of which are incorporated by reference herein. The amino acid sequence of recombinant human IL-2 suitable for use in the invention is given in Table 2 (SEQ ID NO:3). For example, the term IL-2 encompasses human, recombinant forms of IL-2 such as aldesleukin (PROLEUKIN, available commercially from multiple suppliers in 22 million IU per single use vials), as well as the form of recombinant IL-2 commercially supplied by CellGenix, Inc., Portsmouth, NH, USA (CELLGRO GMP) or ProSpec-Tany TechnoGene Ltd., East Brunswick, NJ, USA (Cat. No. CYT-209-b) and other commercial equivalents from other vendors. Aldesleukin (des-alanyl-1, serine-125 human IL-2) is a nonglycosylated human recombinant form of IL-2 with a molecular weight of approximately 15 kDa. The amino acid sequence of aldesleukin suitable for use in the invention is given in Table 2 (SEQ ID NO:4). The term IL-2 also encompasses pegylated forms of IL-2, as described herein, including the pegylated IL2 prodrug bempegaldesleukin (NKTR-214, pegylated human recombinant IL-2 as in SEQ ID NO:4 in which an average of 6 lysine residues are N6substituted with [(2,7- bis{[methylpoly(oxyethylene)]carbamoyl}-9H-fluoren-9-yl)methoxy]carbonyl), which is available from Nektar Therapeutics, South San Francisco, CA, USA, or which may be prepared by methods known in the art, such as the methods described in Example 19 of International Patent Application Publication No. WO 2018 / 132496 A1 or the method described in Example 1 of U.S. Patent Application Publication No. US 2019 / 0275133 A1, the disclosures of which are incorporated by reference herein. Bempegaldesleukin (NKTR-214) and other pegylated IL-2 molecules suitable for use in the invention are described in U.S. Patent Application Publication No. US 2014 / 0328791 A1 and International Patent Application Publication No. WO 2012 / 065086 Al, the disclosures of which are incorporated by reference herein. Alternative forms of conjugated IL-2 suitable for use in theinvention 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 by reference herein. Formulations of IL-2 suitable for use in the invention are described in U.S. Patent No.6,706,289, the disclosure of which is incorporated by reference herein.

[0396] In some embodiments, an IL-2 form suitable 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 invention are described in U.S. Patent Application Publication Nos. US 2020 / 0181220 A1 and US 2020 / 0330601 A1, the disclosures of which are incorporated by reference herein. In some embodiments, and IL-2 form suitable for use in the invention is an interleukin 2 (IL-2) conjugate comprising: an isolated and purified IL-2 polypeptide; and a conjugating 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 at E62. In some embodiments, the 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, the 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 an unnatural amino acid. In some embodiments, the unnatural amino acid comprises N6-azidoethoxy-L-lysine (AzK), N6-propargylethoxy-L-lysine (PraK), BCN-L-lysine, norbornene lysine, TCO-lysine, methyltetrazine lysine, allyloxycarbonyllysine, 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-L-phenylalanine, p-azido-L-phenylalanine, p-acyl-L- phenylalanine, p-benzoyl-L-phenylalanine, p-bromophenylalanine, p-amino-L-phenylalanine, isopropyl-L-phenylalanine, O-allyltyrosine, O-methyl-L-tyrosine, O-4-allyl-L-tyrosine, 4-propyl-L- tyrosine, phosphonotyrosine, tri-O-acetyl-GlcNAcp-serine, L-phosphoserine, phosphonoserine, L-3-(2-naphthyl)alanine, 2-amino-3-((2-((3-(benzyloxy)-3-oxopropyl)amino)ethyl)selanyl)propanoic acid, 2-amino-3-(phenylselanyl)propanoic, or selenocysteine. In some embodiments, the IL-2 conjugate has a decreased affinity to IL-2 receptor α (IL-2Rα) subunit relative to a wild-type IL-2 polypeptide. In some embodiments, the decreased affinity is about 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 95%, 99%, or greater than 99% decrease in binding affinity to IL-2Rα relative to a wild-type IL-2 polypeptide. In some embodiments, the decreased affinity is about 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, 1000-fold, or more relative to a wild-type IL-2 polypeptide. In some embodiments, the conjugating moiety impairs or blocks the binding of IL-2 with IL-2Rα. In some embodiments, the conjugating moiety comprises a water-soluble polymer. In some embodiments, the additional conjugating 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(hydroxyalkylmethacrylamide), poly(hydroxyalkylmethacrylate), poly(saccharides), poly(α-hydroxy acid), poly(vinyl alcohol), polyphosphazene, polyoxazolines (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 a linear PEG or a 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 polyamine. In some embodiments, the conjugating moiety comprises a protein. In some embodiments, the additional conjugating moiety comprises a protein. In some embodiments, each of the proteins independently comprises an albumin, a transferrin, or a 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 IgG. In some embodiments, the conjugating moiety comprises a polypeptide. In some embodiments, the additional conjugating moiety comprises a polypeptide. In some embodiments, each of the polypeptides independently comprises a XTEN peptide, a glycine-rich homoamino acid polymer (HAP), 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 conjugating moiety is directly bound to the isolated and purified IL-2 polypeptide. In some embodiments, the conjugating moiety is indirectly bound to the isolated and purified IL-2polypeptide through a linker. In some embodiments, the linker comprises a homobifunctional linker. In some embodiments, the homobifunctional linker comprises Lomant's reagent dithiobis (succinimidylpropionate) DSP, 3′3′-dithiobis(sulfosuccinimidyl proprionate) (DTSSP), disuccinimidyl suberate (DSS), bis(sulfosuccinimidyl)suberate (BS), disuccinimidyl tartrate (DST), disulfosuccinimidyl tartrate (sulfo DST), ethylene glycobis(succinimidylsuccinate) (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)propionamido)butane (DPDPB), bismaleimidohexane (BMH), aryl halide-containing compound (DFDNB), such as e.g.1,5-difluoro-2,4-dinitrobenzene or 1,3-difluoro-4,6-dinitrobenzene, 4,4′-difluoro-3,3′-dinitrophenylsulfone (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′-hexamethylene-bis(iodoacetamide). In some embodiments, the linker comprises a heterobifunctional linker. In some embodiments, the heterobifunctional linker comprises 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)toluamido]hexanoate (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 (MBs), m-maleimidobenzoyl-N- hydroxysulfosuccinimide ester (sulfo-MBs), N-succinimidyl(4-iodoacteyl)aminobenzoate (sIAB), sulfosuccinimidyl(4-iodoacteyl)aminobenzoate (sulfo-sIAB), succinimidyl-4-(p- maleimidophenyl)butyrate (sMPB), sulfosuccinimidyl-4-(p-maleimidophenyl)butyrate (sulfo-sMPB), N-(γ-maleimidobutyryloxy)succinimide ester (GMBs), N-(γ-maleimidobutyryloxy)sulfosuccinimide ester (sulfo-GMBs), 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 cross-linkers such as 4-(4-N- maleimidophenyl)butyric acid hydrazide (MPBH), 4-(N-maleimidomethyl)cyclohexane-1-carboxyl- hydrazide-8 (M2C2H), 3-(2-pyridyldithio)propionyl hydrazide (PDPH), N-hydroxysuccinimidyl-4- azidosalicylic acid (NHs-AsA), N-hydroxysulfosuccinimidyl-4-azidosalicylic acid (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′- nitrophenyl amino)hexanoate (sANPAH), sulfosuccinimidyl-6-(4′-azido-2′- nitrophenylamino)hexanoate (sulfo-sANPAH), N-5-azido-2-nitrobenzoyloxysuccinimide (ANB-NOs), sulfosuccinimidyl-2-(m-azido-o-nitrobenzamido)-ethyl-1,3′-dithiopropionate (sAND), N-succinimidyl- 4(4-azidophenyl)1,3′-dithiopropionate (sADP), N-sulfosuccinimidyl(4-azidophenyl)-1,3′- dithiopropionate (sulfo-sADP), sulfosuccinimidyl 4-(ρ-azidophenyl)butyrate (sulfo-sAPB), sulfosuccinimidyl 2-(7-azido-4-methylcoumarin-3-acetamide)ethyl-1,3′-dithiopropionate (sAED), sulfosuccinimidyl 7-azido-4-methylcoumain-3-acetate (sulfo-sAMCA), p-nitrophenyl diazopyruvate (pNPDP), p-nitrophenyl-2-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-azidobenzoyl hydrazide (ABH), 4-(ρ- azidosalicylamido)butylamine (AsBA), or p-azidophenyl glyoxal (APG). In some embodiments, the linker comprises a cleavable linker, optionally comprising 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 comprises a maleimide group, optionally comprising maleimidocaproyl (mc), succinimidyl-4-(N-maleimidomethyl)cyclohexane-1- carboxylate (sMCC), or sulfosuccinimidyl-4-(N-maleimidomethyl)cyclohexane-1-carboxylate (sulfo- sMCC). In some embodiments, the linker further comprises a spacer. In some embodiments, the spacer comprises p-aminobenzyl alcohol (PAB), p-aminobenzyoxycarbonyl (PABC), a derivative, or an analog thereof. In some embodiments, the conjugating moiety is capable of extending the serum half-life of the IL-2 conjugate. In some embodiments, the additional conjugating 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 invention is a fragment of any of the IL-2 forms described herein. In some embodiments, the IL-2 form suitable for use in the invention is pegylated as disclosed in U.S. Patent Application Publication No. US 2020 / 0181220 A1 and U.S. Patent Application Publication No. US 2020 / 0330601 A1. In some embodiments, the IL-2 form suitable for use in the invention is an IL-2 conjugate comprising: an IL-2 polypeptide comprising an N6-azidoethoxy-L-lysine (AzK) covalently attached to a conjugating moiety comprising a 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 the AzK substitutes for an amino acid at position K35, F42, F44, K43, E62, P65, R38, T41, E68, Y45, V69, or L72 in reference to the amino acid positions 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 someembodiments, the IL-2 form suitable for use in the invention lacks IL-2R alpha chain engagement but retains normal binding to the intermediate affinity IL-2R beta-gamma signaling complex.

[0397] In some embodiments, the IL-2 form suitable for use in the invention is an IL-2 conjugate comprising: an IL-2 polypeptide comprising an N6-azidoethoxy-L-lysine (AzK) covalently attached to a conjugating moiety comprising a 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 the AzK substitutes for an amino acid at position K35, F42, F44, K43, E62, P65, R38, T41, E68, Y45, V69, or L72 in reference to the amino acid positions within SEQ ID NO:5. In some embodiments, the IL-2 form suitable for use in the invention is an IL-2 conjugate comprising: an IL-2 polypeptide comprising an N6-azidoethoxy-L-lysine (AzK) covalently attached to a conjugating moiety comprising a 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 the AzK substitutes for an amino acid at position K35, F42, F44, K43, E62, P65, R38, T41, E68, Y45, V69, or L72 in reference to the amino acid positions within SEQ ID NO:5. In some embodiments, the IL-2 form suitable for use in the invention is an IL-2 conjugate comprising: an IL-2 polypeptide comprising an N6-azidoethoxy-L- lysine (AzK) covalently attached to a conjugating moiety comprising a 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 the AzK substitutes for an amino acid at position K35, F42, F44, K43, E62, P65, R38, T41, E68, Y45, V69, or L72 in reference to the amino acid positions within SEQ ID NO:5.

[0398] In some embodiments, an IL-2 form suitable for use in the invention is nemvaleukin alfa, also known as ALKS-4230 (SEQ ID NO:6), which is available from Alkermes, Inc. Nemvaleukin alfa is also known as human interleukin 2 fragment (1-59), variant (Cys125>Ser51), fused via peptidyl linker (60GG61) to human interleukin 2 fragment (62-132), fused via peptidyl linker (133GSGGGS138) to human interleukin 2 receptor α-chain fragment (139-303), produced in Chinese hamster ovary (CHO) cells, glycosylated; human interleukin 2 (IL-2) (75-133)-peptide [Cys125(51)>Ser]-mutant (1-59), fused via a G2peptide linker (60-61) to human interleukin 2 (IL-2) (4-74)-peptide (62-132) and via a GSG3S peptide linker (133-138) to human interleukin 2 receptor α-chain (IL2R subunit alpha, IL2Rα, IL2RA) (1-165)-peptide (139-303), produced in Chinese hamster ovary (CHO) cells, glycoform alfa. The amino acid sequence of nemvaleukin alfa is given in SEQ ID NO:6. In some embodiments, nemvaleukin alfa exhibits the following post-translational modifications: disulfide bridges at positions: 31-116, 141-285, 184-242, 269-301, 166-197 or 166-199, 168-199 or 168-197 (using the numbering in SEQ ID NO:6), and glycosylation sites at positions: N187, N206, T212 using thenumbering in 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 invention, is described in U.S. Patent Application Publication No. US 2021 / 0038684 A1 and U.S. Patent No.10,183,979, the disclosures of which are incorporated by reference herein. In some embodiments, an IL-2 form suitable for use in the 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 invention has the amino acid sequence given in SEQ ID NO:6 or conservative amino acid substitutions thereof. In some embodiments, an IL-2 form suitable for use in the invention is a fusion protein comprising amino acids 24-452 of SEQ ID NO: 7, or variants, fragments, or derivatives thereof. In some embodiments, an IL-2 form suitable for use in the 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 variants, fragments, or derivatives thereof. Other IL-2 forms suitable for use in the present invention are described in U.S. Patent No.10,183,979, the disclosure of which is incorporated by reference herein. Optionally, in some embodiments, an IL-2 form suitable for use in the invention is a fusion protein comprising a first fusion partner that is 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 the receptor antagonist activity of IL-Rα, and wherein the second fusion partner comprises all or a portion of an immunoglobulin comprising an Fc region, wherein 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 as compared to a fusion of the first fusion partner to the second fusion partner in the absence of the mucin domain polypeptide linker. TABLE 2. Amino acid sequences of interleukins.

[0399] In some embodiments, an IL-2 form suitable for use in the invention includes a antibody cytokine engrafted protein comprises a heavy chain variable region (VH), comprising complementarity determining regions HCDR1, HCDR2, HCDR3; a light chain variable region (VL), comprising LCDR1, LCDR2, LCDR3; and an IL-2 molecule or a fragment thereof engrafted into a CDR of the VHor the VL, wherein the antibody cytokine engrafted protein preferentially expands T effector cells over regulatory T cells. In some embodiments, the antibody cytokine engrafted proteincomprises a heavy chain variable region (VH), comprising complementarity determining regions HCDR1, HCDR2, HCDR3; a light chain variable region (VL), comprising LCDR1, LCDR2, LCDR3; and an IL-2 molecule or a fragment thereof engrafted into a CDR of the VHor the VL, wherein the IL-2 molecule is a mutein, and wherein the antibody cytokine engrafted protein preferentially expands T effector cells over regulatory T cells. In some embodiments, the IL-2 regimen comprises administration of an antibody described in U.S. Patent Application Publication No. US 2020 / 0270334 A1, the disclosures of which are incorporated by reference herein. In some embodiments, the antibody cytokine engrafted protein comprises a heavy chain variable region (VH), comprising complementarity determining regions HCDR1, HCDR2, HCDR3; a light chain variable region (VL), comprising LCDR1, LCDR2, LCDR3; and an IL-2 molecule or a fragment thereof engrafted into a CDR of the VHor the VL, wherein the IL-2 molecule is a mutein, wherein the antibody cytokine engrafted protein preferentially expands T effector cells over regulatory T cells, and wherein the antibody further comprises an IgG class heavy chain and an IgG class light chain selected from the group consisting of: a IgG class light chain comprising SEQ ID NO:39 and a IgG class heavy chain comprising SEQ ID NO:38; a IgG class light chain comprising SEQ ID NO:37 and a IgG class heavy chain comprising SEQ ID NO:29; a IgG class light chain comprising SEQ ID NO:39 and a IgG class heavy chain comprising SEQ ID NO:29; and a IgG class light chain comprising SEQ ID NO:37 and a IgG class heavy chain comprising SEQ ID NO:38.

[0400] In some embodiments, an IL-2 molecule or a fragment thereof is engrafted into HCDR1 of the VH, wherein the IL-2 molecule is a mutein. In some embodiments, an IL-2 molecule or a fragment thereof is engrafted into HCDR2 of the VH, wherein the IL-2 molecule is a mutein. In some embodiments, an IL-2 molecule or a fragment thereof is engrafted into HCDR3 of the VH, wherein the IL-2 molecule is a mutein. In some embodiments, an IL-2 molecule or a fragment thereof is engrafted into LCDR1 of the VL, wherein the IL-2 molecule is a mutein. In some embodiments, an IL-2 molecule or a fragment thereof is engrafted into LCDR2 of the VL, wherein the IL-2 molecule is a mutein. In some embodiments, an IL-2 molecule or a fragment thereof is engrafted into LCDR3 of the VL, wherein the IL-2 molecule is a mutein.

[0401] The insertion of the IL-2 molecule can be at or near the N-terminal region of the CDR, in the middle region of the CDR or at or near the C-terminal region of the CDR. In some embodiments, the antibody cytokine engrafted protein comprises an IL-2 molecule incorporated into a CDR, wherein the IL2 sequence does not frameshift the CDR sequence. In some embodiments, the antibody cytokine engrafted protein comprises an IL-2 molecule incorporated into a CDR, wherein the IL-2 sequence replaces all or part of a CDR sequence. The replacement by the IL-2 molecule canbe the N-terminal region of the CDR, in the middle region of the CDR or at or near the C-terminal region the CDR. A replacement by the IL-2 molecule can be as few as one or two amino acids of a CDR sequence, or the entire CDR sequences.

[0402] In some embodiments, an IL-2 molecule is engrafted directly into a CDR without a peptide linker, with no additional amino acids between the CDR sequence and the IL-2 sequence. In some embodiments, an IL-2 molecule is engrafted indirectly into a CDR with a peptide linker, with one or more additional amino acids between the CDR sequence and the IL-2 sequence.

[0403] In some embodiments, the IL-2 molecule described herein is an IL-2 mutein. In some instances, the IL-2 mutein comprising an R67A substitution. In some embodiments, the IL-2 mutein comprises the amino acid sequence SEQ ID NO:14 or SEQ ID NO:15. In some embodiments, the IL-2 mutein comprises an amino acid sequence in Table 1 in U.S. Patent Application Publication No. US 2020 / 0270334 A1, the disclosure of which is incorporated by reference herein.

[0404] In some embodiments, the antibody cytokine engrafted 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 engrafted 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 engrafted protein comprises an HCDR1 selected from the group consisting of 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 engrafted 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 engrafted protein comprises a VHregion comprising the amino acid sequence of SEQ ID NO:28. In some embodiments, the antibody cytokine engrafted protein comprises a heavy chain comprising the amino acid sequence of SEQ ID NO:29. In some embodiments, the antibody cytokine engrafted protein comprises a VLregion comprising the amino acid sequence of SEQ ID NO:36. In some embodiments, the antibody cytokine engrafted protein comprises a light chain comprising the amino acid sequence of SEQ ID NO:37. In some embodiments, the antibody cytokine engrafted protein comprises a VHregion comprising the amino acid sequence of SEQ ID NO:28 and a VLregion comprising the amino acid sequence of SEQ ID NO:36. In some embodiments, the antibody cytokine engrafted protein comprises a heavy chain region comprising the amino acid sequence of SEQ ID NO:29 and a light chain region comprising the amino acid sequence of SEQ ID NO:37. In some embodiments, the antibody cytokine engrafted 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 IDNO:39. In some embodiments, the antibody cytokine engrafted protein comprises a heavy chain region comprising the amino acid sequence of SEQ ID NO:38 and a light chain region comprising the amino acid sequence of SEQ ID NO:37. In some embodiments, the antibody cytokine engrafted protein comprises a heavy chain region comprising the amino acid sequence of SEQ ID NO:38 and a light chain region comprising the amino acid sequence of SEQ ID NO:39. In some embodiments, the antibody cytokine engrafted protein comprises IgG.IL2F71A.H1 or IgG.IL2R67A.H1 of U.S. Patent Application Publication No.2020 / 0270334 A1, or variants, derivatives, or fragments thereof, or conservative amino acid substitutions thereof, or proteins with at least 80%, at least 90%, at least 95%, or at least 98% sequence identity thereto. In some embodiments, the antibody components of the antibody cytokine engrafted protein described herein comprise immunoglobulin sequences, framework sequences, or CDR sequences of palivizumab. In some embodiments, the antibody cytokine engrafted protein described herein has a longer serum half-life that a wild-type IL-2 molecule such as, but not limited to, aldesleukin or a comparable molecule. In some embodiments, the antibody cytokine engrafted protein described herein has a sequence as set forth in Table 3. TABLE 3: Sequences of exemplary palivizumab antibody-IL-2 engrafted proteins

[0405] The term “IL-4” (also referred to herein as “IL4”) refers to the cytokine known as interleukin 4, which is produced by Th2 T cells and by eosinophils, basophils, and mast cells. IL-4 regulates the differentiation of naïve helper T cells (Th0 cells) to Th2 T cells. Steinke and Borish, Respir. Res.2001, 2, 66-70. Upon activation by IL-4, Th2 T cells subsequently produce additional IL-4 in a positive feedback loop. IL-4 also stimulates B cell proliferation and class II MHC expression, and induces class switching to IgE and IgG1expression from B cells. Recombinant human IL-4 suitable for use in the invention is commercially available from multiple suppliers, including ProSpec-Tany TechnoGene Ltd., East Brunswick, NJ, USA (Cat. No. CYT-211) and ThermoFisher Scientific, Inc., Waltham, MA, USA (human IL-15 recombinant protein, Cat. No. Gibco CTP0043). The amino acid sequence of recombinant human IL-4 suitable for use in the invention is given in Table 2 (SEQ ID NO:9).

[0406] The term “IL-7” (also referred to herein as “IL7”) refers to a glycosylated tissue- derived cytokine known as interleukin 7, which may be obtained from stromal and epithelial cells, as well as from dendritic cells. Fry and Mackall, Blood 2002, 99, 3892-904. IL-7 can stimulate the development of T cells. IL-7 binds to the IL-7 receptor, a heterodimer consisting of IL-7 receptor alpha and common gamma chain receptor, which in a series of signals important for T cell development within the thymus and survival within the periphery. Recombinant human IL-7 suitable for use in the invention is commercially available from multiple suppliers, including ProSpec-Tany TechnoGene Ltd., East Brunswick, NJ, USA (Cat. No. CYT-254) and ThermoFisher Scientific, Inc., Waltham, MA, USA (human IL-15 recombinant protein, Cat. No. Gibco PHC0071). The amino acid sequence of recombinant human IL-7 suitable for use in the invention is given in Table 2 (SEQ ID NO:10).

[0407] The term “IL-15” (also referred to herein as “IL15”) refers to the T cell growth factor known as interleukin-15, and includes all forms of IL-2 including human and mammalian forms, conservative amino acid substitutions, glycoforms, biosimilars, and variants thereof. IL-15 is described, e.g., in Fehniger and Caligiuri, Blood 2001, 97, 14-32, the disclosure of which is incorporated by reference herein. 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 mass of 12.8 kDa. Recombinant human IL-15 is commercially available from multiple suppliers, including ProSpec-Tany TechnoGene Ltd., East Brunswick, NJ, USA (Cat. No. CYT-230-b) and ThermoFisher Scientific, Inc., Waltham, MA, USA(human IL-15 recombinant protein, Cat. No.34-8159-82). The amino acid sequence of recombinant human IL-15 suitable for use in the invention is given in Table 2 (SEQ ID NO:11).

[0408] The term “IL-21” (also referred to herein as “IL21”) refers to the pleiotropic cytokine protein known as interleukin-21, and includes all forms of IL-21 including human and mammalian forms, conservative amino acid substitutions, glycoforms, biosimilars, and variants thereof. IL-21 is described, e.g., in Spolski and Leonard, Nat. Rev. Drug. Disc.2014, 13, 379-95, the disclosure of which is incorporated by reference herein. IL-21 is primarily produced by natural killer T cells and activated human CD4+T cells. Recombinant human IL-21 is a single, non-glycosylated polypeptide chain containing 132 amino acids with a molecular mass of 15.4 kDa. Recombinant human IL-21 is commercially available from multiple suppliers, including ProSpec-Tany TechnoGene Ltd., East Brunswick, NJ, USA (Cat. No. CYT-408-b) and ThermoFisher Scientific, Inc., Waltham, MA, USA (human IL-21 recombinant protein, Cat. No.14-8219-80). The amino acid sequence of recombinant human IL-21 suitable for use in the invention is given in Table 2 (SEQ ID NO:21).

[0409] When “an anti-tumor effective amount”, “a tumor-inhibiting effective amount”, or “therapeutic amount” is indicated, the precise amount of the compositions of the present invention to be administered can be determined by a physician with consideration of individual differences in age, weight, tumor size, extent of infection or metastasis, and condition of the patient (subject). It can generally be stated that a pharmaceutical composition comprising the tumor infiltrating lymphocytes (e.g. secondary TILs or genetically modified cytotoxic lymphocytes) described herein may be administered at a dosage of 104to 1011cells / kg body weight (e.g., 105to 106, 105to 1010, 105to 1011, 106to 1010, 106to 1011,107to 1011, 107to 1010, 108to 1011, 108to 1010, 109to 1011, or 109to 1010cells / kg body weight), including all integer values within those ranges. TILs (including in some cases, genetically modified cytotoxic lymphocytes) compositions may also be administered multiple times at these dosages. The tumor TILs (inlcuding, in some cases, genetically engineered TILs) can be administered by using infusion techniques that are commonly known in immunotherapy (see, e.g., Rosenberg et al., New Eng. J. of Med.1988, 319, 1676,). The optimal dosage and treatment regime for a particular patient can readily be determined by one skilled in the art of medicine by monitoring the patient for signs of disease and adjusting the treatment accordingly.

[0410] The term “hematological malignancy”, “hematologic malignancy” or terms of correlative meaning refer to mammalian cancers and tumors of the hematopoietic and lymphoid tissues, including but not limited to tissues of the blood, bone marrow, lymph nodes, and lymphatic system. Hematological malignancies are also referred to as “liquid tumors.” Hematological malignancies include, but are not limited to, acute lymphoblastic leukemia (ALL), chroniclymphocytic lymphoma (CLL), small lymphocytic lymphoma (SLL), acute myelogenous leukemia (AML), chronic myelogenous leukemia (CML), multiple myeloma, acute monocytic leukemia (AMoL), Hodgkin’s lymphoma, and non-Hodgkin’s lymphomas. The term “B cell hematological malignancy” refers to hematological malignancies that affect B cells.

[0411] 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, leukemias, myelomas, and lymphomas, as well as other hematological malignancies. TILs obtained from liquid tumors may also be referred to herein as marrow infiltrating lymphocytes (MILs). TILs obtained from liquid tumors, including liquid tumors circulating in peripheral blood, may also be referred to herein as PBLs. The terms MIL, TIL, and PBL are used interchangeably herein and differ only based on the tissue type from which the cells are derived.

[0412] The term “microenvironment,” as used herein, may refer to the solid or hematological tumor microenvironment as a whole or to an individual subset of cells within the microenvironment. The tumor microenvironment, as used herein, refers to a complex mixture of “cells, soluble factors, signaling molecules, extracellular matrices, and mechanical cues that promote neoplastic transformation, support tumor growth and invasion, protect the tumor from host immunity, foster therapeutic resistance, and provide niches for dominant metastases to thrive,” as described in Swartz, et al., Cancer Res., 2012, 72, 2473. Although tumors express antigens that should be recognized by T cells, tumor clearance by the immune system is rare because of immune suppression by the microenvironment.

[0413] In some embodiments, the invention includes a method of treating a cancer with a population of TILs, wherein a patient is pre-treated with non-myeloablative chemotherapy prior to an infusion of TILs according to the invention. In some embodiments, the population of TILs may be provided wherein a patient is pre-treated with nonmyeloablative chemotherapy prior to an infusion of TILs according to the present invention. In some embodiments, the non-myeloablative chemotherapy is cyclophosphamide 60 mg / kg / d for 2 days (days 27 and 26 prior to TIL infusion) and fludarabine 25 mg / m2 / d for 5 days (days 27 to 23 prior to TIL infusion). In some embodiments, after non-myeloablative chemotherapy and TIL infusion (at day 0) according to the invention, the patient receives an intravenous infusion of IL-2 intravenously at 720,000 IU / kg every 8 hours to physiologic tolerance.

[0414] Experimental findings indicate that lymphodepletion prior to adoptive transfer of tumor-specific T lymphocytes plays a key role in enhancing treatment efficacy by eliminatingregulatory T cells and competing elements of the immune system (“cytokine sinks”). Accordingly, some embodiments of the invention utilize a lymphodepletion step (sometimes also referred to as “immunosuppressive conditioning”) on the patient prior to the introduction of the TILs of the invention.

[0415] The term “effective amount” or “therapeutically effective amount” refers to that amount of a compound or combination of compounds as described herein that is sufficient to effect the intended application including, but not limited to, disease treatment. A therapeutically effective amount may vary depending upon the intended application (in vitro or in vivo), or the subject and disease condition being treated (e.g., the weight, age and gender of the subject), the severity of the disease condition, or the manner of administration. The term also applies to a dose that will induce a particular response in target cells (e.g., the reduction of platelet adhesion and / or cell migration). The specific dose will vary depending on the particular compounds chosen, the dosing regimen to be followed, whether the compound is administered in combination with other compounds, timing of administration, the tissue to which it is administered, and the physical delivery system in which the compound is carried.

[0416] The terms “treatment”, “treating”, “treat”, and the like, refer to obtaining a desired pharmacologic and / or physiologic effect. The effect may be prophylactic in terms of completely or partially preventing a disease or symptom thereof and / or may be therapeutic in terms of a partial or complete cure for a disease and / or adverse effect attributable to the disease. “Treatment”, as used herein, covers any treatment of a disease in a mammal, particularly in a human, and includes: (a) preventing the disease from occurring in a subject which may be predisposed to the disease but has not yet been diagnosed as having it; (b) inhibiting the disease, i.e., arresting its development or progression; and (c) relieving the disease, i.e., causing regression of the disease and / or relieving one or more disease symptoms. “Treatment” is also meant to encompass delivery of an agent in order to provide for a pharmacologic effect, even in the absence of a disease or condition. For example, “treatment” encompasses delivery of a composition that can elicit an immune response or confer immunity in the absence of a disease condition, e.g., in the case of a vaccine.

[0417] 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 instance, the nucleic acid is typically recombinantly produced, having two or more sequences from unrelated genes arranged to make 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 theprotein comprises two or more subsequences that are not found in the same relationship to each other in nature (e.g., a fusion protein).

[0418] The terms “sequence identity,” “percent identity,” and “sequence percent identity” (or synonyms thereof, 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 nucleotides or amino acid residues that are the same, when compared and aligned (introducing gaps, if necessary) for maximum correspondence, not considering any conservative amino acid substitutions as part of the sequence identity. The percent identity can be measured using sequence comparison software or algorithms or by visual inspection. Various algorithms and software are known in the art that can be used to obtain alignments of amino acid or nucleotide sequences. Suitable programs to determine percent sequence identity include for example the BLAST suite of programs available from the U.S. Government’s National Center for Biotechnology Information BLAST web site. Comparisons between two sequences can be carried using either the BLASTN or BLASTP algorithm. BLASTN is used to compare nucleic acid sequences, while BLASTP is used to compare amino acid sequences. ALIGN, ALIGN-2 (Genentech, South San Francisco, California) or MegAlign, available from DNASTAR, are additional publicly available software programs that can be used to align sequences. One skilled in the art can determine appropriate parameters for maximal alignment by particular alignment software. In certain embodiments, the default parameters of the alignment software are used.

[0419] As used herein, the term “variant” encompasses but is not limited to antibodies or fusion proteins which comprise an amino acid sequence which differs from the amino acid sequence of a reference antibody by way of one or more substitutions, deletions and / or additions at certain positions within or adjacent to the amino acid sequence of the reference antibody. The variant may comprise one or more conservative substitutions in its amino acid sequence as compared to the amino acid sequence of a reference antibody. Conservative substitutions may involve, e.g., the substitution of similarly charged or uncharged amino acids. The variant retains the ability to specifically bind to the antigen of the reference antibody. The term variant also includes pegylated antibodies or proteins.

[0420] By “tumor infiltrating lymphocytes” or “TILs” herein is meant a population of cells originally obtained as white blood cells that have left the bloodstream of a subject and migrated into a tumor. TILs include, but are not limited to, CD8+cytotoxic T cells (lymphocytes), Th1 and Th17 CD4+T cells, natural killer cells, dendritic cells and M1 macrophages. TILs include both primary and secondary TILs. “Primary TILs” are those that are obtained from patient tissue samples as outlinedherein (sometimes referred to as “freshly harvested”), and “secondary TILs” are any TIL cell populations that have been expanded or proliferated as discussed herein, including, but not limited to bulk TILs, expanded TILs (“REP TILs”) as well as “reREP TILs” as discussed herein. reREP TILs can include for example second expansion TILs or second additional expansion TILs (such as, for example, those described in Step D of Figure 8, including TILs referred to as reREP TILs).

[0421] TILs can generally be defined either biochemically, using cell surface markers, or functionally, by their ability to infiltrate tumors and effect treatment. TILs can be generally categorized by expressing one or more of the following biomarkers: CD4, CD8, 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 a patient. TILs may further be characterized by potency – for example, TILs may be considered potent if, for example, interferon (IFN) release is greater than about 50 pg / mL, greater than about 100 pg / mL, greater than about 150 pg / mL, or greater than about 200 pg / mL. TILs may be considered potent if, for example, interferon (IFNγ) release is greater than about 50 pg / mL, greater than about 100 pg / mL, greater than about 150 pg / mL, or greater than about 200 pg / mL, greater than about 300 pg / mL, greater than about 400 pg / mL, greater than about 500 pg / mL, greater than about 600 pg / mL, greater than about 700 pg / mL, greater than about 800 pg / mL, greater than about 900 pg / mL, greater than about 1000 pg / mL.

[0422] The term “deoxyribonucleotide” encompasses natural and synthetic, unmodified and modified deoxyribonucleotides. Modifications include changes to the sugar moiety, to the base moiety and / or to the linkages between deoxyribonucleotide in the oligonucleotide.

[0423] The term “RNA” defines a molecule comprising at least one ribonucleotide residue. The term “ribonucleotide” defines a nucleotide with a hydroxyl group at the 2' position of a b-D- 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, as well as altered RNA that differs from naturally occurring RNA by the addition, deletion, substitution and / or alteration of one or more nucleotides. Nucleotides of the RNA molecules described herein may also comprise non-standard nucleotides, such as non-naturally occurring nucleotides or chemically synthesized nucleotides or deoxynucleotides. These altered RNAs can be referred to as analogs or analogs of naturally-occurring RNA.

[0424] The terms “pharmaceutically acceptable carrier” or “pharmaceutically acceptable excipient” are intended to include any and all solvents, dispersion media, coatings, antibacterial andantifungal agents, isotonic and absorption delaying agents, and inert 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 therapeutic compositions of the invention is contemplated. Additional active pharmaceutical ingredients, such as other drugs, can also be incorporated into the described compositions and methods.

[0425] The terms “about” and “approximately” mean within a statistically meaningful range of a value. Such a range can be within an order of magnitude, preferably within 50%, more preferably within 20%, more preferably still within 10%, and even more preferably within 5% of a given value or range. The allowable variation encompassed by the terms “about” or “approximately” depends on the particular system under study, and can be readily appreciated by one of ordinary skill in the art. Moreover, 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 desired, reflecting tolerances, conversion factors, rounding off, measurement error and the like, and other factors known to those of skill in the art. In general, a dimension, size, formulation, parameter, shape or other quantity or characteristic is “about” or “approximate” whether or not expressly stated to be such. It is noted that embodiments of very different sizes, shapes and dimensions may employ the described arrangements.

[0426] The transitional terms “comprising,” “consisting essentially of,” and “consisting of,” when used in the appended claims, in original and amended form, define the claim scope with respect to what unrecited additional 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 element, method, step or material. The term “consisting of” excludes any element, step or material other than those specified in the claim and, in the latter instance, impurities ordinarily associated with the specified material(s). The term “consisting essentially of” limits the scope of a claim to the specified elements, steps or material(s) and those that do not materially affect the basic and novel characteristic(s) of the claimed invention. All compositions, methods, and kits described herein that embody the present invention can, in alternate embodiments, be more specifically defined by any of the transitional terms “comprising,” “consisting essentially of,” and “consisting of.”

[0427] The terms “antibody” and its plural form “antibodies” refer to whole immunoglobulins and any antigen-binding fragment (“antigen-binding portion”) or single chains thereof. An “antibody” further refers to a glycoprotein comprising at least two heavy (H) chains and two light (L) chains inter-connected by disulfide bonds, or an antigen-binding portion thereof. Each heavy chain is comprised of a heavy chain variable region (abbreviated herein as VH) and a heavy chain constant region. The heavy chain constant region is comprised of three domains, CH1, CH2 and CH3. Each light chain is comprised of a light chain variable region (abbreviated herein as VL) and a light chain constant region. The light chain constant region is comprised of one domain, CL. The VHand VLregions of an antibody may be further subdivided into regions of hypervariability, which are referred to as complementarity determining regions (CDR) or hypervariable regions (HVR), and which can be interspersed with regions that are more conserved, termed framework regions (FR). Each VHand VLis composed of three CDRs and four FRs, arranged from amino-terminus to carboxy- terminus in the following order: FR1, CDR1, FR2, CDR2, FR3, CDR3, FR4. The variable regions of the heavy and light chains contain a binding domain that interacts with an antigen epitope or epitopes. The constant regions of the antibodies 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.

[0428] The term “antigen” refers to a substance that induces an immune response. In some embodiments, an antigen is a molecule capable of being bound by an antibody or a TCR if presented by major histocompatibility complex (MHC) molecules. The term “antigen”, as used herein, also encompasses T cell epitopes. An antigen is additionally capable of being recognized by the immune system. In some embodiments, an antigen is capable of inducing a humoral immune response or a cellular immune response leading to the activation of B lymphocytes and / or T lymphocytes. In some cases, this may require that the antigen contains or is linked to a Th cell epitope. An antigen can also have one or more epitopes (e.g., B- and T-epitopes). In some embodiments, an antigen will preferably react, typically in a highly specific and selective manner, with its corresponding antibody or TCR and not with the multitude of other antibodies or TCRs which may be induced by other antigens.

[0429] The terms “monoclonal antibody,” “mAb,” “monoclonal antibody composition,” or their plural forms refer to a preparation of antibody molecules of single molecular composition. A monoclonal antibody composition displays a single binding specificity and affinity for a particular epitope. Monoclonal antibodies specific to certain receptors can be made using knowledge and skill in the art of injecting test subjects with suitable antigen and then isolating hybridomas expressingantibodies having the desired sequence or functional characteristics. DNA encoding the monoclonal antibodies is readily isolated and sequenced using conventional procedures (e.g., by using oligonucleotide probes that are capable of binding specifically to genes encoding the heavy and light chains of the monoclonal antibodies). The hybridoma cells serve as a preferred source of such DNA. Once isolated, the DNA may be placed into expression vectors, which are then transfected into host cells such as E. coli cells, simian COS cells, Chinese hamster ovary (CHO) cells, or myeloma cells that do not otherwise produce immunoglobulin protein, to obtain the synthesis of monoclonal antibodies in the recombinant host cells. Recombinant production of antibodies will be described in more detail below.

[0430] The terms “antigen-binding portion” or “antigen-binding fragment” of an antibody (or simply “antibody portion” or “fragment”), as used herein, 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 within the term “antigen-binding portion” of an antibody include (i) a Fab fragment, a monovalent fragment consisting of the VL, VH, CLand CH1 domains; (ii) a F(ab′)2 fragment, a bivalent fragment comprising two Fab fragments linked by a disulfide bridge at the hinge region; (iii) a Fd fragment consisting of the VHand CH1 domains; (iv) a Fv fragment consisting of the VLand VHdomains of a single arm of an antibody, (v) a domain antibody (dAb) fragment (Ward, et al., Nature, 1989, 341, 544-546), which may consist of a VHor a VLdomain; and (vi) an isolated complementarity determining region (CDR). Furthermore, although the two domains of the Fv fragment, VLand VH, are coded for by separate genes, they can be joined, using recombinant methods, by a synthetic linker that enables them to be made as a single protein chain in which the VLand VHregions pair to form monovalent molecules known as 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 within the terms “antigen-binding portion” or “antigen-binding fragment” of an antibody. These antibody fragments are obtained using conventional techniques known to those with skill in the art, and the fragments are screened for utility in the same manner as are intact antibodies. In some embodiments, a scFv protein domain comprises a VHportion and a VLportion. A scFv molecule is denoted as either VL-L-VHif the VLdomain is the N-terminal part of the scFv molecule, or as VH-L-VLif the VHdomain is the N-terminal part of the scFv molecule. Methods for making scFv molecules and designing suitable peptide linkers are described in U.S. Pat. No.4,704,692, U.S. Pat. No.4,946,778, R. Raag and M. Whitlow, “Single Chain Fvs.” FASEB Vol 9:73-80 (1995) and R. E. Bird and B. W. Walker, Single Chain Antibody VariableRegions, TIBTECH, Vol 9: 132-137 (1991), the disclosures of which are incorporated by reference herein.

[0431] The term “human antibody,” as used herein, 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). The term “human antibody”, as used herein, 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.

[0432] The term “human monoclonal antibody” refers to antibodies displaying a single binding specificity which have variable regions in which both the framework and CDR regions are derived from human germline immunoglobulin sequences. In some embodiments, the human monoclonal antibodies are produced by a hybridoma which includes a B cell obtained from a transgenic nonhuman animal, e.g., a transgenic mouse, having a genome comprising a human heavy chain transgene and a light chain transgene fused to an immortalized cell.

[0433] The term “recombinant human antibody”, as used herein, includes all human antibodies that are prepared, expressed, created or isolated by recombinant means, such as (a) antibodies isolated from an animal (such as a mouse) that is transgenic or transchromosomal for human immunoglobulin genes or a hybridoma prepared therefrom (described further below), (b) antibodies isolated from a host cell transformed to express the human antibody, e.g., from a transfectoma, (c) antibodies isolated from a recombinant, combinatorial human antibody library, and (d) antibodies prepared, expressed, created or isolated by any other means that involve splicing of 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. In certain embodiments, however, such recombinant human antibodies can be subjected to in vitro mutagenesis (or, when an animal transgenic for human Ig sequences is used, in vivo somatic mutagenesis) and thus the amino acid sequences of the VH and VL regions of the recombinant antibodies are sequences that, while derived from and related to human germline VHand VLsequences, may not naturally exist within the human antibody germline repertoire in vivo.

[0434] As used herein, “isotype” refers to the antibody class (e.g., IgM or IgG1) that is encoded by the heavy chain constant region genes.

[0435] The phrases “an antibody recognizing an antigen” and “an antibody specific for an antigen” are used interchangeably herein with the term “an antibody which binds specifically to an antigen.”

[0436] The term “human antibody derivatives” refers to any modified form of the human antibody, including a conjugate of the antibody and another active pharmaceutical ingredient or antibody. The terms “conjugate,” “antibody-drug conjugate”, “ADC,” or “immunoconjugate” refers to an antibody, or a fragment thereof, conjugated to another therapeutic moiety, which can be conjugated to antibodies described herein using methods available in the art.

[0437] The terms “humanized antibody,” “humanized antibodies,” and “humanized” are intended to refer to antibodies in which CDR sequences derived from the germline of another mammalian species, such as a mouse, have been grafted onto human framework sequences. Additional framework region modifications may be made within the human framework sequences. Humanized forms of non-human (for example, murine) antibodies are chimeric antibodies that contain minimal sequence derived from non-human immunoglobulin. For the most part, humanized antibodies are human immunoglobulins (recipient antibody) in which residues from a hypervariable region of the recipient are replaced by residues from a 15 hypervariable region of a non-human species (donor antibody) such as mouse, rat, rabbit or nonhuman primate having the desired specificity, affinity, and capacity. In some instances, Fv framework region (FR) residues of the human immunoglobulin are replaced by corresponding non-human residues. Furthermore, humanized antibodies may comprise residues that are not found in the recipient antibody or in the donor antibody. These modifications are made to further refine antibody performance. In general, the 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 optionally also will 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 may also be modified to employ any Fc variant which is known to impart an improvement (e.g., reduction) in effector function and / or FcR binding. The Fc variants may include, for example, any one of the amino acid substitutions disclosed in International Patent Application Publication Nos. WO 1988 / 07089 A1,WO 1996 / 14339 A1, WO 1998 / 05787 A1, WO 1998 / 23289 A1, WO 1999 / 51642 A1, WO 99 / 58572 A1, WO 2000 / 09560 A2, WO 2000 / 32767 A1, WO 2000 / 42072 A2, WO 2002 / 44215 A2, WO 2002 / 060919 A2, WO 2003 / 074569 A2, WO 2004 / 016750 A2, WO 2004 / 029207 A2, WO 2004 / 035752 A2, WO 2004 / 063351 A2, WO 2004 / 074455 A2, WO 2004 / 099249 A2, WO 2005 / 040217 A2, WO 2005 / 070963 A1, WO 2005 / 077981 A2, WO 2005 / 092925 A2, WO 2005 / 123780 A2, WO 2006 / 019447 A1, WO 2006 / 047350 A2, and WO 2006 / 085967 A2; and U.S. Patent 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 by reference herein.

[0438] The term “chimeric antibody” is intended to refer to antibodies in which the variable region sequences are derived from one species and the constant region sequences are derived from another species, such as an antibody in which the variable region sequences are derived from a mouse antibody and the constant region sequences are derived from a human antibody.

[0439] A “diabody” is a small antibody fragment with two antigen-binding sites. The fragments comprise a heavy chain variable domain (VH) connected to a light chain variable domain (VL) in the same polypeptide chain (VH-VLor VL-VH). By using a linker that is too short to allow pairing between the two domains on the same chain, the domains are forced to pair with the complementary domains of another chain and create two antigen-binding sites. Diabodies are described more fully in, e.g., 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.

[0440] The term “glycosylation” refers to a modified derivative of an antibody. An aglycoslated antibody lacks glycosylation. Glycosylation can be altered to, for example, increase the affinity of the antibody for antigen. Such carbohydrate modifications can be accomplished by, for example, altering one or more sites of glycosylation within the antibody sequence. For example, one or more amino acid substitutions can be made that result in elimination of one or more variable region framework glycosylation sites to thereby eliminate glycosylation at that site. Aglycosylation may increase the affinity of the antibody for antigen, as described in U.S. Patent Nos.5,714,350 and 6,350,861. Additionally or alternatively, an antibody can be made that has an altered type of glycosylation, such as a hypofucosylated antibody having reduced amounts of fucosyl residues or an antibody having increased bisecting GlcNac structures. Such altered glycosylation patterns have been demonstrated to increase the ability of antibodies. Such carbohydrate modifications can be accomplished by, for example, expressing the antibody in a host cell with altered glycosylation machinery. Cells with altered glycosylation machinery have been described in the art and can beused as host cells in which to express recombinant antibodies of the invention to thereby produce an antibody 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 the Ms704, Ms705, and Ms709 cell lines lack fucose on their carbohydrates. The Ms704, Ms705, and Ms709 FUT8− / − cell lines were created by the targeted disrupƟon of the FUT8 gene in CHO / DG44 cells using two replacement vectors (see e.g., U.S. Patent Publication No.2004 / 0110704 or Yamane- Ohnuki, et al., Biotechnol. Bioeng., 2004, 87, 614-622). As another example, European Patent No. EP 1,176,195 describes a cell line with a functionally disrupted FUT8 gene, which encodes a fucosyl transferase, such that antibodies expressed in such a cell line exhibit hypofucosylation by reducing or eliminating the alpha 1,6 bond-related enzyme, and also describes cell lines which have a low enzyme activity for adding fucose to the N-acetylglucosamine that binds to the Fc region of the antibody or does not have the enzyme activity, for example the rat myeloma cell line YB2 / 0 (ATCC CRL 1662). International Patent Publication WO 03 / 035835 describes a variant CHO cell line, Lec 13 cells, with reduced ability to attach fucose to Asn(297)-linked carbohydrates, also resulting in hypofucosylation of antibodies expressed in that host cell (see also Shields, et al., J. Biol. Chem. 2002, 277, 26733-26740. International Patent Publication WO 99 / 54342 describes cell lines engineered to express glycoprotein-modifying glycosyl transferases (e.g., beta(1,4)-N- acetylglucosaminyltransferase III (GnTIII)) such that antibodies expressed in the engineered cell lines exhibit increased bisecting GlcNac structures which results in increased ADCC activity of the antibodies (see also Umana, et al., Nat. Biotech.1999, 17, 176-180). Alternatively, the fucose residues of the antibody may be cleaved off using a fucosidase enzyme. For example, the fucosidase alpha-L-fucosidase removes fucosyl residues from antibodies as described in Tarentino, et al., Biochem.1975, 14, 5516-5523.

[0441] “Pegylation” refers to a modified antibody, or a fragment thereof, that typically is reacted with polyethylene glycol (PEG), such as a reactive ester or aldehyde derivative of PEG, under conditions in which one or more PEG groups become attached to the antibody or antibody fragment. Pegylation may, for example, increase the biological (e.g., serum) half life of the antibody. Preferably, the 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” is intended to encompass any of the forms of PEG that have been used to derivatize other proteins, such as mono (C1-C10)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,as described for example in European Patent Nos. EP 0154316 and EP 0401384 and U.S. Patent No. 5,824,778, the disclosures of each of which are incorporated by reference herein.

[0442] The term “biosimilar” means a biological product, including a monoclonal antibody or protein, that is highly similar to a U.S. licensed reference biological product notwithstanding minor differences in clinically inactive components, and for which there are no clinically meaningful differences between the biological product and the reference product in terms of the safety, purity, and potency of the product. Furthermore, a similar biological or “biosimilar” medicine is a biological medicine that is similar to another biological medicine that has already been authorized for use by the European Medicines Agency. The term “biosimilar” is also used synonymously by other national and regional regulatory agencies. Biological products or biological medicines are medicines that are made by or derived from a biological source, such as a bacterium or yeast. They can consist of relatively small molecules such as human insulin or erythropoietin, or complex molecules such as monoclonal antibodies. For example, if the reference IL-2 protein is aldesleukin (PROLEUKIN), a protein approved by drug regulatory authorities with reference to aldesleukin is a “biosimilar to” aldesleukin or is a “biosimilar thereof” of aldesleukin. In Europe, a similar biological or “biosimilar” medicine is a biological medicine that is similar to another biological medicine that has already been authorized for use by the European Medicines Agency (EMA). The relevant legal basis for similar biological applications in Europe is Article 6 of Regulation (EC) No 726 / 2004 and Article 10(4) of Directive 2001 / 83 / EC, as amended and therefore in Europe, the biosimilar may be authorized, approved for authorization or subject of an application for authorization under Article 6 of Regulation (EC) No 726 / 2004 and Article 10(4) of Directive 2001 / 83 / EC. The already authorized original biological medicinal product may be referred to as a “reference medicinal product” in Europe. Some of the requirements for a product to be considered a biosimilar are outlined in the CHMP Guideline on Similar Biological Medicinal Products. In addition, product specific guidelines, including guidelines relating to monoclonal antibody biosimilars, are provided on a product-by- product basis by the EMA and published on its website. A biosimilar as described herein may be similar to the reference medicinal product by way of quality characteristics, biological activity, mechanism of action, safety profiles and / or efficacy. In addition, the biosimilar may be used or be intended for use to treat the same conditions as the reference medicinal product. Thus, a biosimilar as described herein may be deemed to have similar or highly similar quality characteristics to a reference medicinal product. Alternatively, or in addition, a biosimilar as described herein may be deemed to have similar or highly similar biological activity to a reference medicinal product. Alternatively, or in addition, a biosimilar as described herein may be deemed to have a similar or highly similar safety profile to a reference medicinal product. Alternatively, or in addition, abiosimilar as described herein may be deemed to have similar or highly similar efficacy to a reference medicinal product. As described herein, a biosimilar in Europe is compared to a reference medicinal product which has been authorized by the EMA. However, in some instances, the biosimilar may be compared to a biological medicinal product which has been authorized outside the European Economic Area (a non-EEA authorized “comparator”) in certain studies. Such studies include for example certain clinical and in vivo non-clinical studies. As used herein, the term “biosimilar” also relates to a biological medicinal product which has been or may be compared to a non-EEA authorized comparator. Certain biosimilars are proteins such as antibodies, antibody fragments (for example, antigen binding portions) and fusion proteins. A protein biosimilar may have an amino acid sequence that has minor modifications in the amino acid structure (including for example deletions, additions, and / or substitutions of amino acids) which do not significantly affect the function of the polypeptide. The biosimilar may comprise an amino acid sequence having a sequence identity of 97% or greater to the amino acid sequence of its reference medicinal product, e.g., 97%, 98%, 99% or 100%. The biosimilar may comprise one or more post-translational modifications, for example, although not limited to, glycosylation, oxidation, deamidation, and / or truncation which is / are different to the post-translational modifications of the reference medicinal product, provided that the differences do not result in a change in safety and / or efficacy of the medicinal product. The biosimilar may have an identical or different glycosylation pattern to the reference medicinal product. Particularly, although not exclusively, the biosimilar may have a different glycosylation pattern if the differences address or are intended to address safety concerns associated with the reference medicinal product. Additionally, the biosimilar may deviate from the reference medicinal product in for example its strength, pharmaceutical form, formulation, excipients and / or presentation, providing safety and efficacy of the medicinal product is not compromised. The biosimilar may comprise differences in for example pharmacokinetic (PK) and / or pharmacodynamic (PD) profiles as compared to the reference medicinal product but is still deemed sufficiently similar to the reference medicinal product as to be authorized or considered suitable for authorization. In certain circumstances, the biosimilar exhibits different binding characteristics as compared to the reference medicinal product, wherein the different binding characteristics are considered by a Regulatory Authority such as the EMA not to be a barrier for authorization as a similar biological product. The term “biosimilar” is also used synonymously by other national and regional regulatory agencies. II. Gene-Editing Processes A. Overview: TIL Expansion + Gene-Editing

[0443] In some embodiments of the present invention directed to methods for expanding TIL populations (e.g., PD-1-, CD39- and / or CD103-expressing TIL populations). In some embodiments, the methods comprise one or more steps of gene-editing at least a portion of the TILs in order to enhance their therapeutic effect. As used herein, “gene-editing,” “gene editing,” and “genome editing” refer to a type of genetic modification in which DNA is permanently modified in the genome of a cell, e.g., DNA is inserted, deleted, modified or replaced within the cell’s genome. In some embodiments, gene-editing causes the expression of a DNA sequence to be silenced (sometimes referred to as a gene knockout) or inhibited / reduced (sometimes referred to as a gene knockdown). In other embodiments, gene-editing causes the expression of a DNA sequence to be enhanced (e.g., by causing over-expression). In accordance with embodiments of the present invention, gene-editing technology is used to enhance the effectiveness of a therapeutic population of TILs.

[0444] In some embodiments, the population of TILs is genetically modified to silence or reduce expression of one or more cell surface receptors. In exemplary embodiments, the cell surface receptors are PD-1, CD39 and / or CD103. As used herein “Programmed cell death protein 1,” “PD-1,” “cluster of differentiation 279,” and “CD279” all refer to a type I membrane protein expressed on immune cells (T cells and pro-B cells) that is a member of the extended CD28 / CTLA-4 family of T cell regulators. PD-1 has two ligands, PD-L1 and PD-L2, which are members of the B7 family. PD-1 and its ligands negatively regulate immune responses. PD-L1, for example, is highly expressed in several cancers and inhibition of the interaction between PD-1 / PD-L1 is believed to enhance T-cell responses and thereby promote anti-tumor activity. As used herein, “CD39”, “ENTPD1”, “ATPDase”, “NTPDase-1”, “SPG64”, and “ectonucleoside triphosphate diphosphohydrolase 1” all refer to a cell surface enzyme that catalyzes the hydrolysis of γ- and β- phosphate residues of triphospho- and diphosphonucleosides to the monophosphonucleoside derivative. High expression or activity of CD39 can prevent the immune system from inhibiting the progression of cancer. Indeed, both PD-1 and CD39 are commonly used markers of T cell exhaustion. As used herein, “CD103,” “Integrin Subunit Alpha E,” “Integrin, Alpha E (Antigen CD103, Human Mucosal Lymphocyte Antigen 1; Alpha Polypeptide),” “Human Mucosal Lymphocyte Antigen 1, Alpha Polypeptide,” “Mucosal Lymphocyte 1 Antigen,” “Integrin Alpha-IEL,” “HUMINAE,” “HML-1 Antigen,” and “CD103 Antigen” all refer to an I-domain-containing alpha integrin that undergoes post-translational cleavage in the extracellular domain, yielding disulfide-linked heavy and light chains and is preferentially expressed in lymphocytes. Enhanced expression of CD103 coincides with PD-1 and CD39, suggesting that this factor is phenotypically associated with these markers of T cell exhaustion and plays a role in the same phenotypes observed in high-expression of these otherproteins. Thus, without being bound by any particular theory of operation, it is believed that, just as PD-1 blockade increases anti-tumor activity in vivo, TILs genetically modified to silence or reduce PD- 1, CD39 and / or CD103 expression exhibit anti-tumor activity. As such TILs in some embodiments are capable of evading PD-1 mediated checkpoint inhibition, TILs can be modified to silence or reduce expression of CD39, and / or CD103 using any suitable methods known in the art including the genetic modification methods described herein. Exemplary gene modification technique include, for example, CRISPR, TALE and zinc finger methods described herein.

[0445] In some embodiments, the genetically modified TIL population is first preselected for CD39 and / or CD103 expression and the CD39-, and / or CD103-expressing TIL population is subsequently genetically modified to silence or reduce PD-1, CD39, and / or CD103 expression. Without being bound by any particular theory of operation, it is believed that such TIL populations that are subsequently genetically modified to silence or reduce PD-1, CD39, and / or CD103 expression exhibit enhanced anti-tumor activity as compared to control TIL populations (e.g., TIL populations that are not pre-selected for CD39, and / or CD103 expression and / or subsequently modified to reduce PD-1, CD39, and / or CD103 expression). TILs are preselected for CD39 and / or CD103 expression using any suitable method including, for example, the CD39, and / or CD103 preselection methods provided herein.

[0446] In some embodiments, the genetically modified TIL population (after preselection for CD39, and / or CD103 expression and subsequent genetic modification to silence or reduce PD-1, CD39, and / or CD103 expression) is expanded to create a therapeutic population of TILs that are genetically modified to silence or reduce PD-1, CD39, and / or CD103 expression. Any suitable expansion method can be used to expand the genetically modified TIL population, including the expansion methods provided herein.

[0447] A method for expanding tumor infiltrating lymphocytes (TILs) into a therapeutic population of TILs may be carried out in accordance with any embodiment of the methods described herein, wherein the method further comprises gene-editing at least a portion of the TILs. According to additional embodiments, a method for expanding TILs into a therapeutic population of TILs is carried out in accordance with any embodiment of the methods described in U.S. Patent Application Publication No.20180228841 A1 (U.S. Pat. No.10,517,894), U.S. Patent Application Publication No. 20200121719 A1, U.S. Patent Application Publication No.20180282694 A1 (U.S. Pat. No. 10,894,063), WO 2020096986, WO 2020096988, PCT / US21 / 30655 or U.S. Patent Application Publication No.20210100842 A1, all of which are incorporated by reference herein in their entireties, wherein the method further comprises gene-editing at least a portion of the TILs. Thus,some embodiments of the present invention provide a therapeutic population of TILs that has been preselected for CD39 and / or CD103 expression and expanded in accordance with any embodiment described herein, wherein at least a portion of the therapeutic population has been gene-edited, e.g., at least a portion of the therapeutic population of TILs that is transferred to the infusion bag is permanently gene-edited. B. Timing of Gene-Editing During TIL Expansion

[0448] In some embodiments, TIL populations are genetically modified in the course of the expansion methods provided herein. The expansion methods (e.g., Gen2 and Gen3 processes described herein or the process depicted in Figure 34) generally include a first expansion and a second expansion. In certain embodiments, TILs are pre-selected for CD39 and / or CD103 expression prior to the first expansion of the expansion methods. In some embodiments, this CD39 and / or CD103 expressing population are genetically modified to silence or minimize PD-1, CD39 and / or CD103 expression prior to undergoing the first expansion (e.g., a Gen2 and Gen3 process first expansion as described herein or the first expansion depicted in Figure 34). In some embodiments, the CD39 and / or CD103 expressing population undergoes a first expansion and the cells produced in the first expansion are genetically modified to silence or reduce PD-1, CD39 and / or CD103 expansion prior to undergoing the second expansion (e.g., a Gen2 and Gen3 process second expansion as described herein or the second expansion depicted in Figure 34). In some embodiments, the CD39 and / or CD103 expressing population undergoes a first expansion and second expansion and the TILs produced as a result of the second expansion are genetically modified to silence or reduce PD-1, CD39 and / or CD103 expansion.

[0449] According to some embodiments, a method for expanding tumor infiltrating lymphocytes (TILs) into a therapeutic population of TILs comprises: (a) obtaining and / or receiving a first population of TILs in a sample that contains a mixture of tumor and TIL cells from a cancer in a patient or subject; (b) selecting CD39 and / or CD103 positive TILs from the first population of TILs in (a) to obtain a population of CD39 and / or CD103 expressing TILs; (c) performing a priming first expansion by culturing the CD39 and / or CD103 expressing TIL population in a first cell culture medium comprising IL-2, OKT-3, and antigen presenting cells (APCs) to produce a second population of TILs, wherein the priming first expansion is performed in a container comprising a first gas-permeable surface area, wherein the priming first expansion isperformed for first period of about 1 to 11 days to obtain the second population of TILs, wherein the second population of TILs is greater in number than the first population of TILs; (d) performing a rapid second expansion by culturing the second population of TILs in a second culture medium comprising IL-2, OKT-3, and APCs, to produce a third population of TILs, wherein the rapid second expansion is performed for a second period of about 14 days or less to obtain the third population of TILs, wherein the third population of TILs is a therapeutic population of TILs; (e) harvesting the therapeutic population of TILs; and (f) genetically modifying the first population of TILs, the population of CD39 and / or CD103 expressing TILs, the second population of TILs and / or the third population of TILs at any time during the method after selection of CD39 and / or CD103positive TILs from the first population of TILs such that the harvested therapeutic population of TILs comprises genetically modified TILs comprising a genetic modification that reduces expression of PD-1, CD39 and / or CD103.

[0450] As stated in step (f) of the embodiment described above, the gene modification process may be carried out on any TIL population in the method, which means that the gene editing may be carried out on TILs before, during, or after any of the steps in the expansion method; for example, during any of steps (c)-(d) outlined in the method above. According to certain embodiments, TILs are collected during the expansion method, and the collected TILs are subjected to a gene-editing process, and, in some cases, subsequently reintroduced back into the expansion method (e.g., back into the culture medium) to continue the expansion process, so that at least a portion of the therapeutic population of TILs are permanently gene-edited. In some embodiments, the gene modification process may be carried out before expansion by activating TILs, performing a gene- editing step on the activated TILs, and expanding the gene-edited TILs according to the processes described herein.

[0451] It should be noted that alternative embodiments of the expansion process may differ from the method shown above; e.g., alternative embodiments may not have the same steps (a)-(f), or may have a different number of steps. Regardless of the specific embodiment, the gene-editing process may be carried out at any time during the TIL expansion method. For example, alternative embodiments may include more than two expansions, and it is possible that the gene modification step may be conducted on the TILs during a third or fourth expansion, etc.

[0452] According to some embodiments, the gene modification process is carried out on TILs from one or more of the population of CD39 and / or CD103 expressing TILs, the second population ofTILs, and the third population of TILs. For example, gene modification may be carried out on the population of CD39 and / or CD103 expressing TILs, or on a portion of TILs collected from the population of CD39 and / or CD103 expressing TILs, and following the gene-editing process those TILs may subsequently be placed back into the expansion process (e.g., back into the culture medium). Alternatively, gene modification may be carried out on TILs from the second or third population, or on a portion of TILs collected from the second or third population, respectively, and following the gene modification process those TILs may subsequently be placed back into the expansion process (e.g., back into the culture medium). According to other embodiments, gene modification is performed while the TILs are still in the culture medium and while the expansion is being carried out, i.e., they are not necessarily “removed” from the expansion in order to conduct gene-editing.

[0453] According to other embodiments, the gene modification process is carried out on TILs from the first expansion, or TILs from the second expansion, or both. For example, during the first expansion or second expansion, gene modification may be carried out on TILs that are collected from the culture medium, and following the gene-editing process those TILs may subsequently be placed back into the expansion method, e.g., by reintroducing them back into the culture medium.

[0454] According to other embodiments, the gene modification process is carried out on at least a portion of the TILs after the first expansion and before the second expansion. For example, after the first expansion, gene-editing may be carried out on TILs that are collected from the culture medium, and following the gene modification process those TILs may subsequently be placed back into the expansion method, e.g., by reintroducing them back into the culture medium for the second expansion.

[0455] According to alternative embodiments, the gene-editing process is carried out before step (c), before step (d), or before step (e).

[0456] In other embodiments, a method for expanding tumor infiltrating lymphocytes (TILs) into a therapeutic population of TILs comprises: (a) obtaining a first population of TILs in multiple tumor fragments obtained from a tumor sample resected from a patient; (b) selecting CD39 and / or CD103 positive TILs from the first population of TILs in (a) to obtain a population of CD39 and / or CD103 expressing TILs; (c) adding the population of CD39 and / or CD103 expressing TILs into a closed system;(d) performing a first expansion by culturing the population of CD39 and / or CD103 expressing TILs in a cell culture medium comprising IL-2, and optionally OKT-3 (e.g., OKT-3 may be present in the culture medium beginning on the start date of the expansion process), to produce a second population of TILs, wherein the first expansion is performed in a closed container providing a first gas-permeable surface area, wherein the first expansion is performed for about 3-14 days to obtain the second population of TILs, and wherein the transition from step (c) to step (d) occurs without opening the system; (e) performing a second expansion by supplementing the cell culture medium of the second population of TILs with additional IL-2, optionally OKT-3, and antigen presenting cells (APCs), to produce a third population of TILs, wherein the second expansion is performed for about 7-14 days to obtain the third population of TILs, wherein the third population of TILs is a therapeutic population of TILs, wherein the second expansion is performed in a closed container providing a second gas-permeable surface area, and wherein the transition from step (d) to step (e) occurs without opening the system; (f) harvesting the third population of TILs obtained from step (e), wherein the transition from step (e) to step (f) occurs without opening the system; (g) transferring the harvested TIL population from step (f) to an infusion bag, wherein the transfer from step (f) to (g) occurs without opening the system; and (h) genetically modifying the first population of TILs, the population of CD39 and / or CD103 expressing TILs, the second population of TILs and / or the third population of TILs at any time during the method after selection of CD39 and / or CD103 positive TILs from the first population of TILs such that the harvested third population of TILs comprises genetically modified TILs comprising a genetic modification that reduces expression of PD-1, CD39 and / or CD103.

[0457] As stated in step (h) of the embodiment described above, the gene-modifying process may be carried out at any time during the TIL expansion method after selection of PD-1 positive TILs from the first population of TILs and prior to the transfer to the infusion bag in step (g). According to certain embodiments, TILs are collected during the expansion method (e.g., the expansion method is “paused” for at least a portion of the TILs), and the collected TILs are subjected to a gene-editing process, and, in some cases, subsequently reintroduced back into the expansion method (e.g., back into the culture medium) to continue the expansion process, so that at least a portion of the therapeutic population of TILs that are eventually transferred to the infusion bag are permanently gene-edited. In some embodiments, the gene-editing process may be carried out before expansionby activating TILs, performing a gene-editing step on the activated TILs, and expanding the gene- edited TILs according to the processes described herein.

[0458] It should be noted that alternative embodiments of the expansion process may differ from the method shown above; e.g., alternative embodiments may not have the same steps (a)-(h), or may have a different number of steps. Regardless of the specific embodiment, the gene-editing process may be carried out at any time during the TIL expansion method after selection of PD-1 positive TILs from the first population of TILs. For example, alternative embodiments may include more than two expansions, and it is possible that gene-editing may be conducted on the TILs during a third or fourth expansion, etc.

[0459] According to some embodiments, the gene-editing process is carried out on TILs from one or more of the population of CD39 and / or CD103 expressing TILs, the second population of TILs, and the third population of TILs. For example, gene-editing may be carried out on the population of CD39 and / or CD103 expressing TILs, or on a portion of TILs collected from the population of CD39 and / or CD103 expressing TILs, and following the gene-editing process those TILs may subsequently be placed back into the expansion process (e.g., back into the culture medium). Alternatively, gene- editing may be carried out on TILs from the second or third population, or on a portion of TILs collected from the second or third population, respectively, and following the gene-editing process those TILs may subsequently be placed back into the expansion process (e.g., back into the culture medium). According to other embodiments, gene-editing is performed while the TILs are still in the culture medium and while the expansion is being carried out, i.e., they are not necessarily “removed” from the expansion in order to conduct gene-editing.

[0460] According to other embodiments, the gene-editing process is carried out on TILs from the first expansion, or TILs from the second expansion, or both. For example, during the first expansion or second expansion, gene-editing may be carried out on TILs that are collected from the culture medium, and following the gene-editing process those TILs may subsequently be placed back into the expansion method, e.g., by reintroducing them back into the culture medium.

[0461] According to other embodiments, the gene-editing process is carried out on at least a portion of the TILs after the first expansion and before the second expansion. For example, after the first expansion, gene-editing may be carried out on TILs that are collected from the culture medium, and following the gene-editing process those TILs may subsequently be placed back into the expansion method, e.g., by reintroducing them back into the culture medium for the second expansion.

[0462] According to alternative embodiments, the gene-editing process is carried out before step (d), before step (e), before step (f), or before step (g).

[0463] It should be noted with regard to OKT-3, according to certain embodiments, that the cell culture medium may comprise OKT-3 beginning on the start day (Day 0), or on Day 1 of the first expansion, such that the gene-editing is carried out on TILs after they have been exposed to OKT-3 in the cell culture medium on Day 0 and / or Day 1. According to other embodiments, the cell culture medium comprises OKT-3 during the first expansion and / or during the second expansion, and the gene-editing is carried out before the OKT-3 is introduced into the cell culture medium. Alternatively, the cell culture medium may comprise OKT-3 during the first expansion and / or during the second expansion, and the gene-editing is carried out after the OKT-3 is introduced into the cell culture medium.

[0464] It should also be noted with regard to a 4-1BB agonist, according to certain embodiments, that the cell culture medium may comprise a 4-1BB agonist beginning on the start day (Day 0), or on Day 1 of the first expansion, such that the gene-editing is carried out on TILs after they have been exposed to a 4-1BB agonist in the cell culture medium on Day 0 and / or Day 1. According to other embodiments, the cell culture medium comprises a 4-1BB agonist during the first expansion and / or during the second expansion, and the gene-editing is carried out before the 4-1BB agonist is introduced into the cell culture medium. Alternatively, the cell culture medium may comprise a 4- 1BB agonist during the first expansion and / or during the second expansion, and the gene-editing is carried out after the 4-1BB agonist is introduced into the cell culture medium.

[0465] It should also be noted with regard to IL-2, according to certain embodiments, that the cell culture medium may comprise IL-2 beginning on the start day (Day 0), or on Day 1 of the first expansion, such that the gene-editing is carried out on TILs after they have been exposed to IL-2 in the cell culture medium on Day 0 and / or Day 1. According to other embodiments, the cell culture medium comprises IL-2 during the first expansion and / or during the second expansion, and the gene-editing is carried out before the IL-2 is introduced into the cell culture medium. Alternatively, the cell culture medium may comprise IL-2 during the first expansion and / or during the second expansion, and the gene-editing is carried out after the IL-2 is introduced into the cell culture medium.

[0466] As discussed above, one or more of OKT-3, 4-1BB agonist and IL-2 may be included in the cell culture medium beginning on Day 0 or Day 1 of the first expansion. According to some embodiments, OKT-3 is included in the cell culture medium beginning on Day 0 or Day 1 of the firstexpansion, and / or a 4-1BB agonist is included in the cell culture medium beginning on Day 0 or Day 1 of the first expansion, and / or IL-2 is included in the cell culture medium beginning on Day 0 or Day 1 of the first expansion. According to an example, the cell culture medium comprises OKT-3 and a 4- 1BB agonist beginning on Day 0 or Day 1 of the first expansion. According to another example, the cell culture medium comprises OKT-3, a 4-1BB agonist and IL-2 beginning on Day 0 or Day 1 of the first expansion. Of course, one or more of OKT-3, 4-1BB agonist and IL-2 may be added to the cell culture medium at one or more additional time points during the expansion process, as set forth in various embodiments described herein.

[0467] According to some embodiments, a method for expanding tumor infiltrating lymphocytes (TILs) into a therapeutic population of TILs comprises: (a) obtaining a first population of TILs from a tumor resected from a patient by processing a tumor sample obtained from the patient into multiple tumor fragments; (b) selecting CD39 and / or CD103 positive TILs from the first population of TILs in (a) to obtain a population of CD39 and / or CD103 expressing TILs; (c) adding the population of CD39 and / or CD103 expressing TILs into a closed system; (d) performing a first expansion by culturing the CD39 and / or CD103 expressing TILs in a cell culture medium comprising IL-2 and optionally comprising OKT-3 and / or a 4-1BB agonist antibody for about 3 to 11 days to produce a second population of TILs, wherein the first expansion is performed in a closed container providing a first gas-permeable surface area; (e) stimulating the second population of TILs by adding OKT-3 and culturing for about 1 to 3 days, wherein the transition from step (d) to step (e) occurs without opening the system; (f) sterile electroporating the second population of TILs to effect transfer of at least one gene editor into a portion of cells of the second population of TILs; (g) resting the second population of TILs for about 1 day; (h) performing a second expansion by supplementing the cell culture medium of the second population of TILs with additional IL-2, optionally OKT-3 antibody, optionally an OX40 antibody, and antigen presenting cells (APCs), to produce a third population of TILs, wherein the second expansion is performed for about 7 to 11 days to obtain a third population of TILs, wherein the second expansion is performed in a closed container providing a second gas-permeable surface area, and wherein the transition from step (g) to step (h) occurs without opening the system;(i) harvesting the therapeutic population of TILs obtained from step (h) to provide a harvested TIL population, wherein the transition from step (h) to step (i) occurs without opening the system, wherein the harvested population of TILs is a therapeutic population of TILs; and (j) transferring the harvested TIL population to an infusion bag, wherein the transfer from step (i) to (j) occurs without opening the system, wherein the sterile electroporation of the at least one gene editor into the portion of cells of the second population of TILs modifies a plurality of cells in the portion or a third population of TILs expanded from such a portion of TILs to include a genetic modification that silences or reduces expression of endogenous PD-1, CD39 and / or CD103.

[0468] According to some embodiments, the foregoing method further comprises cryopreserving the harvested TIL population using a cryopreservation medium. In some embodiments, the cryopreservation medium is a dimethylsulfoxide-based cryopreservation medium. In other embodiments, the cryopreservation medium is CS10.

[0469] In other embodiments, a method for expanding tumor infiltrating lymphocytes (TILs) into a therapeutic population of TILs comprises: (a) obtaining and / or receiving a first population of TILs in a sample that contains a mixture of tumor and TIL cells from a cancer in a patient or subject; (b) selecting CD39 and / or CD103 positive TILs from the first population of TILs in (a) to obtain a population of CD39 and / or CD103 expressing TILs; (c) performing a priming first expansion by culturing the PD-l expressing TIL population in a first cell culture medium comprising IL-2, anti-CD3 agonist antibody (e.g., OKT-3), and antigen presenting cells (APCs) to produce a second population of TILs, wherein the priming first expansion is performed in a container comprising a first gas-permeable surface area, wherein the priming first expansion is performed for first period of about 14 days or less to obtain the second population of TILs, wherein the second population of TILs is greater in number than the first population of TILs; (d) restimulating the second population of TILs with anti-CD3 agonist antibody (e.g., OKT- 3); (e) genetically modifying the second population of TILs to produce a modified second population of TILs, wherein the modified second population of TILs comprises a genetic modification that reduces expression of PD-1, CD39 and / or CD103;(f) performing a rapid second expansion by culturing the modified second population of TILs in a second culture medium comprising IL-2, anti-CD3 agonist antibody (e.g., OKT-3), and APCs, to produce a third population of TILs, wherein the rapid second expansion is performed for a second period of about 14 days or less to obtain the third population of TILs, wherein the third population of TILs is a therapeutic population of TILs comprising the genetic modification that reduces expression of PD-1, CD39 and / or CD103; and (g) harvesting the third population of TILs.

[0470] In some embodiments, the priming first expansion is performed for a first period of about 5 days, about 7 days, or about 11 days.

[0471] In some embodiments, the second population of TILs is restimulated for about 2 days. In some embodiments, the anti-CD3 agonist antibody used for the restimulation is part of an anti- CD3 / anti-CD28 antibody bead. In other embodiments, the antiCD3 agonist antibody is OKT-3.

[0472] In some embodiments, the rapid second expansion is performed for a period of about 7 to 11 days. In some embodiments, the rapid second expansion includes a culture split and scale up after about 5 days of the rapid second expansion. In such embodiments, the subcultures are seeded into new flasks with fresh medium and IL-2 and cultured for about another 6 days.

[0473] In some embodiments, the genetically modifying step comprises electroporation and the delivery of at least one gene editor system selected from the group consisting of a Clustered Regularly Interspersed Short Palindromic Repeat (CRISPR) system, a Transcription Activator-Like Effector (TALE) system, or a zinc finger system, wherein the at least one gene editor system reduces expression of PD-1, CD39 and / or CD103 in the modified second population of TILs.

[0474] According to some embodiments, the foregoing method may be used to provide an autologous harvested TIL population for the treatment of a human subject with cancer. C. Gene Editing Methods

[0475] As discussed above, embodiments of the present invention provide tumor infiltrating lymphocytes (TILs) that have been genetically modified via gene-editing to enhance their therapeutic effect (e.g., silence or reduce expression of endogenous PD-1, CD39 and / or CD103). Embodiments of the present invention embrace genetic editing through nucleotide insertion (RNA or DNA) into a population of TILs for both promotion of the expression of one or more proteins and inhibition of the expression of one or more proteins, as well as combinations thereof. Embodiments of thepresent invention also provide methods for expanding TILs into a therapeutic population, wherein the methods comprise gene-editing the TILs. There are several gene-editing technologies that may be used to genetically modify a population of TILs, which are suitable for use in accordance with the present invention.

[0476] In some embodiments, a method of genetically modifying a population of TILs includes the step of stable incorporation of genes for production of one or more proteins. In some embodiments, a method of genetically modifying a population of TILs includes the step of retroviral transduction. In some embodiments, a method of genetically modifying a population of TILs includes the step of lentiviral transduction. Lentiviral transduction systems are known in the art and are described, e.g., in Levine, et al., Proc. Nat’l Acad. Sci.2006, 103, 17372-77; Zufferey, et al., Nat. Biotechnol.1997, 15, 871-75; Dull, et al., J. Virology 1998, 72, 8463-71, and U.S. Patent No. 6,627,442, the disclosures of each of which are incorporated by reference herein. In some embodiments, a method of genetically modifying a population of TILs includes the step of gamma- retroviral transduction. Gamma-retroviral transduction systems are known in the art and are described, e.g., Cepko and Pear, Cur. Prot. Mol. Biol.1996, 9.9.1-9.9.16, the disclosure of which is incorporated by reference herein. In some embodiments, a method of genetically modifying a population of TILs includes the step of transposon-mediated gene transfer. Transposon-mediated gene transfer systems are known in the art and include systems wherein the transposase is provided as DNA expression vector or as an expressible RNA or a protein such that long-term expression of the transposase does not occur in the transgenic cells, for example, a transposase provided as an mRNA (e.g., an mRNA comprising a cap and poly-A tail). Suitable transposon-mediated gene transfer systems, including the salmonid-type Tel-like transposase (SB or Sleeping Beauty transposase), such as SB10, SB11, and SB100x, and engineered enzymes with increased enzymatic activity, are described in, e.g., Hackett, et al., Mol. Therapy 2010, 18, 674-83 and U.S. Patent No.6,489,458, the disclosures of each of which are incorporated by reference herein.

[0477] In some embodiments, a method of genetically modifying a population of TILs includes the step of stable incorporation of genes for production or inhibition (e.g., silencing) of one or more proteins. In some embodiments, a method of genetically modifying a population of TILs includes the step of electroporation. Electroporation methods are known in the art and are described, e.g., in Tsong, Biophys. J.1991, 60, 297-306, and U.S. Patent Application Publication No. 2014 / 0227237 A1, the disclosures of each of which are incorporated by reference herein. Other electroporation methods known in the art, such as those described in U.S. Patent Nos.5,019,034; 5,128,257; 5,137,817; 5,173,158; 5,232,856; 5,273,525; 5,304,120; 5,318,514; 6,010,613 and6,078,490, the disclosures of which are incorporated by reference herein, may be used. In some embodiments, the electroporation method is a sterile electroporation method. In some embodiments, the electroporation method is a pulsed electroporation method. In some embodiments, the electroporation method is a pulsed electroporation method comprising the steps of treating TILs with pulsed electrical fields to alter, manipulate, or cause defined and controlled, permanent or temporary changes in the TILs, comprising the step of applying a sequence of at least three single, operator-controlled, independently programmed, DC electrical pulses, having field strengths equal to or greater than 100 V / cm, to the TILs, wherein the sequence of at least three DC electrical pulses has one, two, or three of the following characteristics: (1) at least two of the at least three pulses differ from each other in pulse amplitude; (2) at least two of the at least three pulses differ from each other in pulse width; and (3) a first pulse interval for a first set of two of the at least three pulses is different from a second pulse interval for a second set of two of the at least three pulses. In some embodiments, the electroporation method is a pulsed electroporation method comprising the steps of treating TILs with pulsed electrical fields to alter, manipulate, or cause defined and controlled, permanent or temporary changes in the TILs, comprising the step of applying a sequence of at least three single, operator-controlled, independently programmed, DC electrical pulses, having field strengths equal to or greater than 100 V / cm, to the TILs, wherein at least two of the at least three pulses differ from each other in pulse amplitude. In some embodiments, the electroporation method is a pulsed electroporation method comprising the steps of treating TILs with pulsed electrical fields to alter, manipulate, or cause defined and controlled, permanent or temporary changes in the TILs, comprising the step of applying a sequence of at least three single, operator-controlled, independently programmed, DC electrical pulses, having field strengths equal to or greater than 100 V / cm, to the TILs, wherein at least two of the at least three pulses differ from each other in pulse width. In some embodiment, the electroporation method is a pulsed electroporation method comprising the steps of treating TILs with pulsed electrical fields to alter, manipulate, or cause defined and controlled, permanent or temporary changes in the TILs, comprising the step of applying a sequence of at least three single, operator-controlled, independently programmed, DC electrical pulses, having field strengths equal to or greater than 100 V / cm, to the TILs, wherein a first pulse interval for a first set of two of the at least three pulses is different from a second pulse interval for a second set of two of the at least three pulses. In some embodiments, the electroporation method is a pulsed electroporation method comprising the steps of treating TILs with pulsed electrical fields to induce pore formation in the TILs, comprising the step of applying a sequence of at least three DC electrical pulses, having field strengths equal to or greater than 100 V / cm, to TILs, wherein the sequence of at least three DC electrical pulses has one,two, or three of the following characteristics: (1) at least two of the at least three pulses differ from each other in pulse amplitude; (2) at least two of the at least three pulses differ from each other in pulse width; and (3) a first pulse interval for a first set of two of the at least three pulses is different from a second pulse interval for a second set of two of the at least three pulses, such that induced pores are sustained for a relatively long period of time, and such that viability of the TILs is maintained. In some embodiments, a method of genetically modifying a population of TILs includes the step of calcium phosphate transfection. Calcium phosphate transfection methods (calcium phosphate DNA precipitation, cell surface coating, and endocytosis) are known in the art and are described in Graham and van der Eb, Virology 1973, 52, 456-467; Wigler, et al., Proc. Natl. Acad. Sci. 1979, 76, 1373-1376; and Chen and Okayarea, Mol. Cell. Biol.1987, 7, 2745-2752; and in U.S. Patent No.5,593,875, the disclosures of each of which are incorporated by reference herein. In some embodiments, a method of genetically modifying a population of TILs includes the step of liposomal transfection. Liposomal transfection methods, such as methods that employ a 1:1 (w / w) liposome formulation of the cationic lipid N-[1-(2,3-dioleyloxy)propyl]-n,n,n-trimethylammonium chloride (DOTMA) and dioleoyl phophotidylethanolamine (DOPE) in filtered water, are known in the art and are described in Rose, et al., Biotechniques 1991, 10, 520-525 and Felgner, et al., Proc. Natl. Acad. Sci. USA, 1987, 84, 7413-7417 and in U.S. Patent Nos.5,279,833; 5,908,635; 6,056,938; 6,110,490; 6,534,484; and 7,687,070, the disclosures of each of which are incorporated by reference herein. In some embodiments, a method of genetically modifying a population of TILs includes the step of transfection using methods described in U.S. Patent Nos.5,766,902; 6,025,337; 6,410,517; 6,475,994; and 7,189,705; the disclosures of each of which are incorporated by reference herein.

[0478] According to an embodiment, the gene-editing process may comprise the use of a programmable nuclease that mediates the generation of a double-strand or single-strand break at one or more immune checkpoint genes. Such programmable nucleases enable precise genome editing by introducing breaks at specific genomic loci, i.e., they rely on the recognition of a specific DNA sequence within the genome to target a nuclease domain to this location and mediate the generation of a double-strand break at the target sequence. A double-strand break in the DNA subsequently recruits endogenous repair machinery to the break site to mediate genome editing by either non-homologous end-joining (NHEJ) or homology-directed repair (HDR). Thus, the repair of the break can result in the introduction of insertion / deletion mutations that disrupt (e.g., silence, repress, or enhance) the target gene product.

[0479] Major classes of nucleases that have been developed to enable site-specific genomic editing include zinc finger nucleases (ZFNs), transcription activator-like nucleases (TALENs), andCRISPR-associated nucleases (e.g., CRISPR / Cas9). These nuclease systems can be broadly classified into two categories based on their mode of DNA recognition: ZFNs and TALENs achieve specific DNA binding via protein-DNA interactions, whereas CRISPR systems, such as Cas9, are targeted to specific DNA sequences by a short RNA guide molecule that base-pairs directly with the target DNA and by protein-DNA interactions. See, e.g., Cox et al., Nature Medicine, 2015, Vol.21, No.2.

[0480] Non-limiting examples of gene-editing methods that may be used in accordance with TIL expansion methods of the present invention include CRISPR methods, TALE methods, and ZFN methods, embodiments of which are described in more detail below. According to some embodiments, a method for expanding TILs into a therapeutic population may be carried out in accordance with any embodiment of the methods described herein (e.g., process 2A) or as described in PCT / US2017 / 058610, PCT / US2018 / 012605, or PCT / US2018 / 012633, wherein the method further comprises gene-editing at least a portion of the TILs by one or more of a CRISPR method, a TALE method or a ZFN method, in order to generate TILs that can provide an enhanced therapeutic effect. According to some embodiments, gene-edited TILs can be evaluated for an improved therapeutic effect by comparing them to non-modified TILs in vitro, e.g., by evaluating in vitro effector function, cytokine profiles, etc. compared to unmodified TILs.

[0481] In some embodiments of the present invention, electroporation is used for delivery of a gene editing system, such as CRISPR, TALEN, and ZFN systems. In some embodiments of the present invention, the electroporation system is a flow electroporation system. An example of a suitable flow electroporation system suitable for use with some embodiments of the present invention is the commercially-available MaxCyte STX system. There are several alternative commercially-available electroporation instruments which may be suitable for use with the present invention, such as the AgilePulse system or ECM 830 available from BTX-Harvard Apparatus, Cellaxess Elektra (Cellectricon), Nucleofector (Lonza / Amaxa), GenePulser MXcell (BIORAD), iPorator-96 (Primax) or siPORTer96 (Ambion). In some embodiments of the present invention, the electroporation system forms a closed, sterile system with the remainder of the TIL expansion method. In some embodiments of the present invention, the electroporation system is a pulsed electroporation system as described herein, and forms a closed, sterile system with the remainder of the TIL expansion method. D. Immune Checkpoints

[0482] According to particular embodiments of the present invention, a TIL population (i.e., a TIL population that is enriched for CD39 and / or CD103 expression) is gene-edited to silence or reduceexpression of one or more immune checkpoint genes. In exemplary embodiments, the immune checkpoint gene is PD-1.

[0483] Immune checkpoints are molecules expressed by lymphocytes that regulate an immune response via inhibitory or stimulatory pathways. In the case of cancer, immune checkpoint pathways are often activated to inhibit the anti-tumor response, i.e., the expression of certain immune checkpoints by malignant cells inhibits the anti-tumor immunity and favors the growth of cancer cells. See, e.g., Marin-Acevedo et al., Journal of Hematology & Oncology (2018) 11:39. Thus, certain inhibitory checkpoint molecules serve as targets for immunotherapies of the present invention. According to particular embodiments, TILs are gene-edited to block or stimulate certain immune checkpoint pathways and thereby enhance the body’s immunological activity against tumors.

[0484] As used herein, an immune checkpoint gene comprises a DNA sequence encoding an immune checkpoint molecule. According to particular embodiments of the present invention, gene- editing TILs during the TIL expansion method causes expression of one or more immune checkpoint genes to be silenced or reduced in at least a portion of the therapeutic population of TILs. For example, gene-editing may cause the expression of an inhibitory receptor, such as PD-1 or CTLA-4, to be silenced or reduced in order to enhance an immune reaction.

[0485] The most broadly studied checkpoints include programmed cell death receptor-1 (PD-1) and cytotoxic T lymphocyte-associated molecule-4 (CTLA-4), which are inhibitory receptors on immune cells that inhibit key effector functions (e.g., activation, proliferation, cytokine release, cytoxicity, etc.) when they interact with an inhibitory ligand. Numerous checkpoint molecules, in addition to PD-1 and CTLA-4, have emerged as potential targets for immunotherapy, as discussed in more detail below.

[0486] Non-limiting examples of immune checkpoint genes that may be silenced or inhibited by permanently gene-editing TILs of the present invention include PD-1, CTLA-4, LAG-3, HAVCR2 (TIM- 3), Cish, TGFβ, PKA, CBL-B, PPP2CA, PPP2CB, PTPN6, PTPN22, PDCD1, BTLA, CD160, TIGIT, BAFF (BR3), CD96, CRTAM, LAIR1, SIGLEC7, SIGLEC9, CD244, TNFRSF10B, TNFRSF10A, CASP8, CASP10, CASP3, CASP6, CASP7, FADD, FAS, SMAD2, SMAD3, SMAD4, SMAD10, SKI, SKIL, TGIF1, IL10RA, IL10RB, HMOX2, IL6R, IL6ST, EIF2AK4, CSK, PAG1, SIT1, FOXP3, PRDM1, BATF, GUCY1A2, GUCY1A3, GUCY1B2, GUCY1B3, TOX, SOCS1, ANKRD11, and BCOR. For example, immune checkpoint genes that may be silenced or inhibited in TILs of the present invention may be selected from the group comprising PD-1, CTLA-4, LAG-3, TIM-3, Cish, TGFβ, and PKA. BAFF (BR3) is described in Bloom, etal., J. Immunother., 2018, in press. According to another example, immune checkpoint genes that may be silenced or inhibited in TILs of the present invention may be selected from the group comprising PD-1, LAG-3, TIM-3, CTLA-4, TIGIT, CISH, TGFβR2, PRA, CBLB, BAFF (BR3), and combinations thereof.

[0487] According to some embodiments, a method for expanding tumor infiltrating lymphocytes (TILs) into a therapeutic population of TILs comprises: (a) obtaining a first population of TILs in a plurality of tumor fragments produced from a tumor sample resected from a patient; (b) selecting CD39 and / or CD103 positive TILs from the first population of TILs in (a) to obtain a population of CD39 and / or CD103 expressing TILs; (c) adding the population of CD39 and / or CD103 expressing TILs into a closed system; (d) performing a first expansion by culturing the population of CD39 and / or CD103 expressing TILs in a cell culture medium comprising IL-2 and optionally comprising OKT-3 and / or a 4- 1BB agonist antibody for about 3 to 11 days to produce a second population of TILs, wherein the first expansion is performed in a closed container providing a first gas-permeable surface area; (e) stimulating the second population of TILs by adding OKT-3 and culturing for about 1 to 3 days, wherein the transition from step (c) to step (d) occurs without opening the system; (f) sterile electroporating the second population of TILs to effect transfer of at least one gene editor into a plurality of cells in the second population of TILs, wherein the transition from step (e) to step (f) occurs without opening the system; (g) resting the second population of TILs for about 1 day, wherein the transition from step (f) to step (g) occurs without opening the system; (h) performing a second expansion by supplementing the cell culture medium of the second population of TILs with additional IL-2, optionally OKT-3 antibody, optionally an OX40 antibody, and antigen presenting cells (APCs), to produce a third population of TILs, wherein the second expansion is performed for about 7 to 11 days, wherein the second expansion is performed in a closed container providing a second gas-permeable surface area, and wherein the transition from step (g) to step (h) occurs without opening the system;(i) harvesting the third population of TILs obtained from step (g) to provide a harvested TIL population, wherein the transition from step (h) to step (i) occurs without opening the system, wherein the harvested population of TILs is a therapeutic population of TILs; (j) transferring the harvested TIL population to an infusion bag, wherein the transfer from step (i) to (j) occurs without opening the system; and (k) optionally cryopreserving the harvested TIL population using a cryopreservation medium, wherein the electroporation step comprises the delivery of at least one gene editor system selected from the group consisting of a Clustered Regularly Interspersed Short Palindromic Repeat (CRISPR) system, a Transcription Activator-Like Effector (TALE) system, or a zinc finger system. In some embodiments, the at least one gene editor system effects inhibits expression of PD-1, CD39 and / or CD103 and one or more molecules selected from the group consisting of LAG-3, TIM-3, CTLA- 4, TIGIT, CISH, TGFβR2, PRA, CBLB, BAFF (BR3) in the plurality of cells of the second population of TILs. 1. PD-1

[0488] One of the most studied targets for the induction of checkpoint blockade is the programmed death receptor (PD1 or PD-1, also known as PDCD1), a member of the CD28 super family of T-cell regulators. Its ligands, PD-L1 and PD-L2, are expressed on a variety of tumor cells, including melanoma. The interaction of PD-1 with PD-L1 inhibits T-cell effector function, results in T- cell exhaustion in the setting of chronic stimulation, and induces T-cell apoptosis in the tumor microenvironment. PD1 may also play a role in tumor-specific escape from immune surveillance.

[0489] According to particular embodiments, expression of PD1 in TILs is silenced or reduced in accordance with compositions and methods of the present invention. For example, a method for expanding tumor infiltrating lymphocytes (TILs) into a therapeutic population of TILs may be carried out in accordance with any embodiment of the methods described herein (e.g., process 2A, process Gen 3, or the methods shown in Figures 34), wherein the method comprises gene-editing at least a portion of the TILs by silencing or repressing the expression of PD1. As described in more detail below, the gene-editing process may involve the use of a programmable nuclease that mediates the generation of a double-strand or single-strand break at an immune checkpoint gene, such as PD1. For example, a CRISPR method, a TALE method, or a zinc finger method may be used to silence or reduce the expression of PD1 in the TILs. 2. CTLA-4

[0490] CTLA-4 expression is induced upon T-cell activation on activated T-cells, and competes for binding with the antigen presenting cell activating antigens CD80 and CD86. Interaction of CTLA-4 with CD80 or CD86 causes T-cell inhibition and serves to maintain balance of the immune response. However, inhibition of the CTLA-4 interaction with CD80 or CD86 may prolong T-cell activation and thus increase the level of immune response to a cancer antigen.

[0491] According to particular embodiments, expression of CTLA-4 in TILs is silenced or reduced in accordance with compositions and methods of the present invention. For example, a method for expanding tumor infiltrating lymphocytes (TILs) into a therapeutic population of TILs may be carried out in accordance with any embodiment of the methods described herein (e.g., process 2A, process Gen 3, or the methods shown in Figures 34 and 35), wherein the method comprises gene-editing at least a portion of the TILs to silence or repress the expression of CTLA-4 in the TILs. As described in more detail below, the gene-editing process may comprise the use of a programmable nuclease that mediates the generation of a double-strand or single-strand break at an immune checkpoint gene, such as CTLA-4. For example, a CRISPR method, a TALE method, or a zinc finger method may be used to silence or repress the expression of CTLA-4 in the TILs 3. LAG-3

[0492] Lymphocyte activation gene-3 (LAG-3, CD223) is expressed by T cells and natural killer (NK) cells after major histocompatibility complex (MHC) class II ligation. Although its mechanism remains unclear, its modulation causes a negative regulatory effect over T cell function, preventing tissue damage and autoimmunity. LAG-3 and PD-1 are frequently co-expressed and upregulated on TILs, leading to immune exhaustion and tumor growth. Thus, LAG-3 blockade improves anti-tumor responses. See, e.g., Marin-Acevedo et al., Journal of Hematology & Oncology (2018) 11:39.

[0493] According to particular embodiments, expression of LAG-3 in TILs is silenced or reduced in accordance with compositions and methods of the present invention. For example, a method for expanding tumor infiltrating lymphocytes (TILs) into a therapeutic population of TILs may be carried out in accordance with any embodiment of the methods described herein (e.g., process 2A, process Gen 3, or the methods shown in Figures 34 and 35), wherein the method comprises gene-editing at least a portion of the TILs silence or repress the expression of LAG-3 in the TILs. As described in more detail below, the gene-editing process may comprise the use of a programmable nuclease that mediates the generation of a double-strand or single-strand break at an immune checkpoint gene, such as LAG-3. According to particular embodiments, a CRISPR method, a TALE method, or a zinc finger method may be used to silence or repress the expression of LAG-3 in the TILs.4. TIM-3

[0494] T cell immunoglobulin-3 (TIM-3) is a direct negative regulator of T cells and is expressed on NK cells and macrophages. TIM-3 indirectly promotes immunosuppression by inducing expansion of myeloid-derived suppressor cells (MDSCs). Its levels have been found to be particularly elevated on dysfunctional and exhausted T-cells, suggesting an important role in malignancy.

[0495] According to particular embodiments, expression of TIM-3 in TILs is silenced or reduced in accordance with compositions and methods of the present invention. For example, a method for expanding tumor infiltrating lymphocytes (TILs) into a therapeutic population of TILs may be carried out in accordance with any embodiment of the methods described herein (e.g., process 2A, process Gen 3, or the methods shown in Figures 34 and 35), wherein the method comprises gene-editing at least a portion of the TILs to silence or repress the expression of TIM-3 in the TILs. As described in more detail below, the gene-editing process may comprise the use of a programmable nuclease that mediates the generation of a double-strand or single-strand break at an immune checkpoint gene, such as TIM-3. For example, a CRISPR method, a TALE method, or a zinc finger method may be used to silence or repress the expression of TIM-3 in the TILs. 5. Cish

[0496] Cish, a member of the suppressor of cytokine signaling (SOCS) family, is induced by TCR stimulation in CD8+ T cells and inhibits their functional avidity against tumors. Genetic deletion of Cish in CD8+ T cells may enhance their expansion, functional avidity, and cytokine polyfunctionality, resulting in pronounced and durable regression of established tumors. See, e.g., Palmer et al., Journal of Experimental Medicine, 212 (12): 2095 (2015).

[0497] According to particular embodiments, expression of Cish in TILs is silenced or reduced in accordance with compositions and methods of the present invention. For example, a method for expanding tumor infiltrating lymphocytes (TILs) into a therapeutic population of TILs may be carried out in accordance with any embodiment of the methods described herein (e.g., process 2A, process Gen 3, or the methods shown in Figures 34 and 35), wherein the method comprises gene-editing at least a portion of the TILs to silence or repress the expression of Cish in the TILs. As described in more detail below, the gene-editing process may comprise the use of a programmable nuclease that mediates the generation of a double-strand or single-strand break at an immune checkpoint gene, such as Cish. For example, a CRISPR method, a TALE method, or a zinc finger method may be used to silence or repress the expression of Cish in the TILs.6. TGFβ

[0498] The TGFβ signaling pathway has multiple functions in regulating cell growth, differentiation, apoptosis, motility and invasion, extracellular matrix production, angiogenesis, and immune response. TGFβ signaling deregulation is frequent in tumors and has crucial roles in tumor initiation, development and metastasis. At the microenvironment level, the TGFβ pathway contributes to generate a favorable microenvironment for tumor growth and metastasis throughout carcinogenesis. See, e.g., Neuzillet et al., Pharmacology & Therapeutics, Vol.147, pp.22-31 (2015).

[0499] According to particular embodiments, expression of TGFβ in TILs is silenced or reduced in accordance with compositions and methods of the present invention. For example, a method for expanding tumor infiltrating lymphocytes (TILs) into a therapeutic population of TILs may be carried out in accordance with any embodiment of the methods described herein (e.g., process 2A, process Gen 3, or the methods shown in Figures 34 and 35), wherein the method comprises gene-editing at least a portion of the TILs to silence or reduce the expression of TGFβ in the TILs. As described in more detail below, the gene-editing process may comprise the use of a programmable nuclease that mediates the generation of a double-strand or single-strand break at an immune checkpoint gene, such as TGFβ. For example, a CRISPR method, a TALE method, or a zinc finger method may be used to silence or repress the expression of TGFβ in the TILs.

[0500] In some embodiments, TGFβR2 (TGF beta receptor 2) may be suppressed by silencing TGFβR2 using a CRISPR / Cas9 system or by using a TGFβR2 dominant negative extracellular trap, using methods known in the art. 7. PKA

[0501] Protein Kinase A (PKA) is a well-known member of the serine-threonine protein kinase superfamily. PKA, also known as cAMP-dependent protein kinase, is a multi-unit protein kinase that mediates signal transduction of G-protein coupled receptors through its activation upon cAMP binding. It is involved in the control of a wide variety of cellular processes from metabolism to ion channel activation, cell growth and differentiation, gene expression and apoptosis. Importantly, PKA has been implicated in the initiation and progression of many tumors. See, e.g., Sapio et al., EXCLI Journal; 2014; 13: 843–855.

[0502] According to particular embodiments, expression of PKA in TILs is silenced or reduced in accordance with compositions and methods of the present invention. For example, a method for expanding tumor infiltrating lymphocytes (TILs) into a therapeutic population of TILs may be carriedout in accordance with any embodiment of the methods described herein (e.g., process 2A, process Gen 3, or the methods shown in Figures 34 and 35), wherein the method comprises gene-editing at least a portion of the TILs to silence or repress the expression of PKA in the TILs. As described in more detail below, the gene-editing process may comprise the use of a programmable nuclease that mediates the generation of a double-strand or single-strand break at an immune checkpoint gene, such as PKA. For example, a CRISPR method, a TALE method, or a zinc finger method may be used to silence or repress the expression of PKA in the TILs 8. CBLB

[0503] CBLB (or CBL-B) is a E3 ubiquitin-protein ligase and is a negative regulator of T cell activation. Bachmaier, et al., Nature, 2000, 403, 211–216; Wallner, et al., Clin. Dev. Immunol.2012, 692639.

[0504] According to particular embodiments, expression of CBLB in TILs is silenced or reduced in accordance with compositions and methods of the present invention. For example, a method for expanding tumor infiltrating lymphocytes (TILs) into a therapeutic population of TILs may be carried out in accordance with any embodiment of the methods described herein (e.g., process 2A, process Gen 3, or the methods shown in Figures 34 and 35), wherein the method comprises gene-editing at least a portion of the TILs to silence or repressing the expression of CBLB in TILs. As described in more detail below, the gene-editing process may comprise the use of a programmable nuclease that mediates the generation of a double-strand or single-strand break at an immune checkpoint gene, such as CBLB. For example, a CRISPR method, a TALE method, or a zinc finger method may be used to silence or repress the expression of PKA in the TILs. In some embodiments, CBLB is silenced using a TALEN knockout. In some embodiments, CBLB is silenced using a TALE-KRAB transcriptional inhibitor knock in. More details on these methods can be found in Boettcher and McManus, Mol. Cell Review, 2015, 58, 575-585. 9. TIGIT

[0505] T-cell immunoreceptor with Ig and ITIM (immunoreceptor tyrosine-based inhibitory motif) domain or TIGIT is a transmembrane glycoprotein receptor with an Ig-like V-type domain and an ITIM in its cytoplasmic domain. Khalil, et al., Advances in Cancer Research, 2015, 128, 1-68; Yu, et al., Nature Immunology, 2009, Vol.10, No.1, 48-57. TIGIT is expressed by some T cells and Natural Killer Cells. Additionally, TIGIT has been shown to be overexpressed on antigen-specific CD8+ T cells and CD8+ TILs, particularly from individuals with melanoma. Studies have shown that the TIGIT pathway contributes to tumor immune evasion and TIGIT inhibition has been shown to increase T-cell activation and proliferation in response to polyclonal and antigen-specific stimulation. Khalil, et al., Advances in Cancer Research, 2015, 128, 1-68. Further, coblockade of TIGIT with either PD-1 or TIM3 has shown synergistic effects against solid tumors in mouse models. Id.; see also Kurtulus, et al., The Journal of Clinical Investigation, 2015, Vol.125, No.11, 4053-4062.

[0506] According to particular embodiments, expression of TIGIT in TILs is silenced or reduced in accordance with compositions and methods of the present invention. For example, a method for expanding tumor infiltrating lymphocytes (TILs) into a therapeutic population of TILs may be carried out in accordance with any embodiment of the methods described herein (e.g., process 2A, process Gen 3, or the methods shown in Figures 34 and 35), wherein the method comprises gene-editing at least a portion of the TILs to silence or repress the expression of TIGIT in the TILs. As described in more detail below, the gene-editing process may comprise the use of a programmable nuclease that mediates the generation of a double-strand or single-strand break at an immune checkpoint gene, such as TIGIT. For example, a CRISPR method, a TALE method, or a zinc finger method may be used to silence or repress the expression of TIGIT in the TILs. 10. TOX

[0507] Thymocyte selection associated high mobility group (HMG) box (TOX) is a transcription factor containing an HMG box DNA binding domain. TOX is a member of the HMG box superfamily that is thought to bind DNA in a sequence-independent but structure-dependent manner.

[0508] TOX was identified as a critical regulator of tumor-specific CD8+T cell dysfunction or T cell exhaustion and was found to transcriptionally and epigenetically program CD8+T cell exhaustion, as described, for example in Scott, et al., Nature, 2019, 571, 270-274 and Khan, et al., Nature, 2019, 571, 211-218, both of which are herein incorporated by reference in their entireties. TOX was also found to be critical factor for progression of T cell dysfunction and maintenance of exhausted T cells during chronic infection, as described in Alfei, et al., Nature, 2019, 571, 265-269, which is herein incorporated by reference in its entirety. TOX is highly expressed in dysfunctional or exhausted T cells from tumors and chronic viral infection. Ectopic expression of TOX in effector T cells in vitro induced a transcriptional program associated with T cell exhaustion, whereas deletion of TOX in T cells abrogated the T exhaustion program.

[0509] According to particular embodiments, expression of TOX in TILs is silenced or reduced in accordance with compositions and methods of the present invention. For example, a method for expanding tumor infiltrating lymphocytes (TILs) into a therapeutic population of TILs may be carried out in accordance with any embodiment of the methods described herein (e.g., process 2A, processGen 3, or the methods shown in Figures 34 and 35), wherein the method comprises gene-editing at least a portion of the TILs silence or repress the expression of TOX. As described in more detail below, the gene-editing process may comprise the use of a programmable nuclease that mediates the generation of a double-strand or single-strand break at an immune checkpoint gene, such as TOX. For example, a CRISPR method, a TALE method, or a zinc finger method may be used to silence or repress the expression of TOX in the TILs. E. Overexpression of Co-Stimulatory Receptors or Adhesion Molecules

[0510] According to additional embodiments, gene-editing TILs during the TIL expansion method causes expression of one or more co-stimulatory receptors, adhesion molecules and / or cytokines to be enhanced in at least a portion of the therapeutic population of TILs. For example, gene-editing may cause the expression of a co-stimulatory receptor, adhesion molecule or cytokine to be enhanced, which means that it is overexpressed as compared to the expression of a co-stimulatory receptor, adhesion molecule or cytokine that has not been genetically modified. Non-limiting examples of co-stimulatory receptor, adhesion molecule or cytokine genes that may exhibit enhanced expression by permanently gene-editing TILs of the present invention include certain chemokine receptors and interleukins, such as CCR2, CCR4, CCR5, CXCR2, CXCR3, CX3CR1, IL-2, IL-4, IL-7, IL-10, IL-15, IL-21, the NOTCH 1 / 2 intracellular domain (ICD), and / or the NOTCH ligand mDLL1. 1. CCRs

[0511] For adoptive T cell immunotherapy to be effective, T cells need to be trafficked properly into tumors by chemokines. A match between chemokines secreted by tumor cells, chemokines present in the periphery, and chemokine receptors expressed by T cells is important for successful trafficking of T cells into a tumor bed.

[0512] According to particular embodiments, gene-editing methods of the present invention may be used to increase the expression of certain chemokine receptors in the TILs, such as one or more of CCR2, CCR4, CCR5, CXCR2, CXCR3 and CX3CR1. Over-expression of CCRs may help promote effector function and proliferation of TILs following adoptive transfer.

[0513] According to particular embodiments, expression of one or more of CCR2, CCR4, CCR5, CXCR2, CXCR3 and CX3CR1 in TILs is enhanced in accordance with compositions and methods of the present invention. For example, a method for expanding tumor infiltrating lymphocytes (TILs) into a therapeutic population of TILs may be carried out in accordance with any embodiment of the methods described herein (e.g., process 2A, process Gen 3, or the methods shown in Figures 34 and35), wherein the method comprises gene-editing at least a portion of the TILs to express at least one immunomodulatory composition at the cell surface of and enhance the expression of one or more of CCR2, CCR4, CCR5, CXCR2, CXCR3 and CX3CR1 in the TILs.

[0514] As described in more detail below, the gene-editing process may comprise the use of a programmable nuclease that mediates the generation of a double-strand or single-strand break at a chemokine receptor gene. For example, a CRISPR method, a TALE method, or a zinc finger method may be used to enhance the expression of certain chemokine receptors in the TILs.

[0515] In some embodiments, CCR4 and / or CCR5 adhesion molecules are inserted into a TIL population using a gamma-retroviral or lentiviral method as described herein. In some embodiments, CXCR2 adhesion molecule are inserted into a TIL population using a gamma-retroviral or lentiviral method as described in Forget, et al., Frontiers Immunology 2017, 8, 908 or Peng, et al., Clin. Cancer Res.2010, 16, 5458, the disclosures of which are incorporated by reference herein.

[0516] According to some embodiments, a method for expanding tumor infiltrating lymphocytes (TILs) into a therapeutic population of TILs comprises: (a) obtaining a first population of TILs in a plurality of tumor fragments produced from a tumor sample resected from a patient; (b) adding the plurality of tumor fragments into a closed system; (c) performing a first expansion by culturing the first population of TILs in a cell culture medium comprising IL-2 and optionally comprising OKT-3 and / or a 4-1BB agonist antibody for about 3 to 11 days to produce a second population of TILs, wherein the first expansion is performed in a closed container providing a first gas-permeable surface area; (d) stimulating the second population of TILs by adding OKT-3 and culturing for about 1 to 3 days to obtain the second population of TILs, wherein the transition from step (c) to step (d) occurs without opening the system; (e) sterile electroporating the second population of TILs to effect transfer of at least one gene editor into a plurality of cells in the second population of TILs, wherein the transition from step (d) to step (e) occurs without opening the system; (f) resting the second population of TILs for about 1 day, wherein the transition from step (e) to step (f) occurs without opening the system;(g) performing a second expansion by supplementing the cell culture medium of the second population of TILs with additional IL-2, optionally OKT-3 antibody, optionally an OX40 antibody, and antigen presenting cells (APCs), to produce a third population of TILs, wherein the second expansion is performed for about 7 to 11 days, wherein the second expansion is performed in a closed container providing a second gas-permeable surface area, and wherein the transition from step (f) to step (g) occurs without opening the system; (h) harvesting the third population of TILs obtained from step (g) to provide a harvested TIL population, wherein the transition from step (g) to step (h) occurs without opening the system, wherein the harvested population of TILs is a therapeutic population of TILs; (i) transferring the harvested TIL population to an infusion bag, wherein the transfer from step (h) to (i) occurs without opening the system; and (j) optionally cryopreserving the harvested TIL population using a cryopreservation medium, wherein the electroporation step comprises the delivery of at least one gene editor system selected from the group consisting of a Clustered Regularly Interspersed Short Palindromic Repeat (CRISPR) system, a Transcription Activator-Like Effector (TALE) system, or a zinc finger system, wherein the at least one gene editor system effects inhibition of expression of PD-1 and, optionally, LAG-3, in the plurality of cells of the second population of TILs, and further wherein the at least one gene editor system effects expression of a CXCR2 adhesion molecule at the cell surface of the plurality of cells of the second population of TILs or the CXCR2 adhesion molecule is inserted by a gammaretroviral or lentiviral method into the first population of TILs, second population of TILs, or harvested population of TILs.

[0517] According to some embodiments, a method for expanding tumor infiltrating lymphocytes (TILs) into a therapeutic population of TILs comprises: (a) obtaining a first population of TILs in a plurality of tumor fragments produced from a tumor sample resected from a patient; (b) adding the plurality of tumor fragments into a closed system; (c) performing a first expansion by culturing the first population of TILs in a cell culture medium comprising IL-2 and optionally comprising OKT-3 and / or a 4-1BB agonist antibody for about 3 to 11 days to produce a second population of TILs, wherein the first expansion is performed in a closed container providing a first gas-permeable surface area;(d) stimulating the second population of TILs by adding OKT-3 and culturing for about 1 to 3 days, and wherein the transition from step (c) to step (d) occurs without opening the system; (e) sterile electroporating the second population of TILs to effect transfer of at least one gene editor into a plurality of cells in the second population of TILs, and wherein the transition from step (d) to step (e) occurs without opening the system; (f) resting the second population of TILs for about 1 day, and wherein the transition from step (e) to step (f) occurs without opening the system; (g) performing a second expansion by supplementing the cell culture medium of the second population of TILs with additional IL-2, optionally OKT-3 antibody, optionally an OX40 antibody, and antigen presenting cells (APCs), to produce a third population of TILs, wherein the second expansion is performed for about 7 to 11 days, wherein the second expansion is performed in a closed container providing a second gas-permeable surface area, and wherein the transition from step (f) to step (g) occurs without opening the system; (h) harvesting the third population of TILs obtained from step (g) to provide a harvested TIL population, wherein the transition from step (g) to step (h) occurs without opening the system, wherein the harvested population of TILs is a therapeutic population of TILs; (i) transferring the harvested TIL population to an infusion bag, wherein the transfer from step (h) to (i) occurs without opening the system; and (j) optionally cryopreserving the harvested TIL population using a cryopreservation medium, wherein the electroporation step comprises the delivery of at least one gene editor system selected from the group consisting of a Clustered Regularly Interspersed Short Palindromic Repeat (CRISPR) system, a Transcription Activator-Like Effector (TALE) system, or a zinc finger system, which at least one gene editor system effects inhibition of expression of PD-1 and, optionally, LAG-3, in the plurality of cells of the second population of TILs and further wherein the at least one gene editor system effects expression of a CCR4 and / or CCR5 adhesion molecule at the cell surface of the plurality of cells of the second population of TILs or the CCR4 and / or CCR5 adhesion molecule is inserted by a gammaretroviral or lentiviral method into the first population of TILs, second population of TILs, or harvested population of TILs.

[0518] According to some embodiments, a method for expanding tumor infiltrating lymphocytes (TILs) into a therapeutic population of TILs comprises:(a) obtaining a first population of TILs in a plurality of tumor fragments produced from a tumor sample resected from a patient; (b) adding the plurality of tumor fragments into a closed system; (c) performing a first expansion by culturing the first population of TILs in a cell culture medium comprising IL-2 and optionally comprising OKT-3 and / or a 4-1BB agonist antibody for about 3 to 11 days to produce a second population of TILs, wherein the first expansion is performed in a closed container providing a first gas-permeable surface area; (d) stimulating the second population of TILs by adding OKT-3 and culturing for about 1 to 3 days to obtain the second population of TILs, wherein the transition from step (c) to step (d) occurs without opening the system; (e) sterile electroporating the second population of TILs to effect transfer of at least one gene editor into a plurality of cells in the second population of TILs, wherein the transition from step (d) to step (e) occurs without opening the system; (f) resting the second population of TILs for about 1 day, wherein the transition from step (e) to step (f) occurs without opening the system; (g) performing a second expansion by supplementing the cell culture medium of the second population of TILs with additional IL-2, optionally OKT-3 antibody, optionally an OX40 antibody, and antigen presenting cells (APCs), to produce a third population of TILs, wherein the second expansion is performed for about 7 to 11 days, wherein the second expansion is performed in a closed container providing a second gas-permeable surface area, and wherein the transition from step (f) to step (g) occurs without opening the system; (h) harvesting the third population of TILs obtained from step (g) to provide a harvested TIL population, wherein the transition from step (g) to step (h) occurs without opening the system, wherein the harvested population of TILs is a therapeutic population of TILs; (i) transferring the harvested TIL population to an infusion bag, wherein the transfer from step (h) to (i) occurs without opening the system; and (j) optionally cryopreserving the harvested TIL population using a cryopreservation medium, wherein the electroporation step comprises the delivery of at least one gene editor system selected from the group consisting of a Clustered Regularly Interspersed Short Palindromic Repeat (CRISPR) system, a Transcription Activator-Like Effector (TALE) system, or a zinc finger system, which at leastone gene editor system effects inhibition of expression of PD-1 and, optionally, LAG-3, in the plurality of cells of the second population of TILs, and further wherein the at least one gene editor system effects expression of an adhesion molecule selected from the group consisting of CCR2, CCR4, CCR5, CXCR2, CXCR3, CX3CR1, and combinations thereof, at the cell surface of the plurality of cells of the second population of TILs or the adhesion molecule is inserted by a gammaretroviral or lentiviral method into the first population of TILs, second population of TILs, or harvested population of TILs. 2. Interleukins

[0519] According to additional embodiments, gene-editing methods of the present invention may be used to increase the expression of certain interleukins, such as one or more of IL-2, IL-4, IL-7, IL- 10, IL-15, and IL-21. Certain interleukins have been demonstrated to augment effector functions of T cells and mediate tumor control.

[0520] According to particular embodiments, expression of one or more of IL-2, IL-4, IL-7, IL-10, IL-15, and IL-21 in TILs is enhanced in accordance with compositions and methods of the present invention. For example, a method for expanding tumor infiltrating lymphocytes (TILs) into a therapeutic population of TILs may be carried out in accordance with any embodiment of the methods described herein (e.g., process 2A, process Gen 3, or the methods shown in Figures 34 and 35), wherein the method comprises gene-editing at least a portion of the TILs by enhancing the expression of one or more of IL-2, IL-4, IL-7, IL-10, IL-15, and IL-21. As described in more detail below, the gene-editing process may comprise the use of a programmable nuclease that mediates the generation of a double-strand or single-strand break at an interleukin gene. For example, a CRISPR method, a TALE method, or a zinc finger method may be used to enhance the expression of certain interleukins in the TILs.

[0521] According to some embodiments, a method for expanding tumor infiltrating lymphocytes (TILs) into a therapeutic population of TILs comprises: (a) obtaining a first population of TILs in a plurality of tumor fragments produced from a tumor resected from a patient; (b) adding the plurality of tumor fragments into a closed system; (c) performing a first expansion by culturing the first population of TILs in a cell culture medium comprising IL-2 and optionally comprising OKT-3 and / or a 4-1BB agonist antibody for about3 to 11 days to produce a second population of TILs, wherein the first expansion is performed in a closed container providing a first gas-permeable surface area; (d) stimulating the second population of TILs by adding OKT-3 and culturing for about 1 to 3 days to obtain the second population of TILs, wherein the transition from step (c) to step (d) occurs without opening the system; (e) sterile electroporating the second population of TILs to effect transfer of at least one gene editor, wherein the transition from step (d) to step (e) occurs without opening the system; (f) resting the second population of TILs for about 1 day into a plurality of cells in the second population of TILs, wherein the transition from step (e) to step (f) occurs without opening the system; (g) performing a second expansion by supplementing the cell culture medium of the second population of TILs with additional IL-2, optionally OKT-3 antibody, optionally an OX40 antibody, and antigen presenting cells (APCs), to produce a third population of TILs, wherein the second expansion is performed for about 7 to 11, wherein the second expansion is performed in a closed container providing a second gas-permeable surface area, and wherein the transition from step (f) to step (g) occurs without opening the system; (h) harvesting the therapeutic population of TILs obtained from step (g) to provide a harvested TIL population, wherein the transition from step (g) to step (h) occurs without opening the system, wherein the harvested population of TILs is a therapeutic population of TILs; (i) transferring the harvested TIL population to an infusion bag, wherein the transfer from step (h) to (i) occurs without opening the system; and (j) optionally cryopreserving the harvested TIL population using a cryopreservation medium, wherein the electroporation step comprises the delivery of at least one gene editor system selected from the group consisting of a Clustered Regularly Interspersed Short Palindromic Repeat (CRISPR) system, a Transcription Activator-Like Effector (TALE) system, or a zinc finger system, which at least one gene editor system effects inhibition of expression of PD-1 and, optionally, LAG-3, in the plurality of cells of the second population of TILs and further wherein the at least one gene editor system effects expression of an interleukin selected from the group consisting of IL-2, IL-4, IL-7, IL- 10, IL-15, IL-21, and combinations thereof, at the cell surface of the plurality of cells of the second population of TILs or the interleukin is inserted by a gammaretroviral or lentiviral method into the first population of TILs, second population of TILs, or harvested population of TILs.3. Gene Editing Methods

[0522] As discussed above, embodiments of the present invention provide tumor infiltrating lymphocytes (TILs) that have been genetically modified via gene-editing to enhance their therapeutic effect. Embodiments of the present invention embrace genetic editing through nucleotide insertion (RNA or DNA) into a population of TILs for both promotion of the expression of one or more proteins and inhibition of the expression of one or more proteins, as well as combinations thereof. Embodiments of the present invention also provide methods for expanding TILs into a therapeutic population, wherein the methods comprise gene-editing the TILs. There are several gene-editing technologies that may be used to genetically modify a population of TILs, which are suitable for use in accordance with the present invention. In some embodiments, electroporation is employed as part of the gene editing methods.

[0523] In some embodiments, a method of genetically modifying a population of TILs includes the step of stable incorporation of genes for production of one or more proteins. In some embodiments, a method of genetically modifying a population of TILs includes the step of retroviral transduction. In some embodiments, a method of genetically modifying a population of TILs includes the step of lentiviral transduction. Lentiviral transduction systems are known in the art and are described, e.g., in Levine, et al., Proc. Nat’l Acad. Sci.2006, 103, 17372-77; Zufferey, et al., Nat. Biotechnol.1997, 15, 871-75; Dull, et al., J. Virology 1998, 72, 8463-71, and U.S. Patent No. 6,627,442, the disclosures of each of which are incorporated by reference herein. In some embodiments, a method of genetically modifying a population of TILs includes the step of gamma- retroviral transduction. Gamma-retroviral transduction systems are known in the art and are described, e.g., Cepko and Pear, Cur. Prot. Mol. Biol.1996, 9.9.1-9.9.16, the disclosure of which is incorporated by reference herein. In some embodiments, a method of genetically modifying a population of TILs includes the step of transposon-mediated gene transfer. Transposon-mediated gene transfer systems are known in the art and include systems wherein the transposase is provided as DNA expression vector or as an expressible RNA or a protein such that long-term expression of the transposase does not occur in the transgenic cells, for example, a transposase provided as an mRNA (e.g., an mRNA comprising a cap and poly-A tail). Suitable transposon-mediated gene transfer systems, including the salmonid-type Tel-like transposase (SB or Sleeping Beauty transposase), such as SB10, SB11, and SB100x, and engineered enzymes with increased enzymatic activity, are described in, e.g., Hackett, et al., Mol. Therapy 2010, 18, 674-83 and U.S. Patent No.6,489,458, the disclosures of each of which are incorporated by reference herein.

[0524] In some embodiments, a method of genetically modifying a population of TILs includes the step of stable incorporation of genes for production or inhibition (e.g., silencing) of one or more proteins. In some embodiments, a method of genetically modifying a population of TILs includes the step of electroporation. Electroporation methods are known in the art and are described, e.g., in Tsong, Biophys. J.1991, 60, 297-306, and U.S. Patent Application Publication No. 2014 / 0227237 A1, the disclosures of each of which are incorporated by reference herein. Other electroporation methods known in the art, such as those described in U.S. Patent Nos.5,019,034; 5,128,257; 5,137,817; 5,173,158; 5,232,856; 5,273,525; 5,304,120; 5,318,514; 6,010,613 and 6,078,490, the disclosures of which are incorporated by reference herein, may be used. In some embodiments, the electroporation method is a sterile electroporation method. In some embodiments, the electroporation method is a pulsed electroporation method. In some embodiments, the electroporation method is a pulsed electroporation method comprising the steps of treating TILs with pulsed electrical fields to alter, manipulate, or cause defined and controlled, permanent or temporary changes in the TILs, comprising the step of applying a sequence of at least three single, operator-controlled, independently programmed, DC electrical pulses, having field strengths equal to or greater than 100 V / cm, to the TILs, wherein the sequence of at least three DC electrical pulses has one, two, or three of the following characteristics: (1) at least two of the at least three pulses differ from each other in pulse amplitude; (2) at least two of the at least three pulses differ from each other in pulse width; and (3) a first pulse interval for a first set of two of the at least three pulses is different from a second pulse interval for a second set of two of the at least three pulses. In some embodiments, the electroporation method is a pulsed electroporation method comprising the steps of treating TILs with pulsed electrical fields to alter, manipulate, or cause defined and controlled, permanent or temporary changes in the TILs, comprising the step of applying a sequence of at least three single, operator-controlled, independently programmed, DC electrical pulses, having field strengths equal to or greater than 100 V / cm, to the TILs, wherein at least two of the at least three pulses differ from each other in pulse amplitude. In some embodiments, the electroporation method is a pulsed electroporation method comprising the steps of treating TILs with pulsed electrical fields to alter, manipulate, or cause defined and controlled, permanent or temporary changes in the TILs, comprising the step of applying a sequence of at least three single, operator-controlled, independently programmed, DC electrical pulses, having field strengths equal to or greater than 100 V / cm, to the TILs, wherein at least two of the at least three pulses differ from each other in pulse width. In some embodiments, the electroporation method is a pulsed electroporation method comprising the steps of treating TILs with pulsed electrical fields to alter, manipulate, or cause defined and controlled, permanent or temporary changes in the TILs,comprising the step of applying a sequence of at least three single, operator-controlled, independently programmed, DC electrical pulses, having field strengths equal to or greater than 100 V / cm, to the TILs, wherein a first pulse interval for a first set of two of the at least three pulses is different from a second pulse interval for a second set of two of the at least three pulses. In some embodiments, the electroporation method is a pulsed electroporation method comprising the steps of treating TILs with pulsed electrical fields to induce pore formation in the TILs, comprising the step of applying a sequence of at least three DC electrical pulses, having field strengths equal to or greater than 100 V / cm, to TILs, wherein the sequence of at least three DC electrical pulses has one, two, or three of the following characteristics: (1) at least two of the at least three pulses differ from each other in pulse amplitude; (2) at least two of the at least three pulses differ from each other in pulse width; and (3) a first pulse interval for a first set of two of the at least three pulses is different from a second pulse interval for a second set of two of the at least three pulses, such that induced pores are sustained for a relatively long period of time, and such that viability of the TILs is maintained.

[0525] In some embodiments, a method of genetically modifying a population of TILs includes the step of calcium phosphate transfection. Calcium phosphate transfection methods (calcium phosphate DNA precipitation, cell surface coating, and endocytosis) are known in the art and are described in Graham and van der Eb, Virology 1973, 52, 456-467; Wigler, et al., Proc. Natl. Acad. Sci.1979, 76, 1373-1376; and Chen and Okayarea, Mol. Cell. Biol.1987, 7, 2745-2752; and in U.S. Patent No.5,593,875, the disclosures of each of which are incorporated by reference herein. In some embodiments, a method of genetically modifying a population of TILs includes the step of liposomal transfection. Liposomal transfection methods, such as methods that employ a 1:1 (w / w) liposome formulation of the cationic lipid N-[1-(2,3-dioleyloxy)propyl]-n,n,n-trimethylammonium chloride (DOTMA) and dioleoyl phophotidylethanolamine (DOPE) in filtered water, are known in the art and are described in Rose, et al., Biotechniques 1991, 10, 520-525 and Felgner, et al., Proc. Natl. Acad. Sci. USA, 1987, 84, 7413-7417 and in U.S. Patent Nos.5,279,833; 5,908,635; 6,056,938; 6,110,490; 6,534,484; and 7,687,070, the disclosures of each of which are incorporated by reference herein. In some embodiments, a method of genetically modifying a population of TILs includes the step of transfection using methods described in U.S. Patent Nos.5,766,902; 6,025,337; 6,410,517; 6,475,994; and 7,189,705; the disclosures of each of which are incorporated by reference herein.

[0526] According to some embodiments, the gene-editing process may comprise the use of a programmable nuclease that mediates the generation of a double-strand or single-strand break at one or more immune checkpoint genes. Such programmable nucleases enable precise genomeediting by introducing breaks at specific genomic loci, i.e., they rely on the recognition of a specific DNA sequence within the genome to target a nuclease domain to this location and mediate the generation of a double-strand break at the target sequence. A double-strand break in the DNA subsequently recruits endogenous repair machinery to the break site to mediate genome editing by either non-homologous end-joining (NHEJ) or homology-directed repair (HDR). Thus, the repair of the break can result in the introduction of insertion / deletion mutations that disrupt (e.g., silence, repress, or enhance) the target gene product.

[0527] Major classes of nucleases that have been developed to enable site-specific genomic editing include zinc finger nucleases (ZFNs), transcription activator-like nucleases (TALENs), and CRISPR-associated nucleases (e.g., CRISPR / Cas9). These nuclease systems can be broadly classified into two categories based on their mode of DNA recognition: ZFNs and TALENs achieve specific DNA binding via protein-DNA interactions, whereas CRISPR systems, such as Cas9, are targeted to specific DNA sequences by a short RNA guide molecule that base-pairs directly with the target DNA and by protein-DNA interactions. See, e.g., Cox et al., Nature Medicine, 2015, Vol.21, No.2.

[0528] Non-limiting examples of gene-editing methods that may be used in accordance with TIL expansion methods of the present invention include CRISPR methods, TALE methods, and ZFN methods, embodiments of which are described in more detail below. According to some embodiments, a method for expanding TILs into a therapeutic population may be carried out in accordance with any embodiment of the methods described herein (e.g., process 2A) or as described in PCT / US2017 / 058610, PCT / US2018 / 012605, or PCT / US2...

Claims

WHAT IS CLAIMED IS:

1. A method of treating a cancer in a patient or subject in need thereof comprising administering a population of modified tumor infiltrating lymphocytes (TILs), the method comprising the steps of: (a) obtaining and / or receiving a first population of TILs from a tumor resected from the subject or patient by processing a tumor sample obtained from the subject into multiple tumor fragments or processing a tumor sample obtained from the subject into a tumor digest; (b) selecting, from the first population of TILs in (a), a population of TILs that expresses at least CD39 and CD103; (c) optionally adding the population of TILs into a closed system; (d) performing a first expansion by culturing the population of the TILs in a cell culture medium comprising IL-2 to produce a second population of TILs, wherein the first expansion is optionally performed in a closed container providing a first gas-permeable surface area, wherein the first expansion is performed for about 3-14 days to obtain the second population of TILs, and wherein the transition from step (c) to step (d) optionally occurs without opening the system; (e) performing a second expansion by supplementing the cell culture medium of the second population of TILs with additional IL-2, OKT-3, and antigen presenting cells (APCs), to produce a third population of TILs, wherein the second expansion is performed for about 7- 14 days to obtain the third population of TILs, wherein the third population of TILs is a therapeutic population of TILs, wherein the second expansion is optionally performed in a closed container providing a second gas-permeable surface area, and wherein the transition from step (d) to step (e) optionally occurs without opening the system; (f) harvesting the third population of TILs obtained from step (e), wherein the transition from step (e) to step (f) optionally occurs without opening the system; (g) transferring the harvested third TILs population from step (f) to an infusion bag, wherein the transfer from step (f) to (g) optionally occurs without opening the system; (h) cryopreserving the infusion bag comprising the harvested third TILs population from step (g) using a cryopreservation process; (i) administering a therapeutically effective dosage of the third population of TILs from the infusion bag in step (h) to the subject; and(j) optionally genetically modifying the population of TILs at any time prior to the administering step (i) such that the administered third population of TILs comprises genetically modified TILs comprising a genetic modification that reduces or increases the expression of one or more T cell exhaustion markers and / or one or more factors phenotypically associated with said T cell exhaustion markers.

2. A method of treating a cancer in a patient or subject in need thereof comprising administering a population of tumor infiltrating lymphocytes (TILs), the method comprising the steps of: (a) obtaining a first population of TILs from a tumor resected from a subject by processing a tumor sample obtained from the subject into multiple tumor fragments or processing a tumor sample obtained from the subject into a tumor digest; (b) selecting, from the first population of TILs in (a), a population of TILs that expresses at least CD39 and CD103; (c) optionally adding the population of TILs into a closed system; (d) performing a first expansion by culturing population of the TILs in a cell culture medium comprising IL-2 to produce a second population of TILs, wherein the first expansion is optionally performed in a closed container providing a first gas-permeable surface area, wherein the first expansion is performed for about 3-11 days to obtain the second population of TILs, and wherein the transition from step (c) to step (d) optionally occurs without opening the system; (e) performing a second expansion by supplementing the cell culture medium of the second population of TILs with additional IL-2, OKT-3, and antigen presenting cells (APCs), to produce a third population of TILs, wherein the second expansion is performed for about 7- 11 days to obtain the third population of TILs, wherein the second expansion is optionally performed in a closed container providing a second gas-permeable surface area, and wherein the transition from step (d) to step (e) optionally occurs without opening the system; (f) harvesting the third population of TILs obtained from step (e), wherein the transition from step (e) to step (f) optionally occurs without opening the system; (g) transferring the harvested third TILs population from step (f) to an infusion bag, wherein the transfer from step (f) to (g) optionally occurs without opening the system;(h) cryopreserving the infusion bag comprising the harvested TILs population from step (g) using a cryopreservation process; (i) administering a therapeutically effective dosage of the third population of TILs from the infusion bag in step (h) to the subject; and (j) optionally genetically modifying the population of TILs at any time prior to the administering step (i) such that the administered third population of TILs comprises genetically modified TILs comprising a genetic modification that reduces or increases the expression of one or more T cell exhaustion markers and / or one or more factors phenotypically associated with said T cell exhaustion markers.

3. A method of treating a cancer in a patient or subject in need thereof comprising administering a population of tumor infiltrating lymphocytes (TILs), the method comprising the steps of: (a) obtaining and / or receiving a first population of TILs from surgical resection, needle biopsy, core biopsy, small biopsy, or other means for obtaining a sample that contains a mixture of tumor and TIL cells from the cancer in the patient or subject, (b) selecting, from the first population of TILs in (a), a population of TILs that expresses at least CD39 and CD103; (c) optionally adding the population TILs into a closed system; (d) performing a first expansion by culturing the population of TILs in a cell culture medium comprising IL-2 to produce a second population of TILs, wherein the first expansion is optionally performed in a closed container providing a first gas-permeable surface area, wherein the first expansion is performed for about 3-11 days to obtain the second population of TILs, and wherein the transition from step (c) to step (d) optionally occurs without opening the system; (e) performing a second expansion by supplementing the cell culture medium of the second population of TILs with additional IL-2, OKT-3, and antigen presenting cells (APCs), to produce a third population of TILs, wherein the second expansion is performed for about 7- 11 days to obtain the third population of TILs, wherein the second expansion is optionally performed in a closed container providing a second gas-permeable surface area, and wherein the transition from step (d) to step (e) optionally occurs without opening the system;(f) harvesting the third population of TILs obtained from step (e), wherein the transition from step (e) to step (f) optionally occurs without opening the system; (g) transferring the harvested third TILs population from step (f) to an infusion bag, wherein the transfer from step (e) to (f) optionally occurs without opening the system; (h) cryopreserving the infusion bag comprising the harvested TILs population from step (g) using a cryopreservation process; (i) administering a therapeutically effective dosage of the third population of TILs from the infusion bag in step (h) to the subject.; and (j) optionally genetically modifying the population of TILs at any time prior to the administering step (i) such that the administered third population of TILs comprises genetically modified TILs comprising a genetic modification that reduces or increases the expression of one or more T cell exhaustion markers and / or one or more factors phenotypically associated with said T cell exhaustion markers.

4. A method of treating a cancer in a patient or subject in need thereof comprising administering a population of modified tumor infiltrating lymphocytes (TILs), the method comprising the steps of: (a) resecting a tumor from the subject or patient, the tumor comprising a first population of TILs, optionally from surgical resection, needle biopsy, core biopsy, small biopsy, or other means for obtaining a sample that contains a mixture of tumor and TIL cells from the cancer; (b) processing the tumor into multiple tumor fragments; (c) enzymatically digesting the multiple tumor fragments to obtain the first population of TILs; (d) selecting, from the first population of TILs in (a), a population of TILs that expresses at least CD39 and CD103; (e) optionally adding the population of TILs into a closed system; (f) performing a first expansion by culturing the population of TILs in a cell culture medium comprising IL-2 to produce a second population of TILs, wherein the first expansion is optionally performed in a closed container providing a first gas-permeable surface area, wherein the first expansion is performed for about 3-11 days to obtain the second population of TILs, and wherein the transition from step (e) to step (f) optionally occurswithout opening the system; (g) performing a second expansion by supplementing the cell culture medium of the second population of TILs with additional IL-2, OKT-3, and antigen presenting cells (APCs), to produce a third population of TILs, wherein the second expansion is performed for about 7- 11 days to obtain the third population of TILs, wherein the second expansion is optionally performed in a closed container providing a second gas-permeable surface area, and wherein the transition from step (f) to step (g) optionally occurs without opening the system; (h) harvesting the third population of TILs obtained from step (g), wherein the transition from step (g) to step (h) optionally occurs without opening the system; (i) transferring the harvested third TILs population from step (h) to an infusion bag, wherein the transfer from step (h) to (i) optionally occurs without opening the system; (j) cryopreserving the infusion bag comprising the harvested TILs population from step (i) using a cryopreservation process; (k) administering a therapeutically effective dosage of the third population of TILs from the infusion bag in step (g) to the subject or patient with the cancer; and (j) optionally genetically modifying the population of TILs, the second population of TILs and / or the third population of TILs at any time prior to the administering step (k) such that the administered third population of TILs comprises genetically modified TILs comprising a genetic modification that reduces or increases the expression of one or more T cell exhaustion markers and / or one or more factors phenotypically associated with said T cell exhaustion markers.

5. A method of treating a cancer in a patient or subject in need thereof comprising administering a population of tumor infiltrating lymphocytes (TILs), the method comprising the steps of: (a) obtaining and / or receiving a first population of TILs from surgical resection, needle biopsy, core biopsy, small biopsy, or other means for obtaining a sample that contains a mixture of tumor and TIL cells from the subject or patient; (b) selecting, from the first population of TILs in (a), a population of TILs that expresses at least CD39 and CD103;(c) contacting the population of TILs with a first cell culture medium; (d) performing a first expansion of the population of TILs in the first cell culture medium to obtain a second population of TILs, wherein the second population of TILs is at least 5-fold greater in number than the first population of TILs, wherein the first cell culture medium comprises IL-2, optionally OKT-3 (anti-CD3 antibody), and optionally antigen presenting cells (APCs), where the first expansion occurs for a period of 1 to 8 days; (e) performing a rapid second expansion of the second population of TILs in a second cell culture medium to obtain a third population of TILs, wherein the third population of TILs is at least 50-fold greater in number than the second population of TILs after 7-8 days from the start of the rapid expansion; wherein the second cell culture medium comprises IL-2, OKT-3 (anti-CD3 antibody), and APCs; and wherein the rapid expansion is performed over a period of 14 days or less, optionally the rapid second expansion can proceed for 1 day, 2 days, 3 days, 4, days, 5 days, 6 days, 7 days, 8 days, 9 days or 10 days after initiation of the rapid second expansion; (f) harvesting the third population of TILs; (g) administering a therapeutically effective portion of the third population of TILs to the subject or patient with the cancer; and (h) optionally genetically modifying the population of TILs at any time prior to the administering step (g) such that the administered third population of TILs comprises genetically modified TILs comprising a genetic modification that that reduces or increases the expression of one or more T cell exhaustion markers and / or one or more factors phenotypically associated with said T cell exhaustion markers.

6. A method of treating a cancer in a patient or subject in need thereof comprising administering a population of tumor infiltrating lymphocytes (TILs), the method comprising the steps of: (a) resecting a tumor from the cancer in the subject or patient, the tumor comprising a first population of TILs, optionally from surgical resection, needle biopsy, core biopsy, small biopsy, or other means for obtaining a sample that contains a mixture of tumor and TIL cells from the cancer; (b) fragmenting the tumor into tumor fragments or processing th tumor into a tumor digest; (c) selecting, from the first population of TILs in (a), a population of TILs that expresses at least CD39 and CD103;(d) contacting the tumor fragments with a first cell culture medium; (e) performing a first expansion of the population of TILs in the first cell culture medium to obtain a second population of TILs, wherein the second population of TILs is at least 5-fold greater in number than the first population of TILs, wherein the first cell culture medium comprises IL-2, optionally OKT-3 (anti-CD3 antibody), and optionally antigen presenting cells (APCs), where the first expansion occurs for a period of 1 to 8 days; (f) performing a rapid second expansion of the second population of TILs in a second cell culture medium to obtain a third population of TILs, wherein the third population of TILs is at least 50-fold greater in number than the second population of TILs after 7-8 days from the start of the rapid expansion; wherein the second cell culture medium comprises IL-2, OKT-3 (anti-CD3 antibody), and APCs; and wherein the rapid expansion is performed over a period of 14 days or less, optionally the rapid second expansion can proceed for 1 day, 2 days, 3 days, 4, days, 5 days, 6 days, 7 days, 8 days, 9 days or 10 days after initiation of the rapid second expansion; (g) harvesting the third population of TILs; (h) administering a therapeutically effective portion of the third population of TILs to the subject or patient with the cancer; and (i) optionally genetically modifying the population of TILs at any time prior to the administering step (h) such that the administered third population of TILs comprises genetically modified TILs comprising a genetic modification that reduces or increases the expression of one or more T cell exhaustion markers and / or one or more factors phenotypically associated with said T cell exhaustion markers.

7. A method of treating a cancer in a patient or subject in need thereof comprising administering a population of tumor infiltrating lymphocytes (TILs), the method comprising the steps of: (a) obtaining and / or receiving a first population of TILs from surgical resection, needle biopsy, core biopsy, small biopsy, or other means for obtaining a sample that contains a mixture of tumor and TIL cells from the cancer in the patient or subject, (b) selecti selecting, from the first population of TILs in (a), a population of TILs that expresses at least CD39 and CD103; (c) performing a priming first expansion by culturing the TILs population in a first cell culturemedium comprising IL-2, OKT-3, and antigen presenting cells (APCs) to produce a second population of TILs, wherein the priming first expansion is performed in a container comprising a first gas-permeable surface area, wherein the priming first expansion is performed for first period of about 1 to 11 days to obtain the second population of TILs, wherein the second population of TILs is greater in number than the first population of TILs; (d) optionally restimulating the second population of TILs with OKT-3; (e) genetically modifying the second population of TILs to produce a modified second population of TILs, wherein the modified second population of TILs comprises a genetic modification that reduces or increases the expression of one or more T cell exhaustion markers and / or one or more factors phenotypically associated with said T cell exhaustion markers; (f) performing a rapid second expansion by culturing the modified second population of TILs in a second culture medium comprising IL-2, OKT-3, and APCs, to produce a third population of TILs, wherein the rapid second expansion is performed for a second period of about 14 days or less to obtain the therapeutic population of TILs, wherein the third population of TILs is a therapeutic population of TILs comprising the genetic modification that reduces or increases the expression of the one or more T cell exhaustion markers and / or the one or more factors phenotypically associated with said T cell exhaustion markers; (g) harvesting the third population of TILs; (h) administering a therapeutically effective portion of the third population of TILs to the subject or patient with the cancer, and (i) optionally genetically modifying the population of TILs at any time prior to the administering step (h) such that the administered third population of TILs comprises genetically modified TILs comprising a genetic modification that reduces or increases the expression of one or more T cell exhaustion markers and / or one or more factors phenotypically associated with said T cell exhaustion markers.

8. A method for expanding tumor infiltrating lymphocytes (TILs) into a therapeutic population of TILs comprising: (a) obtaining and / or receiving a first population of TILs from a tumor resected from a cancer in a subject by processing a tumor sample obtained from the tumor into multiple tumor fragments or processing a tumor sample obtained from the subject into a tumor digest;(b) selecting, from the first population of TILs in (a), a population of TILs that expresses at least CD39 and CD103; (c) performing a priming first expansion by culturing the TILs population in a first cell culture medium comprising IL-2, OKT-3, and antigen presenting cells (APCs) to produce a second population of TILs, wherein the priming first expansion is performed in a container comprising a first gas-permeable surface area, wherein the priming first expansion is performed for first period of about 1 to 7 / 8 days to obtain the second population of TILs, wherein the second population of TILs is greater in number than the first population of TILs; (d) performing a rapid second expansion by culturing the second population of TILs in a second culture medium comprising IL-2, OKT-3, and APCs, to produce a third population of TILs, wherein the number of APCs added in the rapid second expansion is at least twice the number of APCs added in step (b), wherein the rapid second expansion is performed for a second period of about 1 to 11 days to obtain the therapeutic population of TILs, wherein the third population of TILs is a therapeutic population of TILs, wherein the rapid second expansion is performed in a container comprising a second gas-permeable surface area; (e) harvesting the therapeutic population of TILs obtained from step (d); (f) transferring the harvested TILs population from step (e) to an infusion bag; and (g) optionally genetically modifying the population of TILs at any time prior to the harvesting step (e) such that the therapeutic population of TILs comprises genetically modified TILs comprising a genetic modification that reduces or increases the expression of one or more T cell exhaustion markers and / or one or more factors phenotypically associated with said T cell exhaustion markers.

9. A method of expanding tumor infiltrating lymphocytes (TILs) into a therapeutic population of TILs, the method comprising the steps of: (a) obtaining and / or receiving a first population of TILs from a tumor resected from a cancer in a subject or patient by processing a tumor sample obtained from the tumor into multiple tumor fragments or processing a tumor sample obtained from the subject into a tumor digest; (b) selecting, from the first population of TILs in (a), a population of TILs that expresses at least CD39 and CD103; (c) optionally adding the population of TILs into a closed system;(d) performing a first expansion by culturing the population of TILs in a cell culture medium comprising IL-2 to produce a second population of TILs, wherein the first expansion is optionally performed in a closed container providing a first gas-permeable surface area, wherein the first expansion is performed for about 3-14 days to obtain the second population of TILs, and wherein the transition from step (c) to step (d) optionally occurs without opening the system; (e) performing a second expansion by supplementing the cell culture medium of the second population of TILs with additional IL-2, OKT-3, and antigen presenting cells (APCs), to produce a third population of TILs, wherein the second expansion is performed for about 7- 14 days to obtain the third population of TILs, wherein the third population of TILs is a therapeutic population of TILs, wherein the second expansion is optionally performed in a closed container providing a second gas-permeable surface area, and wherein the transition from step (d) to step (e) optionally occurs without opening the system; (f) harvesting the third population of TILs obtained from step (e), wherein the transition from step (e) to step (f) optionally occurs without opening the system; (g) transferring the harvested third TILs population from step (f) to an infusion bag, wherein the transfer from step (f) to (g) optionally occurs without opening the system; and (h) optionally genetically modifying the population of TILs, at any time prior to the harvesting step (f) such that the third population of TILs comprises genetically modified reduces or increases the expression of one or more T cell exhaustion markers and / or one or more factors phenotypically associated with said T cell exhaustion markers.

10. A method of expanding tumor infiltrating lymphocytes (TILs) into a therapeutic population of TILs, the method comprising the steps of: (a) obtaining a first population of TILs from a tumor resected from a cancer in a subject by processing a tumor sample obtained from the tumor into multiple tumor fragments or processing a tumor sample obtained from the subject into a tumor digest; b) selecting, from the first population of TILs in (a), a population of TILs that expresses at least CD39 and CD103; (c) optionally adding the population of TILs into a closed system; (d) performing a first expansion by culturing population of TILs in a cell culture mediumcomprising IL-2 to produce a second population of TILs, wherein the first expansion is optionally performed in a closed container providing a first gas-permeable surface area, wherein the first expansion is performed for about 3-11 days to obtain the second population of TILs, and wherein the transition from step (c) to step (d) optionally occurs without opening the system; (e) performing a second expansion by supplementing the cell culture medium of the second population of TILs with additional IL-2, OKT-3, and antigen presenting cells (APCs), to produce a third population of TILs, wherein the second expansion is performed for about 7- 11 days to obtain the third population of TILs, wherein the second expansion is optionally performed in a closed container providing a second gas-permeable surface area, and wherein the transition from step (d) to step (e) optionally occurs without opening the system; (f) harvesting the third population of TILs obtained from step (e), wherein the transition from step (e) to step (f) optionally occurs without opening the system; (g) transferring the harvested third TILs population from step (f) to an infusion bag, wherein the transfer from step (f) to (g) optionally occurs without opening the system; and (h) optionally genetically modifying the population of TILs, at any time prior to the harvesting step (f) such that the third population of TILs comprises genetically modified TILs comprising a genetic modification that reduces or increases the expression of one or more T cell exhaustion markers and / or one or more factors phenotypically associated with said T cell exhaustion markers.

11. A method of expanding tumor infiltrating lymphocytes (TILs) into a therapeutic population of TILs, the method comprising the steps of: (a) obtaining and / or receiving a first population of TILs from surgical resection, needle biopsy, core biopsy, small biopsy, or other means for obtaining a sample that contains a mixture of tumor and TIL cells from a cancer in a patient or subject, (b) selecting, from the first population of TILs in (a), a population of TILs that expresses at least CD39 and CD103; (c) optionally adding the population of TILs into a closed system; (d) performing a first expansion by culturing the population of TILs in a cell culture mediumcomprising IL-2 to produce a second population of TILs, wherein the first expansion is optionally performed in a closed container providing a first gas-permeable surface area, wherein the first expansion is performed for about 3-11 days to obtain the second population of TILs, and wherein the transition from step (c) to step (d) optionally occurs without opening the system; (e) performing a second expansion by supplementing the cell culture medium of the second population of TILs with additional IL-2, OKT-3, and antigen presenting cells (APCs), to produce a third population of TILs, wherein the second expansion is performed for about 7- 11 days to obtain the third population of TILs, wherein the second expansion is optionally performed in a closed container providing a second gas-permeable surface area, and wherein the transition from step (d) to step (e) optionally occurs without opening the system; (f) harvesting the third population of TILs obtained from step (e), wherein the transition from step (e) to step (f) optionally occurs without opening the system; (g) transferring the harvested third TILs population from step (f) to an infusion bag, wherein the transfer from step (e) to (f) optionally occurs without opening the system; and (h) optionally genetically modifying the population of TILs at any time prior to the harvesting step (f) such that the third population of TILs comprises genetically modified TILs comprising a genetic modification that reduces or increases the expression of one or more T cell exhaustion markers and / or one or more factors phenotypically associated with said T cell exhaustion markers.

12. A method of expanding tumor infiltrating lymphocytes (TILs) to a therapeutic population of TILs, the method comprising the steps of: (a) resecting a tumor from a cancer in subject or patient, the tumor comprising a first population of TILs, optionally from surgical resection, needle biopsy, core biopsy, small biopsy, or other means for obtaining a sample that contains a mixture of tumor and TIL cells from the cancer; (b) processing the tumor into multiple tumor fragments or into a tumor digest; (c) enzymatically digesting the multiple tumor fragments to obtain the first population of TILs; (d) selecting, from the first population of TILs in (c), a population of TILs that expresses at least one checkpoint inhibitor comprising CD39 and CD103;(e) optionally adding the population of TILs into a closed system; (f) performing a first expansion by culturing the population of TILs in a cell culture medium comprising IL-2 to produce a second population of TILs, wherein the first expansion is optionally performed in a closed container providing a first gas-permeable surface area, wherein the first expansion is performed for about 3-11 days to obtain the second population of TILs, and wherein the transition from step (e) to step (f) optionally occurs without opening the system; (g) performing a second expansion by supplementing the cell culture medium of the second population of TILs with additional IL-2, OKT-3, and antigen presenting cells (APCs), to produce a third population of TILs, wherein the second expansion is performed for about 7- 11 days to obtain the third population of TILs, wherein the second expansion is optionally performed in a closed container providing a second gas-permeable surface area, and wherein the transition from step (f) to step (g) optionally optionally occurs without opening the system; (h) harvesting the third population of TILs obtained from step (g), wherein the transition from step (g) to step (h) optionally occurs without opening the system; (i) transferring the harvested third TILs population from step (h) to an infusion bag, wherein the transfer from step (h) to (i) optionally occurs without opening the system; and (j) optionally genetically modifying the population of TILs at any time prior to the harvesting step (h) such that the third population of TILs comprises genetically modified TILs comprising a genetic modification that reduces or increases the expression of the one or more T cell exhaustion markers and / or the one or more factors phenotypically associated with said T cell exhaustion markers.

13. A method of expanding tumor infiltrating lymphocytes (TILs) into a therapeutic population of TILs, the method comprising the steps of: (a) obtaining and / or receiving a first population of TILs from surgical resection, needle biopsy, core biopsy, small biopsy, or other means for obtaining a sample that contains a mixture of tumor and TIL cells from a cancer in the subject or patient; (b) selecting, from the first population of TILs in (a), a population of TILs that expresses at least CD39 and CD103;(c) contacting the population of TILs with a first cell culture medium; (d) performing a first expansion of the population of TILs in the first cell culture medium to obtain a second population of TILs, wherein the second population of TILs is at least 5-fold greater in number than the first population of TILs, wherein the first cell culture medium comprises IL-2, optionally OKT-3 (anti-CD3 antibody), and optionally antigen presenting cells (APCs), where the priming first expansion occurs for a period of 1 to 8 days; (e) performing a rapid second expansion of the second population of TILs in a second cell culture medium to obtain a third population of TILs, wherein the third population of TILs is at least 50-fold greater in number than the second population of TILs after 7-8 days from the start of the rapid expansion; wherein the second cell culture medium comprises IL-2, OKT-3 (anti-CD3 antibody), and APCs; and wherein the rapid expansion is performed over a period of 14 days or less, optionally the rapid second expansion can proceed for 1 day, 2 days, 3 days, 4, days, 5 days, 6 days, 7 days, 8 days, 9 days or 10 days after initiation of the rapid second expansion; (f) harvesting the third population of TILs; and (g) optionally genetically modifying the population of TILs at any time prior to the harvesting step (f) such that the third population of TILs comprises genetically modified TILs comprising a genetic modification that reduces or increases the expression of one or more T cell exhaustion markers and / or one or more factors phenotypically associated with said T cell exhaustion markers.

14. A method of expanding tumor infiltrating lymphocytes (TILs) into a therapeutic population of TILs, the method comprising the steps of: a) resecting a tumor from the cancer in the subject or patient, the tumor comprising a first population of TILs, optionally from surgical resection, needle biopsy, core biopsy, small biopsy, or other means for obtaining a sample that contains a mixture of tumor and TIL cells from the cancer; (b) fragmenting the tumor into tumor fragments or into a tumor digest; (c) selecting, from the first population of TILs in (a), a population of TILs that expresses at least CD39 and CD103; (c) contacting the tumor fragments with a first cell culture medium;(d) performing an initial expansion (or priming first expansion) of the population of TILs in the first cell culture medium to obtain a second population of TILs, wherein the first cell culture medium comprises IL-2, optionally OKT-3 (anti-CD3 antibody), and optionally antigen presenting cells (APCs), where the priming first expansion occurs for a period of 1 to 8 days; (e) performing a rapid second expansion of the second population of TILs in a second cell culture medium to obtain a third population of TILs; wherein the second cell culture medium comprises IL-2, OKT-3 (anti-CD3 antibody), and APCs; and wherein the rapid expansion is performed over a period of 14 days or less, optionally the rapid second expansion can proceed for 1 day, 2 days, 3 days, 4, days, 5 days, 6 days, 7 days, 8 days, 9 days or 10 days after initiation of the rapid second expansion; (f) harvesting the third population of TILs; and (g) optionally genetically modifying the population of TILs at any time prior to the harvesting (f) such that the harvested third population of TILs comprises genetically modified TILs comprising a genetic modification that reduces or increases the expression of one or more T cell exhaustion markers and / or one or more factors phenotypically associated with said T cell exhaustion markers.

15. A method for expanding tumor infiltrating lymphocytes (TILs) into a therapeutic population of TILs comprising: (a) obtaining and / or receiving a first population of TILs from a tumor resected from a cancer in a subject by processing a tumor sample obtained from the tumor into multiple tumor fragments or processing a tumor sample obtained from the subject into a tumor digest; (b) selecting, from the first population of TILs in (a), a population of TILs that expresses at least CD39 and CD103; (c) performing a priming first expansion by culturing the population of TILs in a cell culture medium comprising IL-2, optionally OKT-3, and optionally comprising antigen presenting cells (APCs), to produce a second population of TILs, wherein the priming first expansion is performed for a first period of about 1 to 11 days to obtain the second population of TILs, wherein the second population of TILs is greater in number than the first population of TILs; (d) performing a rapid second expansion by contacting the second population of TILs with a cell culture medium comprising IL-2, OKT-3, and APCs, to produce a third population of TILs,wherein the rapid second expansion is performed for a second period of about 1 to 11 days to obtain the third population of TILs, wherein the third population of TILs is a therapeutic population of TILs; (e) harvesting the therapeutic population of TILs obtained from step (c); and (g) optionally genetically modifying the population of TILs at any time prior to the harvesting step (e) such that the third population of TILs comprises genetically modified TILs comprising a genetic modification that reduces or increases the expression of one or more T cell exhaustion markers and / or one or more factors phenotypically associated with said T cell exhaustion markers.

16. The method of claim 15, wherein in step (c) the cell culture medium further comprises antigen- presenting cells (APCs), and wherein the number of APCs in the culture medium in step (d) is greater than the number of APCs in the culture medium in step (c).

17. A method for expanding tumor infiltrating lymphocytes (TILs) into a therapeutic population of TILs comprising: (a) obtaining and / or receiving a first population of TILs from surgical resection, needle biopsy, core biopsy, small biopsy, or other means for obtaining a sample that contains a mixture of tumor and TIL cells from a cancer in a patient or subject, (b) selecting, from the first population of TILs in (a), a population of TILs that expresses at least CD39 and CD103; (c) performing a priming first expansion by culturing the TILs population in a first cell culture medium comprising IL-2, OKT-3, and antigen presenting cells (APCs) to produce a second population of TILs, wherein the priming first expansion is performed in a container comprising a first gas-permeable surface area, wherein the priming first expansion is performed for first period of about 1 to 11 days to obtain the second population of TILs, wherein the second population of TILs is greater in number than the first population of TILs; (d) optionally restimulating the second population of TILs with OKT-3; (e) genetically modifying the second population of TILs to produce a modified second population of TILs, wherein the modified second population of TILs comprises a genetic modification that reduces or increases the expression of one or more T cell exhaustion markers and / orone or more factors phenotypically associated with said T cell exhaustion markers; (f) performing a rapid second expansion by culturing the modified second population of TILs in a second culture medium comprising IL-2, OKT-3, and APCs, to produce a third population of TILs, wherein the rapid second expansion is performed for a second period of about 14 days or less to obtain the therapeutic population of TILs, wherein the third population of TILs is a therapeutic population of TILs comprises the genetic modification that reduces or increases the expression of the one or more T cell exhaustion markers and / or the one or more factors phenotypically associated with said T cell exhaustion markers; and (g) harvesting the third population of TILs.

18. The method of any one of claims 1-17, wherein the cancer is selected from the group consisting of melanoma, ovarian cancer, cervical cancer, non-small-cell lung cancer (NSCLC), lung cancer, bladder cancer, breast cancer, triple negative breast cancer, cancer caused by human papilloma virus, head and neck cancer (including head and neck squamous cell carcinoma (HNSCC)), renal cancer, and renal cell carcinoma.

19. A method for expanding tumor infiltrating lymphocytes (TILs) into a therapeutic population of TILs comprising: (a) performing a priming first expansion by culturing a first population of TILs in a cell culture medium comprising IL-2, optionally OKT-3, wherein the population TILs expresses at least CD39 and CD103; and optionally, wherein the TILs comprise antigen presenting cells (APCs), to produce a second population of TILs, wherein the priming first expansion is performed for a first period of about 1 to 11 days to obtain the second population of TILs, wherein the second population of TILs is greater in number than the first population of TILs; (b) performing a rapid second expansion by contacting the second population of TILs with a cell culture medium comprising IL-2, OKT-3, and APCs, to produce a third population of TILs, wherein the rapid second expansion is performed for a second period of about 1 to 11 days to obtain the third population of TILs, wherein the third population of TILs is a therapeutic population of TILs;(c) harvesting the third population of TILs obtained from step (b); and (d) genetically modifying the population of TILs, the second population of TILs and / or the third population of TILs at any time prior to the harvesting step (c) such that the harvested third population of TILs comprises genetically modified TILs comprising a genetic modification that reduces or increases the expression of one or more T cell exhaustion markers and / or one or more factors phenotypically associated with said T cell exhaustion markers.

20. The method of claim 19, wherein in step (a) the cell culture medium further comprises antigen- presenting cells (APCs), and wherein the number of APCs in the culture medium in step (c) is greater than the number of APCs in the culture medium in step (b).

21. A method of expanding T cells comprising: (a) performing a priming first expansion of a first population of TILs obtained from a donor by culturing the first population of TILs to effect growth and to prime an activation of the first population of T cells, wherein the first population of TILs is a population of TILs, wherein the population of TILs expresses at least CD39 and CD103; (b) after the activation of the first population of TILs primed in step (a) begins to decay, performing a rapid second expansion of the first population of TILs by culturing the population of first population of TILs to effect growth and to boost the activation of the first population of T cells to obtain a second population of T cells; (c) harvesting the second population of T cells; and (d) genetically modifying the first population of TILs and / or the second population of TILs such that the harvested second population of TILs comprises genetically modified TILs comprising a genetic modification that reduces or increases the expression of one or more T cell exhaustion markers and / or one or more factors phenotypically associated with said T cell exhaustion markers.

22. A method of expanding T cells comprising: (a) performing a priming first expansion of a first population of T cells from a tumor sample obtained from one or more small biopsies, core biopsies, or needle biopsies of atumor in a donor by culturing the first population of T cells to effect growth and to prime an activation of the first population of T cells, wherein the first population of T cells is a population of T cells that expresses at least CD39 and CD103; (b) after the activation of the first population of T cells primed in step (a) begins to decay, performing a rapid second expansion of the first population of T cells by culturing the first population of T cells to effect growth and to boost the activation of the first population of T cells to obtain a second population of T cells; and (c) harvesting the second population of T cells; and (d) genetically modifying the first population of T cells and / or the second population of TILs such that the harvested second population of T cells comprises genetically modified TILs comprising a genetic modification that reduces or increases the expression of one or more T cell exhaustion markers and / or one or more factors phenotypically associated with said T cell exhaustion markers.

23. The method of any one of claims 1-22, wherein the one or more T cell exhaustion markers is selected from PD-1 and CD39, and a combination thereof.

24. The method of any one of claims 1-23, wherein the one or more factors phenotypically associated with said T cell exhaustion markers is CD103.

25. The method according to any one of claims 1-24, further comprising an additional step comprising: selecting, from the population of TILs, a population of TILs, that expresses at least one protein selected from the group consisting of: OX40, 4-1BB, and combinations thereof.

26. The method of claim 25, wherein the additional selecting step occurs before the first expansion.

27. The method of claim 25, wherein the additional selecting step occurs before the priming first expansion.

28. The method of claim 25, wherein the additional selecting step occurs during the priming first expansion.

29. The method of any one of claims 25-28, wherein the additional selecting step occurs during Day 0, Day 1, Day 2, Day 3, Day 4, and / or Day 5 or the the priming first expansion, wherein no OKT-3 has yet been added.

30. The method of any one of claims 1-15, 17, or 19, further comprising a second selecting, from the second population of TILs, a population of TILs that expresses at least one protein selected from the group consisting of: OX40, 4-1BB and combinations thereof.

31. The method of any one of claims 1-4, 9-13, or 16, wherein the modifying is carried out on the second population of TILs from the first expansion, or the third population of TILs from the second expansion, or both.

32. The method of any one of claims 5, 6, 8, 14-16, or 18-20, wherein the modifying is carried out on the second population of TILs from the priming first expansion, or the third population of TILs from the rapid second expansion, or both.

33. The method of any one of claims 1-4, 9-13, or 16, wherein the modifying is carried out on the second population of TILs from the first expansion and before the second expansion.

34. The method of any one of claims 5, 6, 8, 14-16, or 18-20, wherein the modifying is carried out on the second population of TILs from the priming first expansion and before the rapid second expansion, or both.

35. The method of any one of claims 1-4, 9-13, or 16, wherein the modifying is carried out on the third population of TILs from the second expansion.

36. The method of any one of claims 5, 6, 8, 14-16, or 18-20, wherein the modifying is carried out on the third population of TILs from the rapid second expansion.

37. The method of any one of claims 1-6, 8-16, or 18-22, wherein the modifying is carried out after the harvesting.

38. The method of any one of claims 1-4, 9-13, or 16, wherein the first expansion is performed over a period of about 11 days.

39. The method of any one of claims 5-8 or 14-22, wherein the priming first expansion is performed over a period of about 11 days.

40. The method of any one of claims 1-4, 9-13, or 16, wherein the IL-2 is present at an initial concentration of between 1000 IU / mL and 6000 IU / mL in the cell culture medium in the first expansion.

41. The method of any one of claims 5-8 or 14-22, wherein the IL-2 is present at an initial concentration of between 1000 IU / mL and 6000 IU / mL in the cell culture medium in the priming first expansion.

42. The method of any one of claims 1-4, 9-13, or 16, wherein in the second expansion step, the IL-2 is present at an initial concentration of between 1000 IU / mL and 6000 IU / mL and the OKT-3 antibody is present at an initial concentration of about 30 ng / mL.

43. The method of any one of claims 5-8 or 14-22, wherein in the rapid second expansion step, the IL-2 is present at an initial concentration of between 1000 IU / mL and 6000 IU / mL and the OKT-3 antibody is present at an initial concentration of about 30 ng / mL.

44. The method of any one of claims 1-4, 9-13, or 16, wherein the first expansion is performed using a gas permeable container.

45. The method of any one of claims 5-8 or 14-22, wherein the priming first expansion is performed using a gas permeable container.

46. The method of any one of claims 1-4, 9-13, or 16, wherein the second expansion is performed using a gas permeable container.

47. The method of any one of claims 5-8 or 14-22, wherein the rapid second expansion is performed using a gas permeable container.

48. The method of any one of claims 1-4, 9-13, or 16, wherein the cell culture medium of the first expansion further comprises a cytokine selected from the group consisting of IL-4, IL-7, IL-15, IL- 21, and combinations thereof.

49. The method of of any one of claims 5-8 or 14-22, wherein the cell culture medium of the priming first expansion further comprises a cytokine selected from the group consisting of IL-4, IL-7, IL- 15, IL-21, and combinations thereof.

50. The method of any one of claims 1-4, 9-13, or 16, wherein the cell culture medium of the second expansion further comprises a cytokine selected from the group consisting of IL-4, IL-7, IL-15, IL- 21, and combinations thereof.

51. The method of any one of claims 5-8 or 14-22, wherein the cell culture medium of the rapid second expansion further comprises a cytokine selected from the group consisting of IL-4, IL-7, IL-15, IL-21, and combinations thereof.

52. The method of any one of claims 1-7, further comprising the step of treating the patient with anon-myeloablative lymphodepletion regimen prior to administering the third population of TILs to the patient.

53. The method of claim 52, wherein the non-myeloablative lymphodepletion regimen comprises the steps of administration of cyclophosphamide at a dose of 60 mg / m2 / day for two days followed by administration of fludarabine at a dose of 25 mg / m2 / day for three days.

54. The method of claim 52, wherein the non-myeloablative lymphodepletion regimen comprises the steps of administration of cyclophosphamide at a dose of 60 mg / m2 / day and fludarabine at a dose of 25 mg / m2 / day for two days followed by administration of fludarabine at a dose of 25 mg / m2 / day for three days.

55. The method of claim 52, wherein the non-myeloablative lymphodepletion regimen comprises the steps of administration of cyclophosphamide at a dose of 60 mg / m2 / day and fludarabine at a dose of 25 mg / m2 / day for two days followed by administration of fludarabine at a dose of 25 mg / m2 / day for one day.

56. The method of any one of claims 53-55, wherein the cyclophosphamide is administered with mesna.

57. The method of any one of claims 1-7 or 52-56, further comprising the step of treating the patient with an IL-2 regimen starting on the day after the administration of TILs to the patient.

58. The method of any one of claims 1-7 or 52-56, further comprising the step of treating the patient with an IL-2 regimen starting on the same day as administration of TILs to the patient.

59. The method of claim 57 or 58, wherein the IL-2 regimen is a high-dose IL-2 regimen comprising 600,000 or 720,000 IU / kg of aldesleukin, or a biosimilar or variant thereof, administered as a 15- minute bolus intravenous infusion every eight hours until tolerance.

60. The method of any one of claims 1-7 or 52-59, wherein a therapeutically effective population of TILs is administered and comprises from about 2.3×1010to about 13.7×1010TILs.

61. The method of any one of claims 5-8 or 14-22, wherein the priming first expansion and rapid second expansion are performed over a period of 21 days or less.

62. The method of any one of claims 5-8 or 14-22, wherein the priming first expansion and rapid second expansion are performed over a period of 16 or 17 days or less.

63. The method of any one of claims 5-8 or 14-22, wherein the priming first expansion is performed over a period of 7 or 8 days or less.

64. The method of any one of claims 5-8 or 14-22, wherein the rapid second expansion is performed over a period of 11 days or less.

65. The method of any one of claims 1-4, 9-13, or 16, the first expansion and the second expansion are each individually performed within a period of 11 days.

66. The method of claim 7 or 17, wherein step (a) through step (f) is performed within about 26 days.

67. The method according to any one of claims 1-66, wherein the genetically modified TILs further comprises an additional genetic modification that reduces or increases expression of one or more of the following immune checkpoint genes selected from the group comprising CTLA-4, Cish, TGFβ, PKA, CBL-B, PPP2CA, PPP2CB, PTPN6, PTPN22, PDCD1, BTLA, CD160, TIGIT, CD96, CRTAM, LAIR1, SIGLEC7, SIGLEC9, CD244, TNFRSF10B, TNFRSF10A, CASP8, CASP10, CASP3, CASP6, CASP7, FADD, FAS, SMAD2, SMAD3, SMAD4, SMAD10, SKI, SKIL, TGIF1, IL10RA, IL10RB, HMOX2, IL6R, IL6ST, EIF2AK4, CSK, PAG1, SIT1, FOXP3, PRDM1, BATF, GUCY1A2, GUCY1A3, GUCY1B2, GUCY1B3, and TOX.

68. The method according to claim 67, wherein the one or more immune checkpoint genes is / are selected from the group comprising PD-1, CTLA-4, LAG-3, HAVCR2 (TIM-3), Cish, TGFβ, and PKA.

69. The method according to any of claims 1-68, wherein the genetically modified TILs further comprises an additional genetic modification that causes expression of one or more immune checkpoint genes to be enhanced in at least a portion of the therapeutic population of TILs, the immune checkpoint gene(s) being selected from the group comprising CCR2, CCR4, CXCR2, CX3CR1, IL-2, IL-4, IL-7, IL-10, IL-15, IL-21, the NOTCH 1 / 2 intracellular domain (ICD), and / or the NOTCH ligand mDLL1.

70. The method according to any of claims 1-69, wherein the genetically modifying step is performed using a programmable nuclease that mediates the generation of a double-strand or single-strand break at said one or more immune checkpoint genes.

71. The method according to any of claims 1-70, wherein the genetically modifying is performed using one or more methods selected from a CRISPR method, a TALE method, a zinc finger method, and a combination thereof.

72. The method of claim 71, wherein the method comprises a CRISPR method.

73. The method of claim 72, wherein the CRISPR method is a CRISPR / Cas9 method.

74. The method of claim 71, wherein the genetically modifying comprises a TALE method.

75. The method of claim 71, wherein the genetically modifying comprises a zinc finger method.

76. The methods according to any of claims 1-75, wherein processing a tumor sample obtained from the subject into a tumor digest comprises incubating the tumor sample in an enzymatic media.

77. The methods according to any of claims 1-75, wherein processing a tumor sample obtained from the subject into a tumor digest further comprises disrupting the tumor sample mechanically so as to dissociate the tumor sample.

78. The methods according to any of claims 1-75, wherein processing a tumor sample obtained from the subject into a tumor digest further comprises purifying the disassociated tumor sample using a density gradient separation.

79. The method of claim 76, wherein the enzymatic media comprises DNase.

80. The method of claim 76 or 79, wherein the enzymatic media comprises 30 units / mL of DNase.

81. The method according to any of claims 76 or 79-80, wherein the enzymatic media comprises collagenase.

82. The method according to any of claims 76 or 79-81, wherein the enzymatic media comprises 1.0 mg / mL of collagenase.

83. The method according to any of claims 1-82, wherein the therapeutic population of TILs harvested comprises sufficient TILs for use in administering a therapeutically effective dosage to a subject.

84. The method according to any of claims 1-83, wherein the therapeutically effective dosage comprises from about 1×109to about 9×1010TILs.

85. The method according to any of claims 1-83, wherein the therapeutically effective population of TILs is administered and comprises from about 2.3×1010to about 13.7×1010TILs.

86. The method according to any of claims 1-85, wherein the APCs comprise peripheral blood mononuclear cells (PBMCs).

87. The method according to any of claims 1-86, wherein the therapeutic population of TILs harvested in step (e) exhibits an increased subpopulation of CD8+ cells relative to the first and / or second population of TILs.

88. The method according to any of claims 1-87, wherein the PBMCs are supplemented at a ratio of about 1:25 TIL:PBMCs.

89. The method according to any of claims 1-88, wherein the first expansion and the second expansion are each individually performed within a period of 11-12 days.

90. The method according to any of claims 1-89, wherein steps (a) through (e), (f), or (g) are performed in about 10 days to about 24 days.

91. The method according to any of claims 1-90, wherein steps (a) through (e), (f), or (g) are performed in about 15 days to about 24 days.

92. The method according to any of claims 1-91, wherein steps (a) through (e), (f), or (g)are performed in about 20 days to about 24 days.

93. The method according to any of claims 1-92, wherein steps (a) through (e), (f), or (g) are performed in about 20 days to about 22 days.

94. The method according to any of claims 1-93, wherein the second population of TILs is at least 50- fold greater in number than the first population of TILs.

95. A method of treating a cancer in a patient or subject in need thereof comprising administering a population of tumor infiltrating lymphocytes (TILs), the method comprising the steps of: (a) selecting, from the first population of TILs, obtained and / or received from surgical resection, needle biopsy, core biopsy, small biopsy, or other means for obtaining a sample that contains a mixture of tumor and TIL cells from the cancer in the patient or subject, a population of TILs that expresses at least one protein selected from the group consisting of: CD39, CD103, and combinations thereof; (b) further selecting, from the population ofTILs, a population of TILs that expresses at least one protein selected from the group consisting of: OX40, 4-1BB, and combinations thereof; (c) optionally adding the population of TILs into a closed system; (d) performing a first expansion by culturing the population of TILs in a cell culture medium comprising IL-2 to produce a second population of TILs, wherein the first expansion is optionally performed in a closed container providing a first gas-permeable surface area, wherein the first expansion is performed for about 3-11 days to obtain the second population of TILs, and wherein the transition from step (c) to step (d) optionally occurs without opening the system; and (e) performing a second expansion by supplementing the cell culture medium of the second population of TILs with additional IL-2, OKT-3, and antigen presenting cells (APCs), to produce a third population of TILs, wherein the second expansion is performed for about 7- 11 days to obtain the third population of TILs, wherein the second expansion is optionally performed in a closed container providing a second gas-permeable surface area, and wherein the transition from step (d) to step (e) optionally occurs without opening the system.

96. A method for expanding tumor infiltrating lymphocytes (TILs) into a therapeutic population of TILs comprising: (a) obtaining and / or receiving a first population of TILs from a tumor resected from a cancer in a subject by processing a tumor sample obtained from the tumor into multiple tumor fragments or processing a tumor sample obtained from the subject into a tumor digest; (b) selecting, optionally within a closed system, from the first population of TILs in (a), a population of TILs that expresses at least one protein selected from the group consisting of: CD39, CD103, and combinations thereof; (c) further selecting, from the population of TILs, a population of TILs that expresses at least one protein selected from the group consisting of: OX40, 4-1BB, and combinations thereof; (d) performing a priming first expansion by culturing the TILs population in a first cell culture medium comprising IL-2, OKT-3, and antigen presenting cells (APCs) to produce a second population of TILs, wherein the priming first expansion is performed in a container comprising a first gas-permeable surface area, wherein the priming first expansion is performed for first period of about 1 to 7 / 8 days to obtain the second population of TILs, wherein the second population of TILs is greater in number than the first population of TILs; and (e) performing a rapid second expansion by culturing the second population of TILs in a second culture medium comprising IL-2, OKT-3, and APCs, to produce a third population of TILs, wherein the number of APCs added in the rapid second expansion is at least twice the number of APCs added in step (b), wherein the rapid second expansion is performed for a second period of about 1 to 11 days to obtain the therapeutic population of TILs, wherein the third population of TILs is a therapeutic population of TILs, wherein the rapid second expansion is performed in a container comprising a second gas-permeable surface area.

97. The method of claims 95 or 96, further comprising: (f) harvesting the third population of TILs obtained from step (e), wherein the transition from step (e) to step (f) optionally occurs without opening the system; (g) transferring the harvested third TILs population from step (f) to an infusion bag, wherein the transfer from step (e) to (f) optionally occurs without opening the system;(h) cryopreserving the infusion bag comprising the harvested TILs population from step (g) using a cryopreservation process; (i) administering a therapeutically effective dosage of the third population of TILs from the infusion bag in step (h) to the subject.; and (j) optionally genetically modifying the population of TILs at any time prior to the administering step (i) such that the administered third population of TILs comprises genetically modified TILs comprising a genetic modification that reduces or increases the expression of one or more T cell exhaustion markers and / or one or more factors phenotypically associated with said T cell exhaustion markers.

98. The method of any of claims 95-97, further comprising the methods according to any of claims 1- 94.

99. A population of TILs according to any of the methods of claims 1-98.

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