Cytokine-associated tumor infiltrating lymphocyte compositions and methods

Abstract

Provided herein are compositions and methods for treating cancer using modified TILs, the modified TILs comprising one or more immunomodulatory agents (e.g., cytokines) associated with their cell surface. The immunomodulatory agents associated with the TILs provide a local immunostimulatory effect that can advantageously enhance TIL survival, proliferation, and / or antitumor activity in a patient recipient. Thus, the compositions and methods disclosed herein provide effective cancer therapy.

Description

[Technical Field]

[0001] CROSS-REFERENCE TO RELATED APPLICATIONS This application claims priority to U.S. Provisional Application No. 63 / 304,498, filed January 28, 2022, U.S. Provisional Application No. 63 / 356,933, filed June 29, 2022, U.S. Provisional Application No. 63 / 394,267, filed August 1, 2022, U.S. Provisional Application No. 63 / 382,493, filed November 4, 2022, and U.S. Provisional Application No. 63 / 429,114, filed November 30, 2022, the disclosures of which are incorporated herein by reference in their entireties. [Background technology]

[0002] Adoptive cell therapy using TILs cultured ex vivo by rapid expansion protocol (REP) has been successful in cancer patients after host immunosuppression. However, in some cases, the survival and antitumor activity of transferred TILs may be reduced after transfer to patients.

[0003] The administration of supportive immunostimulatory agents (e.g., cytokines) has been explored to enhance T cell therapy, however, such immunostimulatory agents require high systemic doses that can result in undesirable toxicity.

[0004] Thus, there remains a need for improved TIL therapies for the treatment of cancer. Summary of the Invention

[0005] Provided herein are compositions and methods for treating cancer using modified TILs, where the modified TILs comprise one or more immunomodulatory agents (e.g., cytokines) associated with their cell surface. The immunomodulatory agents associated with the TILs provide a local immunostimulatory effect that can advantageously enhance TIL survival, proliferation, and / or antitumor activity in a patient recipient. Thus, the compositions and methods disclosed herein provide effective cancer therapies.

[0006] In one aspect, provided herein is a method of treating cancer in a patient or subject in need thereof, comprising administering a population of tumor-infiltrating lymphocytes (TILs), optionally wherein the patient or subject has received at least one prior therapy, wherein a portion of the TIL population are TILs each modified to include an immunomodulatory composition associated with its surface membrane.

[0007] In one aspect, provided herein is a method of treating cancer in a patient or subject in need thereof, comprising administering a population of tumor-infiltrating lymphocytes (TILs), the method comprising: (a) obtaining and / or receiving a first population of TILs from a tumor excised from a subject or patient by processing a tumor sample obtained from the subject into a plurality of tumor fragments; (b) adding the first TIL population to the closed system; (c) performing a first expansion by culturing the first TIL population in a cell culture medium containing IL-2 to produce a second TIL population, wherein the first expansion is performed in a sealed container that provides a first gas permeable surface area, and the first expansion is performed for about 3 to 14 days to obtain the second TIL population, and the transition from step (b) to step (c) occurs without opening the system; (d) performing a second expansion by supplementing the cell culture medium of the second TIL population with additional IL-2, OKT-3, and antigen-presenting cells (APCs) to produce a third TIL population, wherein the second expansion is performed for about 7 to 14 days to obtain the third TIL population, the third TIL population being a therapeutic TIL population, and wherein the second expansion is performed in a sealed container that provides a second gas-permeable surface area, and the transition from step (c) to step (d) occurs without opening the system. (e) harvesting the therapeutic TIL population obtained from step (d), wherein the transition from step (d) to step (e) occurs without opening the system; (f) transferring the harvested TIL population from step (e) into an infusion bag, wherein the transition from step (e) to (f) occurs without opening the system; (g) cryopreserving the infusion bag containing the harvested TIL population from step (f) using a cryopreservation process; (h) administering a therapeutically effective dose of the third population of TILs from the infusion bag of step (g) to the subject; (i) at any time before administering step (h), modifying a portion of the first, second, or third TIL populations to produce modified TILs, each of which comprises an immunomodulatory composition associated with its surface membrane.

[0008] In one aspect, provided herein is a method of treating cancer in a patient or subject in need thereof, comprising administering a population of tumor-infiltrating lymphocytes (TILs), the method comprising: (a) obtaining a first population of TILs from a tumor excised from a subject by processing a tumor sample obtained from the subject into a plurality of tumor fragments; (b) adding tumor fragments to the closed system; (c) performing a first expansion by culturing the first TIL population in a cell culture medium containing IL-2 to produce a second TIL population, wherein the first expansion is performed in a sealed container that provides a first gas permeable surface area, and the first expansion is performed for about 3 to 11 days to obtain the second TIL population, and the transition from step (b) to step (c) occurs without opening the system; (d) performing a second expansion by supplementing the cell culture medium of the second TIL population with additional IL-2, OKT-3, and antigen-presenting cells (APCs) to produce a third TIL population, wherein the second expansion is performed for about 7 to 11 days to obtain the third TIL population, and wherein the second expansion is performed in a sealed container that provides a second gas-permeable surface area, and the transition from step (c) to step (d) occurs without opening the system. (e) harvesting the third population of TILs obtained from step (d), wherein the transition from step (d) to step (e) occurs without opening the system; (f) transferring the harvested third population of TILs from step (e) to an infusion bag, wherein the transition from step (e) to (f) occurs without opening the system; (g) cryopreserving the infusion bag containing the harvested TIL population from step (f) using a cryopreservation process; (h) administering a therapeutically effective dose of the third population of TILs from the infusion bag of step (g) to the subject; (i) at any time before administering step (h), modifying a portion of the first, second, or third TIL populations to produce modified TILs, each of which comprises an immunomodulatory composition associated with its surface membrane.

[0009] In one aspect, provided herein is a method of treating cancer in a patient or subject in need thereof, comprising administering a population of tumor-infiltrating lymphocytes (TILs), the method comprising: (a) obtaining and / or receiving a first population of TILs from a surgical resection, needle biopsy, core biopsy, mini-biopsy, or other means for obtaining a sample containing a mixture of tumor cells and TIL cells from a cancer in a patient or subject; (b) adding the first TIL population to the closed system; (c) performing a first expansion by culturing the first TIL population in a cell culture medium containing IL-2 to produce a second TIL population, wherein the first expansion is performed in a sealed container that provides a first gas permeable surface area, and the first expansion is performed for about 3 to 11 days to obtain the second TIL population, and the transition from step (b) to step (c) occurs without opening the system; (d) performing a second expansion by supplementing the cell culture medium of the second TIL population with additional IL-2, OKT-3, and antigen-presenting cells (APCs) to produce a third TIL population, wherein the second expansion is performed for about 7 to 11 days to obtain the third TIL population, and wherein the second expansion is performed in a sealed container that provides a second gas-permeable surface area, and the transition from step (c) to step (d) occurs without opening the system. (e) harvesting the third population of TILs obtained from step (d), wherein the transition from step (d) to step (e) occurs without opening the system; (f) transferring the harvested third population of TILs from step (e) to an infusion bag, wherein the transition from step (e) to (f) occurs without opening the system; (g) cryopreserving the infusion bag containing the harvested TIL population from step (f) using a cryopreservation process; (h) administering a therapeutically effective dose of the third population of TILs from the infusion bag of step (g) to the subject; (i) at any time before administering step (h), modifying a portion of the first, second, or third TIL populations to produce modified TILs, each of which comprises an immunomodulatory composition associated with its surface membrane.

[0010] In another aspect, provided herein is a method of treating cancer in a patient or subject in need thereof, comprising administering a population of tumor-infiltrating lymphocytes (TILs), the method comprising: (a) resecting a tumor from a subject or patient, the tumor optionally comprising a first population of TILs from surgical resection, needle biopsy, core biopsy, mini-biopsy, or other means for obtaining a sample containing a mixture of tumor cells and TIL cells from a cancer; (b) processing the tumor into a plurality of tumor fragments and adding the tumor fragments to a closed system; (c) performing a first expansion by culturing the first TIL population in a cell culture medium containing IL-2 to produce a second TIL population, wherein the first expansion is performed in a sealed container that provides a first gas permeable surface area, and the first expansion is performed for about 3 to 11 days to obtain the second TIL population, and the transition from step (b) to step (c) occurs without opening the system; (d) performing a second expansion by supplementing the cell culture medium of the second TIL population with additional IL-2, OKT-3, and antigen-presenting cells (APCs) to produce a third TIL population, wherein the second expansion is performed for about 7 to 11 days to obtain the third TIL population, and wherein the second expansion is performed in a sealed container that provides a second gas-permeable surface area, and the transition from step (c) to step (d) occurs without opening the system. (e) harvesting the third population of TILs obtained from step (d), wherein the transition from step (d) to step (e) occurs without opening the system; (f) transferring the harvested third population of TILs from step (e) to an infusion bag, wherein the transition from step (e) to (f) occurs without opening the system; (g) cryopreserving the infusion bag containing the harvested TIL population from step (f) using a cryopreservation process; (h) administering a therapeutically effective dose of the third population of TILs from the infusion bag of step (g) to a subject or patient with cancer; (i) at any time before administering step (h), modifying a portion of the first, second, or third TIL populations to produce modified TILs, each of which comprises an immunomodulatory composition associated with its surface membrane.

[0011] In another aspect, provided herein is a method of treating cancer in a patient or subject in need thereof, comprising administering a population of tumor-infiltrating lymphocytes (TILs), the method comprising: (a) obtaining and / or receiving a first population of TILs from a surgical resection, needle biopsy, core biopsy, mini-biopsy, or other means for obtaining a sample containing a mixture of tumor cells and TIL cells from a subject or patient; (c) contacting the first TIL population with a first cell culture medium; (d) performing an initial expansion (or first expansion by priming) of the first TIL population in a first cell culture medium to obtain a second TIL population, wherein the first cell culture medium comprises IL-2, optionally OKT-3 (anti-CD3 antibody), and optionally antigen-presenting cells (APCs), and the first expansion by priming occurs over a period of 1 to 8 days; (e) performing a rapid second expansion of the second TIL population in a second cell culture medium to obtain a third TIL population, wherein the second cell culture medium comprises IL-2, OKT-3 (an anti-CD3 antibody), and APCs, and the rapid expansion is performed over a period of 14 days or less, and 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 a third population of TILs; (g) administering a therapeutically effective portion of the third population of TILs to a subject or patient with cancer; (h) any time before administering step (g), modifying a portion of the first, second, or third TIL population to generate modified TILs, each of which comprises an immunomodulatory composition associated with its surface membrane.

[0012] In another aspect, provided herein is a method of treating cancer in a patient or subject in need thereof, comprising administering a population of tumor-infiltrating lymphocytes (TILs), the method comprising: (a) resecting a tumor from a cancer in a subject or patient, the tumor optionally comprising a first TIL population from surgical resection, needle biopsy, core biopsy, mini-biopsy, or other means for obtaining a sample containing a mixture of tumor cells and TIL cells from the cancer; (b) fragmenting the tumor into tumor fragments; (c) contacting the tumor fragment with a first cell culture medium; (d) performing an initial expansion (or first expansion by priming) of the first TIL population in a first cell culture medium to obtain a second TIL population, wherein the first cell culture medium comprises IL-2, optionally OKT-3 (anti-CD3 antibody), and optionally antigen-presenting cells (APCs), and the first expansion by priming occurs over a period of 1 to 8 days; (e) performing a rapid second expansion of the second TIL population in a second cell culture medium to obtain a third TIL population, wherein the second cell culture medium comprises IL-2, OKT-3 (an anti-CD3 antibody), and APCs, and the rapid expansion is performed over a period of 14 days or less, and 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 a third population of TILs; (g) administering a therapeutically effective portion of the third population of TILs to a subject or patient with cancer; (h) any time before administering step (g), modifying a portion of the first, second, or third TIL population to generate modified TILs, each of which comprises an immunomodulatory composition associated with its surface membrane.

[0013] In one aspect, provided herein is a method for expanding tumor infiltrating lymphocytes (TILs) into a therapeutic TIL population, 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 a plurality of tumor fragments; (b) selecting PD-1 positive TILs from the first TIL population of step (a) to obtain a PD-1 enriched TIL population; (c) performing a first priming expansion of the PD-1-enriched TIL population by culturing it in a cell culture medium comprising IL-2, OKT-3, and antigen-presenting cells (APCs) to produce a second TIL population, wherein the first priming expansion is performed in a container comprising a first gas permeable surface area, and the first priming expansion is performed for a first period of about 1 to 7 / 8 days to obtain a second TIL population, wherein the second TIL population is greater in number than the first TIL population; and producing a second TIL population. (d) performing a second rapid expansion by culturing the second TIL population in a second culture medium comprising IL-2, OKT-3, and APCs to produce a therapeutic TIL population, wherein the number of APCs added to the rapid second expansion is at least twice the number of APCs added in step (b), and the rapid second expansion is performed for a second period of about 1 to 11 days to obtain a therapeutic TIL population; and producing a third TIL population, wherein the third TIL population is a therapeutic TIL population, and the rapid second expansion is performed in a container comprising a second gas permeable surface area. (e) harvesting the therapeutic TIL population obtained from step (d); (f) transferring the harvested TIL population from step (e) into an infusion bag; (g) at any point during the method, modifying a portion of the first, second, or third TIL populations to produce modified TILs, each of which comprises an immunomodulatory composition associated with its surface membrane.

[0014] Provided herein is a method for expanding tumor-infiltrating lymphocytes (TILs) into a therapeutic TIL population, the method comprising: (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 a plurality of tumor fragments; (b) adding the first TIL population to the closed system; (c) performing a first expansion by culturing the first TIL population in a cell culture medium containing IL-2 to produce a second TIL population, wherein the first expansion is performed in a sealed container that provides a first gas permeable surface area, and the first expansion is performed for about 3 to 14 days to obtain the second TIL population, and the transition from step (b) to step (c) occurs without opening the system; (d) performing a second expansion by supplementing the cell culture medium of the second TIL population with additional IL-2, OKT-3, and antigen-presenting cells (APCs) to produce a third TIL population, wherein the second expansion is performed for about 7 to 14 days to obtain the third TIL population, the third TIL population being a therapeutic TIL population, and wherein the second expansion is performed in a sealed container that provides a second gas-permeable surface area, and the transition from step (c) to step (d) occurs without opening the system. (e) harvesting the therapeutic TIL population obtained from step (d), wherein the transition from step (d) to step (e) occurs without opening the system; (f) transferring the harvested TIL population from step (e) into an infusion bag, wherein the transition from step (e) to (f) occurs without opening the system; (g) at any time prior to transfer to the infusion bag in step (f), modifying a portion of the first, second, or third TIL population to produce modified TILs, each of which comprises an immunomodulatory composition associated with its surface membrane.

[0015] Provided herein is a method for expanding tumor-infiltrating lymphocytes (TILs) into a therapeutic TIL population, the method comprising: (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 a plurality of tumor fragments; (b) adding tumor fragments to the closed system; (c) performing a first expansion by culturing the first TIL population in a cell culture medium containing IL-2 to produce a second TIL population, wherein the first expansion is performed in a sealed container that provides a first gas permeable surface area, and the first expansion is performed for about 3 to 11 days to obtain the second TIL population, and the transition from step (b) to step (c) occurs without opening the system; (d) performing a second expansion by supplementing the cell culture medium of the second TIL population with additional IL-2, OKT-3, and antigen-presenting cells (APCs) to produce a third TIL population, wherein the second expansion is performed for about 7 to 11 days to obtain the third TIL population, and wherein the second expansion is performed in a sealed container that provides a second gas-permeable surface area, and the transition from step (c) to step (d) occurs without opening the system. (e) harvesting the third population of TILs obtained from step (d), wherein the transition from step (d) to step (e) occurs without opening the system; (f) transferring the harvested third population of TILs from step (e) to an infusion bag, wherein the transition from step (e) to (f) occurs without opening the system; (g) at any time prior to transfer to the infusion bag in step (f), modifying a portion of the first, second, or third TIL population to produce modified TILs, each of which comprises an immunomodulatory composition associated with its surface membrane.

[0016] In another aspect, provided herein is a method of expanding tumor infiltrating lymphocytes (TILs) into a therapeutic TIL population, the method comprising: (a) obtaining and / or receiving a first population of TILs from a surgical resection, needle biopsy, core biopsy, mini-biopsy, or other means for obtaining a sample containing a mixture of tumor cells and TIL cells from a cancer in a patient or subject; (b) adding the first TIL population to the closed system; (c) performing a first expansion by culturing the first TIL population in a cell culture medium containing IL-2 to produce a second TIL population, wherein the first expansion is performed in a sealed container that provides a first gas permeable surface area, and the first expansion is performed for about 3 to 11 days to obtain the second TIL population, and the transition from step (b) to step (c) occurs without opening the system; (d) performing a second expansion by supplementing the cell culture medium of the second TIL population with additional IL-2, OKT-3, and antigen-presenting cells (APCs) to produce a third TIL population, wherein the second expansion is performed for about 7 to 11 days to obtain the third TIL population, and wherein the second expansion is performed in a sealed container that provides a second gas-permeable surface area, and the transition from step (c) to step (d) occurs without opening the system. (e) harvesting the third population of TILs obtained from step (d), wherein the transition from step (d) to step (e) occurs without opening the system; (f) transferring the harvested third population of TILs from step (e) to an infusion bag, wherein the transition from step (e) to (f) occurs without opening the system; (g) at any time prior to transfer to the infusion bag in step (f), modifying a portion of the first, second, or third TIL population to produce modified TILs, each of which comprises an immunomodulatory composition associated with its surface membrane.

[0017] In one aspect, provided herein is a method of expanding tumor infiltrating lymphocytes (TILs) into a therapeutic TIL population, the method comprising: (a) resecting a tumor from a cancer in a subject or patient, the tumor optionally comprising a first TIL population from surgical resection, needle biopsy, core biopsy, mini-biopsy, or other means for obtaining a sample containing a mixture of tumor cells and TIL cells from the cancer; (b) adding tumor fragments to the closed system; (c) performing a first expansion by culturing the first TIL population in a cell culture medium containing IL-2 to produce a second TIL population, wherein the first expansion is performed in a sealed container that provides a first gas permeable surface area, and the first expansion is performed for about 3 to 11 days to obtain the second TIL population, and the transition from step (b) to step (c) occurs without opening the system; (d) performing a second expansion by supplementing the cell culture medium of the second TIL population with additional IL-2, OKT-3, and antigen-presenting cells (APCs) to produce a third TIL population, wherein the second expansion is performed for about 7 to 11 days to obtain the third TIL population, and wherein the second expansion is performed in a sealed container that provides a second gas-permeable surface area, and the transition from step (c) to step (d) occurs without opening the system. (e) harvesting the third population of TILs obtained from step (d), wherein the transition from step (d) to step (e) occurs without opening the system; (f) transferring the harvested third population of TILs from step (e) to an infusion bag, wherein the transition from step (e) to (f) occurs without opening the system; (g) at any time prior to transfer to the infusion bag in step (f), modifying a portion of the first, second, or third TIL population to produce modified TILs, each of which comprises an immunomodulatory composition associated with its surface membrane.

[0018] In some embodiments of the methods provided herein, the first expansion is divided into a first step and a second step, and the method further includes performing the first step of the first expansion by culturing the first TIL population in cell culture medium containing IL-2 to produce TILs emerging from the tumor fragment or sample, separating TILs remaining in the tumor fragment or sample from TILs emerging from the tumor fragment or sample, and optionally digesting the tumor fragment or sample to produce a tumor digest, and performing the second step of the first expansion by culturing the TILs remaining in the tumor fragment or sample or tumor digest in cell culture medium to produce a second TIL population.

[0019] In one aspect, provided herein is a method of expanding tumor infiltrating lymphocytes (TILs) into a therapeutic TIL population, the method comprising: (a) obtaining and / or receiving a first population of TILs from a surgical resection, needle biopsy, core biopsy, mini-biopsy, or other means for obtaining a sample containing a mixture of tumor cells and TIL cells from a subject or patient; (b) contacting the first TIL population with a first cell culture medium; (c) performing an initial expansion (or first expansion by priming) of the first TIL population in a first cell culture medium to obtain a second TIL population, wherein the first cell culture medium comprises IL-2, optionally OKT-3 (anti-CD3 antibody), and optionally antigen-presenting cells (APCs), and the first expansion by priming occurs over a period of 1 to 8 days; (d) performing a rapid second expansion of the second TIL population in a second cell culture medium to obtain a third TIL population, wherein the second cell culture medium comprises IL-2, OKT-3 (an anti-CD3 antibody), and APCs, and the rapid expansion is performed over a period of 14 days or less, and 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 a third population of TILs; (f) modifying a portion of the first, second, or third TIL populations at any time before or after collection in step (f) to produce modified TILs, each of which comprises an immunomodulatory composition associated with its surface membrane.

[0020] In one aspect, provided herein is a method of expanding tumor infiltrating lymphocytes (TILs) into a therapeutic TIL population, the method comprising: (a) resecting a tumor in a subject or patient, the tumor optionally comprising a first population of TILs from surgical resection, needle biopsy, core biopsy, mini-biopsy, or other means for obtaining a sample of the tumor containing a mixture of tumor cells and TIL cells; (b) fragmenting the tumor into tumor fragments; (c) contacting the tumor fragment with a first cell culture medium; (d) performing an initial expansion (or first expansion by priming) of the first TIL population in a first cell culture medium to obtain a second TIL population, wherein the first cell culture medium comprises IL-2, optionally OKT-3 (anti-CD3 antibody), and optionally antigen-presenting cells (APCs), and the first expansion by priming occurs over a period of 1 to 8 days; (e) performing a rapid second expansion of the second TIL population in a second cell culture medium to obtain a third TIL population, wherein the second cell culture medium comprises IL-2, OKT-3 (an anti-CD3 antibody), and APCs, and the rapid expansion is performed over a period of 14 days or less, and 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 a third population of TILs; (g) modifying a portion of the first, second, or third TIL populations at any time before or after collection in step (f) to produce modified TILs, each of which comprises an immunomodulatory composition associated with its surface membrane.

[0021] In one aspect, provided herein is a method for expanding tumor infiltrating lymphocytes (TILs) into a therapeutic TIL population, 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 a plurality of tumor fragments; (b) performing a first priming expansion by culturing the first TIL population in a cell culture medium comprising IL-2, optionally OKT-3, and optionally antigen-presenting cells (APCs) to produce a second TIL population, wherein the first priming expansion is performed for a first period of about 1 to 7 / 8 days to obtain the second TIL population, and producing a second TIL population, wherein the second TIL population is greater in number than the first TIL population; (c) performing a second rapid expansion of the second TIL population by contacting the second TIL population with a cell culture medium comprising IL-2, OKT-3, and APC to produce a third TIL population, wherein the second rapid expansion is performed for a second period of about 1 to 11 days to obtain a third TIL population, wherein the third TIL population is a therapeutic TIL population; (d) harvesting the therapeutic TIL population obtained from step (c); (e) modifying a portion of the first, second, or third TIL populations at any time before or after collection in step (d) to produce modified TILs, each of which comprises an immunomodulatory composition associated with its surface membrane.

[0022] In some embodiments of this method, in step (b), the cell culture medium further contains antigen-presenting cells (APCs), and 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).

[0023] In one aspect, provided herein is a method of expanding tumor infiltrating lymphocytes (TILs) into a therapeutic TIL population, comprising: (a) performing a first priming expansion by culturing a first TIL population, which can be obtained by processing a tumor sample from a tumor resected from a cancer in a subject into multiple tumor fragments in a cell culture medium comprising IL-2, optionally OKT-3, and optionally antigen-presenting cells (APCs), to produce a second TIL population, wherein the first priming expansion is performed in a container comprising a first gas-permeable surface area, and the first priming expansion is performed for a first period of about 1 to 7 / 8 days to obtain the second TIL population, and producing a second TIL population, wherein the second TIL population is greater in number than the first TIL population; (b) performing a second rapid expansion by contacting the second TIL population with a cell culture medium of the second TIL population comprising additional IL-2, OKT-3, and APCs to produce a third TIL population, wherein the number of APCs in the second rapid expansion is at least twice the number of APCs in step (a), and the rapid second expansion is performed for a second period of about 1 to 11 days to obtain a third TIL population, wherein the third TIL population is a therapeutic TIL population, and the rapid second expansion is performed in a container comprising a second gas permeable surface area; (c) harvesting the therapeutic TIL population obtained from step (b); (d) modifying a portion of the first, second, or third TIL populations at any time before or after collection in step (c) to produce modified TILs, each of which comprises an immunomodulatory composition associated with its surface membrane.

[0024] In one aspect, provided herein is a method for expanding tumor infiltrating lymphocytes (TILs) into a therapeutic TIL population, comprising: (a) performing a first priming expansion by culturing a first TIL population in a cell culture medium comprising IL-2, optionally OKT-3, and optionally antigen-presenting cells (APCs) to produce a second TIL population, wherein the first priming expansion is performed for a first period of about 1 to 7 / 8 days to obtain the second TIL population, and producing a second TIL population, wherein the second TIL population is greater in number than the first TIL population; (b) performing a second rapid expansion of the second TIL population by contacting the second TIL population with a cell culture medium comprising IL-2, OKT-3, and APC to produce a third TIL population, wherein the second rapid expansion is performed for a second period of about 1 to 11 days to obtain a third TIL population, wherein the third TIL population is a therapeutic TIL population; (c) harvesting the therapeutic TIL population obtained from step (b); (d) modifying a portion of the first, second, or third TIL populations at any time before or after collection in step (c) to produce modified TILs, each of which comprises an immunomodulatory composition associated with its surface membrane.

[0025] In some embodiments of this method, in step (a), the cell culture medium further contains antigen-presenting cells (APCs), and 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).

[0026] In some embodiments of the methods provided herein, the priming first expansion is divided into a first step and a second step, and the method further includes performing the first step of priming first expansion by culturing a first TIL population in cell culture medium containing IL-2 to produce TILs that are shed from the tumor fragment or sample, separating TILs remaining in the tumor fragment or sample from TILs that are shed from the tumor fragment or sample, and optionally digesting the tumor fragment or sample to produce a tumor digest, and performing the second step of priming first expansion in cell culture medium with TILs remaining in the tumor fragment or sample or tumor digest to produce a second population of TILs.

[0027] In one aspect, provided herein is a method for expanding tumor infiltrating lymphocytes (TILs) into a therapeutic TIL population, comprising: (a) obtaining and / or receiving a first population of TILs from a tumor sample obtained from one or more mini-biopsies, core biopsies, or needle biopsies of a tumor from a cancer in a subject by culturing the tumor sample in a first cell culture medium containing IL-2 for about 3 days; (b) performing a first priming expansion by culturing the first TIL population in a second cell culture medium comprising IL-2, OKT-3, and antigen-presenting cells (APCs) to produce a second TIL population, wherein the first priming expansion is performed in a container comprising a first gas permeable surface area, and the first priming expansion is performed for a first period of about 7 or 8 days to obtain the second TIL population, and producing a second TIL population, wherein the second TIL population is greater in number than the first TIL population; (c) performing a second rapid expansion by supplementing a second cell culture medium of the second TIL population with additional IL-2, OKT-3, and APCs to produce a third TIL population, wherein the number of APCs added to the second rapid expansion is at least twice the number of APCs added in step (b), and the second rapid expansion is performed for a second period of about 11 days to obtain a third TIL population, the third TIL population being a therapeutic TIL population, and the second rapid expansion is performed in a container comprising a second gas permeable surface area; (d) harvesting the therapeutic TIL population obtained from step (c); (e) transferring the harvested TIL population from step (d) into an infusion bag; (f) at any time prior to transfer to the infusion bag in step (e), modifying a portion of the first, second, or third TIL population to produce modified TILs, each of which comprises an immunomodulatory composition associated with its surface membrane.

[0028] In another aspect, provided herein is a method for expanding tumor infiltrating lymphocytes (TILs) into a therapeutic TIL population, comprising: (a) obtaining and / or receiving a first population of TILs from a tumor sample obtained from one or more mini-biopsies, core biopsies, or needle biopsies of a tumor from a cancer in a subject by culturing the tumor sample in a first cell culture medium containing IL-2 for about 3 days; (b) performing a first priming expansion by culturing the first TIL population in a second cell culture medium comprising IL-2, OKT-3, and antigen-presenting cells (APCs) to produce a second TIL population, wherein the first priming expansion is performed for a first period of about 7 or 8 days to obtain the second TIL population, and producing a second TIL population, wherein the second TIL population is greater in number than the first TIL population; (c) performing a second rapid expansion of the second TIL population by contacting the second TIL population with a third cell culture medium comprising IL-2, OKT-3, and APC to produce a third TIL population, wherein the second rapid expansion is performed for a second period of about 11 days to obtain a third TIL population, wherein the third TIL population is a therapeutic TIL population; (d) harvesting the therapeutic TIL population obtained from step (c); (e) modifying a portion of the first, second, or third TIL populations at any time before or after collection in step (f) to produce modified TILs, each of which comprises an immunomodulatory composition associated with its surface membrane.

[0029] In some embodiments of the methods provided herein, 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 papillomavirus, head and neck cancer (including head and neck squamous cell carcinoma (HNSCC)), renal cancer, and renal cell carcinoma.

[0030] In one aspect, provided herein is a method of expanding T cells, comprising: (a) performing a first priming expansion of a first T cell population obtained from a donor by culturing the first T cell population to result in growth and prime activation of the first T cell population; (b) after the activation of the first T cell population primed in step (a) begins to decay, performing a rapid second expansion of the first T cell population by culturing the first T cell population to result in growth and promote activation of the first T cell population to obtain a second T cell population; (c) harvesting a second population of T cells; (d) modifying a portion of the first or second T cell population at any time before or after collection in step (c) to generate modified T cells, each of which comprises an immunomodulatory composition associated with its surface membrane.

[0031] In another aspect, provided herein is a method of expanding T cells, comprising: (a) a first expansion by priming of a first T cell population from a tumor sample obtained from one or more mini-biopsies, core biopsies, or needle biopsies of a tumor in a donor by culturing the first T cell population to result in growth and prime activation of the first T cell population; (b) after the activation of the first T cell population primed in step (a) begins to decay, performing a rapid second expansion of the first T cell population by culturing the first T cell population to result in growth and promote activation of the first T cell population to obtain a second T cell population; (c) harvesting a second population of T cells; (d) modifying a portion of the first or second T cell population at any time before or after collection in step (e) to generate modified T cells, each of which comprises an immunomodulatory composition associated with its surface membrane.

[0032] In one aspect, provided herein is a method for expanding peripheral blood lymphocytes (PBLs) from peripheral blood, the method comprising: (a) obtaining a sample of peripheral blood mononuclear cells (PBMCs) from the patient's peripheral blood; (b) culturing the PBMCs in a culture comprising a first cell culture medium having IL-2, an anti-CD3 / anti-CD28 antibody, and a first combination of antibiotics for a period of time selected from the group consisting of about 9 days, about 10 days, about 11 days, about 12 days, about 13 days, and about 14 days, thereby resulting in the expansion of peripheral blood lymphocytes (PBLs) from the PBMCs; (c) harvesting PBLs from the culture in step (b); (d) modifying a portion of the PBLs at any time before or after collection in step (c) to produce modified PBLs, each of which comprises an immunomodulatory composition associated with its surface membrane.

[0033] In some embodiments, the patient is pretreated with ibrutinib or another interleukin-2-inducible T-cell kinase (ITK) inhibitor. In certain embodiments, the patient is refractory to treatment with ibrutinib or another ITK inhibitor.

[0034] In some embodiments, an immunomodulatory composition comprises one or more membrane-anchored immunomodulatory fusion proteins, each comprising one or more immunomodulatory agents and a cell membrane anchor moiety.

[0035] In exemplary embodiments, the one or more immunomodulatory agents comprise one or more cytokines, hi some embodiments, the one or more cytokines comprise one or more of IL-2, IL-6, IL-7, IL-9, IL-12, IL-15, IL-18, IL-21, IL-23, IL-27, IFN-gamma, TNFa, IFN-alpha, IFN-beta, GM-CSF, or GCSF, or variants thereof.

[0036] In some embodiments, the one or more cytokines include IL-2 or a variant thereof. In some embodiments, the IL-2 is human IL-2. In an exemplary embodiment, the human IL-2 has the amino acid sequence of SEQ ID NO: 272.

[0037] In some embodiments, the one or more cytokines comprise IL-2 or one or more variants thereof. In certain embodiments, the IL-12 comprises a human IL-12 p35 subunit attached to a human IL-12 p40 subunit. In certain embodiments, the human IL-12 p35 subunit has the amino acid sequence of SEQ ID NO: 267, and the human IL-12 p40 subunit has the amino acid sequence of SEQ ID NO: 268.

[0038] In some embodiments, the one or more cytokines include IL-15 or a variant thereof. In some embodiments, the IL-15 is human IL-15. In an exemplary embodiment, the human IL-15 has the amino acid sequence of SEQ ID NO: 258.

[0039] In some embodiments, the one or more cytokines include IL-18 or a variant thereof. In certain embodiments, the IL-18 is human IL-18. In certain embodiments, the human IL-18 has the amino acid sequence of any one of SEQ ID NOs: 269, 270, and 331-385.

[0040] In some embodiments, the one or more cytokines comprise IL-21 or a variant thereof. In certain embodiments, the IL-21 is human IL-21. In some embodiments, the human IL-21 has the amino acid sequence of SEQ ID NO: 251.

[0041] In some embodiments, the one or more cytokines comprise IL-15 or a variant thereof and IL-21 or a variant thereof. In some embodiments, the IL-15 is human IL-15 and the IL-21 is human IL-21. In certain embodiments, the human IL-15 has the amino acid sequence of SEQ ID NO: 258 and the human IL-21 has the amino acid sequence of SEQ ID NO: 271.

[0042] In some embodiments, the one or more immunomodulatory agents comprise a CD40 agonist. In certain embodiments, the CD40 agonist is an anti-CD40 binding domain or CD40L. In exemplary embodiments, the CD40 agonist is a CD40 binding domain comprising a variable heavy domain (VH) and a variable light domain (VL). In some embodiments, the VH and VL of the CD40 binding domain are selected from a) a VH having the amino acid sequence of SEQ ID NO: 274 and a VL having the amino acid sequence of SEQ ID NO: 275; b) a VH having the amino acid sequence of SEQ ID NO: 277 and a VL having the amino acid sequence of SEQ ID NO: 278; c) a VH having the amino acid sequence of SEQ ID NO: 280 and a VL having the amino acid sequence of SEQ ID NO: 281; and d) a VH having the amino acid sequence of SEQ ID NO: 283 and a VL having the amino acid sequence of SEQ ID NO: 284. In exemplary embodiments, the CD40 binding domain is an scFv.

[0043] In some embodiments, the CD40 agonist is human CD40L having the amino acid sequence of SEQ ID NO:273.

[0044] In some embodiments, one or more membrane-anchored immunomodulatory fusion proteins independently conform, from N-terminus to C-terminus, to the formula: S-IA-LC, where S is a signal peptide, IA is an immunomodulatory agent, L is a linker, and C is a cell membrane anchor moiety.

[0045] In some embodiments, the cell membrane anchor portion comprises a CD8a transmembrane intracellular domain, a B7-1 transmembrane domain, a B7-2 transmembrane domain, or a CD8a transmembrane domain. In exemplary embodiments, the cell membrane anchor portion comprises a B7-1 transmembrane domain. In some embodiments, the cell membrane anchor portion has the amino acid sequence of SEQ ID NO: 239.

[0046] In some embodiments, the immunomodulatory composition comprises two or more different membrane-anchored immunomodulatory fusion proteins, each of which comprises a different immunomodulatory agent. In some embodiments, the different immunomodulatory agents are selected from IL-2, IL-6, IL-7, IL-9, IL-12, IL-15, IL-18, IL-21, IL-23, IL-27, IFN-gamma, TNFα, IFN-alpha, IFN-beta, GM-CSF, GCSF, or variants thereof, and a CD40 agonist. In some embodiments, the different immunomodulatory agents are selected from IL-12 and IL-15, IL-15 and IL-18, IL-15 and IL-21, CD40L and IL-15, IL-15 and IL-21, IL-2 and IL-12, and variants thereof.

[0047] In some embodiments, the modified TIL comprises a first membrane-anchored immunomodulatory fusion protein and a second membrane-anchored immunomodulatory fusion protein.

[0048] In some embodiments, the first membrane-anchored immunomodulatory fusion protein comprises IL-15 or a variant thereof and the second membrane-anchored immunomodulatory fusion protein comprises IL-21 or a variant thereof.

[0049] In an exemplary embodiment, the first membrane-anchored immunomodulatory fusion protein and the second immunomodulatory fusion protein are expressed under the control of the NFAT promoter in the modified TILs.

[0050] In exemplary embodiments, the one or more membrane-anchored immunomodulatory fusion proteins independently conform, from N-terminus to C-terminus, to the formula: S-IA-LC, where S is a signal peptide, IA is an immunomodulatory agent, L is a linker, and C is a cell membrane anchor moiety. In some embodiments, the IA is a cytokine. In exemplary embodiments, the IA is selected from the group consisting of IL-2, IL-6, IL-7, IL-9, IL-12, IL-15, IL-18, IL-21, IL-23, IL-27, IFN-gamma, TNFα, IFN-alpha, IFN-beta, GM-CSF, GCSF, or a variant thereof. In some embodiments, the IA is IL-2 or a variant thereof. In certain embodiments, the IA is IL-12 or a variant thereof. In some embodiments, the IA is IL-15 or a variant thereof. In certain embodiments, the IA is IL-18 or a variant thereof. In certain embodiments, the IA is DR-IL-18. In certain embodiments, the IA is IL-21 or a variant thereof.

[0051] Illustratively, the one or more membrane-anchored immunomodulatory fusion proteins independently conform to the formula, from N-terminus to C-terminus: S1-IA1-L1-C1-L2-S2-IA2-L3-C2, where S1 and S2 are each independently a signal peptide, IA1 and IA2 are each independently an immunomodulator, L1-L3 are each independently a linker, and C1 and C2 are each independently a cell membrane anchor moiety. In some embodiments, S1 and S2 are the same. In certain embodiments, C1 and C2 are the same. In some embodiments, L2 is a cleavable linker. In exemplary embodiments, L2 is a furin-cleavable linker. In some embodiments, IA1 and IA2 are each independently a cytokine.

[0052] In some embodiments, IA1 and IA2 are each independently selected from the group consisting of IL-2, IL-6, IL-7, IL-9, IL-12, IL-15, IL-18, IL-21, IL-23, IL-27, IFN-gamma, TNFα, IFN-alpha, IFN-beta, GM-CSF, GCSF, or variants thereof. In some embodiments, IA1 and IA2 are each independently selected from the group consisting of IL-2 and IL-12, with the proviso that one of IA1 and IA2 is IL-2 and the other is IL-12. In some embodiments, IA1 and IA2 are each independently selected from the group consisting of IL-15 and IL-21, with the proviso that one of IA1 and IA2 is IL-15 and the other is IL-21.

[0053] In certain embodiments, the modification involves introducing a heterologous nucleic acid encoding a fusion protein into a portion of the TIL and expressing the fusion protein on the surface of the modified TIL.

[0054] In certain embodiments, the modification comprises introducing a heterologous nucleic acid encoding a fusion protein into a portion of the TIL and expressing the fusion protein on the surface of the modified TIL. In some embodiments, the heterologous nucleic acid comprises a viral vector (e.g., an adenoviral vector, a retroviral vector, a lentiviral vector, or an adeno-associated vector (AAV)). In some embodiments, the heterologous nucleic acid comprises a piggyBac transposon. In some embodiments, the heterologous nucleic acid comprises an NFAT promoter, an EF-1a promoter, an MND promoter, or an SSFV promoter.

[0055] In some embodiments, the immunomodulatory composition comprises a fusion protein comprising one or more immunomodulatory agents linked to a TIL surface antigen-binding domain. In some embodiments, the one or more immunomodulatory agents comprise one or more cytokines. In some embodiments, the one or more cytokines comprise one or more of IL-2, IL-6, IL-7, IL-9, IL-12, IL-15, IL-18, IL-21, IL-23, IL-27, IFN-gamma, TNFα, IFN-alpha, IFN-beta, GM-CSF, or GCSF, or variants thereof. In some embodiments, the one or more cytokines comprise IL-12 or a variant thereof. In certain embodiments, the one or more cytokines comprise IL-15 or a variant thereof. In certain embodiments, the one or more cytokines comprise IL-18 or a variant thereof (e.g., DR-IL-18). In some embodiments, the one or more cytokines comprise IL-21 or a variant thereof. In certain embodiments, the TIL surface antigen-binding domain comprises an antibody variable heavy domain and a variable light domain. In some embodiments, the TIL surface antigen-binding domain comprises an antibody or fragment thereof. In some embodiments, the TIL surface antigen-binding domain exhibits affinity for one or more of the following TIL surface antigens: CD45, CD4, CD8, CD3, CD11a, CD11b, CD11c, CD18, CD25, CD127, CD19, CD20, CD22, HLA-DR, CD197, CD38, CD27, CD196, CXCR3, CXCR4, CXCR5, CD84, CD229, CCR1, CCR5, CCR4, CCR6, CCR8, CCR10, CD16, CD56, CD137, OX40, or GITR. In some embodiments, the modification comprises incubating the fusion protein with a portion of the TIL under conditions that allow binding of the fusion protein to the portion of the TIL.

[0056] In some embodiments, the immunomodulatory composition comprises nanoparticles comprising multiple immunomodulatory agents. In some embodiments, the multiple immunomodulatory agents are covalently attached via a degradable linker. In certain embodiments, the nanoparticles comprise at least one polymer, cationic polymer, or cationic block copolymer on the nanoparticle surface. In some embodiments, the one or more cytokines comprise one or more of IL-2, IL-6, IL-7, IL-9, IL-12, IL-15, IL-18, IL-21, IL-23, IL-27, IFN-gamma, TNFα, IFN-alpha, IFN-beta, GM-CSF, or GCSF, or variants thereof. In certain embodiments, the one or more cytokines comprise IL-12. In some embodiments, the one or more cytokines comprise IL-15. In some embodiments, the one or more cytokines comprise IL-21. In some embodiments, the nanoparticles are liposomes, protein nanogels, nucleotide nanogels, polymeric nanoparticles, or solid nanoparticles. In some embodiments, the nanoparticles are nanogels. In certain embodiments, the nanoparticles further comprise an antigen-binding domain that binds to one or more of the following antigens: CD45, CD11a (integrin alpha-L), CD18 (integrin beta-2), CD11b, CD11c, CD25, CD8, or CD4. In some embodiments, the modification comprises attaching an immunomodulatory composition to the surface of a portion of the TILs.

[0057] In certain embodiments of the methods provided herein, the modification is performed on TILs from the first expansion, or on TILs from the second expansion, or both. In certain embodiments, the modification is performed on TILs from the first expansion by priming, or on TILs from the rapid second expansion, or both.

[0058] In some embodiments of the methods provided herein, the modification is performed after the first expansion and before the second expansion. In some embodiments, the modification is performed after the first expansion with priming and before the rapid second expansion, or both. In certain embodiments, the modification is performed after the second expansion. In some embodiments, the modification is performed after the rapid second expansion. In some embodiments, the modification is performed after harvesting.

[0059] In certain embodiments, the first expansion is carried out over a period of about 11 days. In some embodiments, the first expansion with priming is carried out over a period of about 11 days.

[0060] In some embodiments of the methods provided herein, IL-2 is present in the cell culture medium at an initial concentration of 1000 IU / mL to 6000 IU / mL during the first expansion. In certain embodiments, IL-2 is present in the cell culture medium at an initial concentration of 1000 IU / mL to 6000 IU / mL during the first expansion by priming.

[0061] In some embodiments, IL-2 in the second expansion step is present at an initial concentration of 1000 IU / mL to 6000 IU / mL and the OKT-3 antibody is present at an initial concentration of about 30 ng / mL. In some embodiments, IL-2 in the rapid second expansion step is present at an initial concentration of 1000 IU / mL to 6000 IU / mL and the OKT-3 antibody is present at an initial concentration of about 30 ng / mL.

[0062] In some embodiments, the first expansion is performed using a gas-permeable container. In certain embodiments, the first expansion by priming is performed using a gas-permeable container. In some embodiments, the second expansion is performed using a gas-permeable container. In certain embodiments, the rapid second expansion is performed using a gas-permeable container.

[0063] In some embodiments of the methods provided herein, the cell culture medium for the first expansion further comprises a cytokine selected from the group consisting of IL-4, IL-7, IL-15, IL-21, and combinations thereof. In certain embodiments, the cell culture medium for the first expansion by priming further comprises a cytokine selected from the group consisting of IL-4, IL-7, IL-15, IL-21, and combinations thereof. In some embodiments, the cell culture medium for the second expansion further comprises a cytokine selected from the group consisting of IL-4, IL-7, IL-15, IL-21, and combinations thereof. In certain embodiments, the cell culture medium for 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.

[0064] In some embodiments of the methods of treatment provided herein, the method further comprises treating the patient with a non-myeloablative lymphodepletion regimen prior to administering the TILs to the patient. In some embodiments, the non-myeloablative lymphodepletion regimen comprises cyclophosphamide at 60 mg / m 2 / day for 2 days, followed by fludarabine at 25 mg / m 2 / day for 3 days. In some embodiments, the non-myeloablative lymphodepletion regimen comprises administering cyclophosphamide at a dose of 60 mg / m 2 Fludarabine at a dose of 25 mg / m 2 / day for 2 days, followed by fludarabine at 25 mg / m 2 / day for 3 days. In certain embodiments, the non-myeloablative lymphodepletion regimen comprises administering cyclophosphamide at a dose of 60 mg / m 2 Fludarabine at a dose of 25 mg / m 2 / day for 2 days, followed by fludarabine at 25 mg / m 2 / day for 1 day. In certain embodiments, cyclophosphamide is administered with mesna.

[0065] In some embodiments of the methods of treatment provided herein, the methods further comprise treating the patient with an IL-2 regimen starting the day after the TILs are administered to the patient. In some embodiments of the methods of treatment provided herein, the methods further comprise treating the patient with an IL-2 regimen starting the same day the TIL population is administered to the patient. In some embodiments, the IL-2 regimen is a high-dose IL-2 regimen comprising 600,000 or 720,000 IU / kg aldesleukin, or a biosimilar or variant thereof, administered as a 15-minute bolus intravenous infusion every 8 hours until tolerated.

[0066] In some embodiments of the methods provided herein, a therapeutically effective population of TILs is administered, and is about 2.3 x 10 10 ~Approx. 13.7×10 10 Includes TILs.

[0067] In some embodiments of the methods provided herein, the priming first expansion and the rapid second expansion are performed over a period of 21 days or less. In some embodiments, the priming first expansion and the 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 some embodiments, the rapid second expansion is performed over a period of 11 days or less.

[0068] In some embodiments of the methods provided herein, the first expansion in step (c) and the second expansion in step (d) are each performed separately within a period of 11 days. In some embodiments of the methods provided herein, steps (a) through (f) are performed in about 10 days to about 22 days.

[0069] In some embodiments of the methods provided herein, the modified TILs further comprise a genetic modification that silences or reduces expression of one or more immune checkpoint genes in at least a portion of the therapeutic TIL population. In some embodiments, the one or more immune checkpoint genes are PD-1, CTLA-4, LAG-3, HAVCR2 (TIM-3), Cish, TGFβ, PKA, CBL-B, PPP2CA, PPP2CB, PTPN6, PTPN22, PDCD1, BTLA, CD160, TIGIT, TET2, CD96, CRTAM, LAIR1, SIGLEC7, SIGLEC9, CD244, TNFRSF10B, TNFRSF10A, CASP8, CASP 10, 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 certain embodiments, the one or more immune checkpoint genes are selected from the group including PD-1, TGIT, CTLA-4, LAG-3, HAVCR2 (TIM-3), Cish, TGFβ, and PKA. In certain embodiments, the immune checkpoint gene is PD-1. In some embodiments, the genetic modification is produced using RNA interference (e.g., shRNA).

[0070] In some embodiments, the modified TILs further comprise a genetic modification that enhances expression of one or more immune checkpoint genes in at least a portion of the therapeutic TIL population, where the immune checkpoint gene(s) are selected from the group including CCR2, CCR4, CCR5, CXCR2, CXCR3, CX3CR1, IL-2, IL-4, IL-7, IL-10, IL-15, IL-21, NOTCH 1 / 2 intracellular domain (ICD), and / or NOTCH ligand mDLL1. In certain embodiments, the genetic modification is produced using a programmable nuclease that mediates the generation of double- or single-stranded breaks in one or more immune checkpoint genes. In some embodiments, the genetic modification is produced using one or more methods selected from RNA interference (e.g., shRNA), CRISPR, TALE, zinc finger, Cas-CLOVER, and combinations thereof. In certain embodiments, the genetic modification is produced using CRISPR. In some embodiments, the CRISPR method is CRISPR / Cas9 method.In certain embodiments, the genetic modification is produced using TALE method.In some embodiments, the genetic modification is produced using zinc finger method.In some embodiments, the genetic modification is produced using Cas-CLOVER method.

[0071] In some embodiments, the modified TILs transiently express an immunomodulatory composition on the cell surface. In some embodiments, the immunomodulatory composition comprises one or more membrane-anchored immunomodulatory fusion proteins, each fusion protein comprising one or more immunomodulatory agents and a cell membrane anchor moiety.

[0072] In exemplary embodiments, the one or more immunomodulatory agents comprise one or more cytokines, hi some embodiments, the one or more cytokines comprise one or more of IL-2, IL-6, IL-7, IL-9, IL-12, IL-15, IL-18, IL-21, IL-23, IL-27, IFN-gamma, TNFa, IFN-alpha, IFN-beta, GM-CSF, or GCSF, or variants thereof.

[0073] In some embodiments, the one or more cytokines include IL-2 or a variant thereof. In some embodiments, the IL-2 is human IL-2. In an exemplary embodiment, the human IL-2 has the amino acid sequence of SEQ ID NO: 272.

[0074] In some embodiments, the one or more cytokines comprise IL-12 or a variant thereof. In certain embodiments, the IL-12 comprises a human IL-12 p35 subunit attached to a human IL-12 p40 subunit. In certain embodiments, the human IL-12 p35 subunit has the amino acid sequence of SEQ ID NO:267, and the human IL-12 p40 subunit has the amino acid sequence of SEQ ID NO:268.

[0075] In some embodiments, the one or more cytokines include IL-15 or a variant thereof. In some embodiments, the IL-15 is human IL-15. In an exemplary embodiment, the human IL-15 has the amino acid sequence of SEQ ID NO: 258.

[0076] In some embodiments, the one or more cytokines include IL-18 or a variant thereof (e.g., DR-IL-18). In certain embodiments, the IL-18 is human IL-18. In certain embodiments, the human IL-18 has the amino acid sequence of any one of SEQ ID NOs: 269, 270, and 331-385.

[0077] In some embodiments, the one or more cytokines comprise IL-21 or a variant thereof. In certain embodiments, the IL-21 is human IL-21. In some embodiments, the human IL-21 has the amino acid sequence of SEQ ID NO: 271.

[0078] In some embodiments, the one or more cytokines comprise IL-15 and IL-21. In some embodiments, the IL-15 is human IL-15 and the IL-21 is human IL-21. In certain embodiments, the human IL-15 has the amino acid sequence of SEQ ID NO: 258 and the human IL-21 has the amino acid sequence of SEQ ID NO: 271.

[0079] In some embodiments, the one or more immunomodulatory agents comprise a CD40 agonist. In certain embodiments, the CD40 agonist is an anti-CD40 binding domain or CD40L. In exemplary embodiments, the CD40 agonist is a CD40 binding domain comprising a variable heavy domain (VH) and a variable light domain (VL). In some embodiments, the VH and VL of the CD40 binding domain are selected from a) a VH having the amino acid sequence of SEQ ID NO: 274 and a VL having the amino acid sequence of SEQ ID NO: 275; b) a VH having the amino acid sequence of SEQ ID NO: 277 and a VL having the amino acid sequence of SEQ ID NO: 278; c) a VH having the amino acid sequence of SEQ ID NO: 280 and a VL having the amino acid sequence of SEQ ID NO: 281; and d) a VH having the amino acid sequence of SEQ ID NO: 283 and a VL having the amino acid sequence of SEQ ID NO: 284. In exemplary embodiments, the CD40 binding domain is an scFv.

[0080] In some embodiments, the CD40 agonist is human CD40L having the amino acid sequence of SEQ ID NO: 273. In some embodiments, the membrane-anchored immunomodulatory fusion protein conforms, from N-terminus to C-terminus, to the formula: S-IA-LC, where S is a signal peptide, IA is an immunomodulatory agent, L is a linker, and C is a cell membrane anchor moiety.

[0081] In some embodiments, the cell membrane anchor portion comprises a CD8a transmembrane intracellular domain, a B7-1 transmembrane domain, a B7-2 transmembrane domain, or a CD8a transmembrane domain. In exemplary embodiments, the cell membrane anchor portion comprises a B7-1 transmembrane domain. In some embodiments, the cell membrane anchor portion has the amino acid sequence of SEQ ID NO: 239.

[0082] In some embodiments, the immunomodulatory composition comprises two or more different membrane-anchored immunomodulatory fusion proteins, each of which comprises a different immunomodulatory agent. In some embodiments, the different immunomodulatory agents are selected from IL-2, IL-6, IL-7, IL-9, IL-12, IL-15, IL-18, IL-21, IL-23, IL-27, IFN-gamma, TNFα, IFN-alpha, IFN-beta, GM-CSF, GCSF, or variants thereof, and a CD40 agonist. In some embodiments, the different immunomodulatory agents are selected from IL-12 and IL-15, IL-15 and IL-18, CD40L, IL-15 and IL-21, and IL-15, and IL-2 and IL-12.

[0083] In some embodiments, the modified TILs are modified by transfecting the TILs with a nucleic acid encoding a fusion protein comprising one or more immunomodulatory agents and a cell membrane anchor moiety to transiently express the fusion protein on the cell surface. In some embodiments, the nucleic acid is RNA. In some embodiments, the RNA is mRNA. In some embodiments, the TILs are transfected with the mRNA by electroporation. In some embodiments, the TILs are transfected with the mRNA by electroporation after the first expansion and before the second expansion. In some embodiments, the TILs are transfected with the mRNA by electroporation before the first expansion. In some embodiments, the method further comprises activating the TILs by incubation with an anti-CD3 agonist before transfecting the TILs with the mRNA. In some embodiments, the anti-CD3 agonist is OKT-3. In some embodiments, the TILs are activated by incubating the TILs with the anti-CD3 agonist for about 1 to 3 days before transfecting the TILs with the mRNA.

[0084] In some embodiments, the modified TILs are transfected with a nucleic acid encoding the fusion protein using a microfluidic device to transiently disrupt the cell membrane of the TILs, thereby allowing transfection of the nucleic acid.

[0085] In some embodiments, artificial antigen-presenting cells (aAPCs) are used in place of APCs. In some embodiments, the aAPCs comprise cells expressing HLA-A / B / C, CD64, CD80, ICOS-L, and CD58. In some embodiments, the aAPCs comprise MOLM-14 cells. In some embodiments, the aAPCs comprise MOLM-13 cells. In some embodiments, the aAPCs comprise MOLM-14 cells that endogenously express HLA-A / B / C, CD64, CD80, ICOS-L, and CD58. In some embodiments, the aAPCs comprise MOLM-14 cells that endogenously express HLA-A / B / C, CD64, CD80, ICOS-L, and CD58, and the MOLM-14 cells are permanently gene-edited to express CD86. In some embodiments, MOLM-14 cells are transduced with one or more viral vectors, wherein the one or more viral vectors comprise a nucleic acid sequence encoding CD86 and a nucleic acid sequence encoding 4-1BBL, and the MOLM-14 cells express CD86 and 4-1BBL. In some embodiments, aAPCs are transiently gene edited to transiently express an immunomodulatory composition on the cell surface, the immunomodulatory composition comprising an immunomodulatory fusion protein. In some embodiments, the aAPCs transiently express an immunomodulatory fusion protein on the cell surface, the immunomodulatory fusion protein comprising a membrane anchor fused to a cytokine. In some embodiments, the aAPCs transiently express a membrane anchor fused to a cytokine selected from the group consisting of IL-2, IL-7, IL-10, IL-12, IL-15, and IL-21 on the cell surface. In some embodiments, the aAPCs transiently express a membrane anchor fused to a cytokine selected from the group consisting of IL-2, IL-12, IL-15, and IL-21 on the cell surface. In some embodiments, the aAPC transiently expresses a membrane anchor on the cell surface fused to a cytokine selected from the group consisting of IL-12, IL-15, and IL-21.

[0086] In some embodiments, the modified TILs are genetically modified to express an immunomodulatory composition on their cell surface. In some embodiments, the immunomodulatory composition comprises one or more membrane-anchored immunomodulatory fusion proteins, each comprising one or more immunomodulatory agents and a cell membrane anchor moiety. In some embodiments, the one or more membrane-anchored immunomodulatory fusion proteins comprise IL-2 or a variant thereof. In certain embodiments, the one or more membrane-anchored immunomodulatory fusion proteins comprise IL-15 or a variant thereof. In exemplary embodiments, the one or more membrane-anchored immunomodulatory fusion proteins comprise IL-18 or a variant thereof (e.g., DR-IL-18). In some embodiments, the one or more membrane-anchored immunomodulatory fusion proteins comprise IL-21 or a variant thereof.

[0087] In certain embodiments, the modified TILs comprise a first membrane-anchored immunomodulatory fusion protein and a second membrane-anchored immunomodulatory fusion protein. In some embodiments, the first membrane-anchored immunomodulatory fusion protein comprises IL-15 and the second membrane-anchored immunomodulatory fusion protein comprises IL-21. In some embodiments, the first membrane-anchored immunomodulatory fusion protein and the second immunomodulatory fusion protein are expressed under the control of an NFAT promoter, an EF-1a promoter, an MND promoter, or an SSFV promoter in the modified TILs.

[0088] In some embodiments, one or more membrane-anchored immunomodulatory fusion proteins independently conform, from N-terminus to C-terminus, to the formula: S-IA-LC, where S is a signal peptide, IA is an immunomodulatory agent, L is a linker, and C is a cell membrane anchor moiety. In some embodiments, the IA is a cytokine. In exemplary embodiments, the IA is selected from the group consisting of IL-2, IL-6, IL-7, IL-9, IL-12, IL-15, IL-18, IL-21, IL-23, IL-27, IFN-gamma, TNFα, IFN-alpha, IFN-beta, GM-CSF, GCSF, or variants thereof. In some embodiments, the IA is IL-2. In certain embodiments, the IA is IL-12. In some embodiments, the IA is IL-15. In certain embodiments, the IA is IL-21. In some embodiments, L is a CD8a transmembrane intracellular domain, a B7-1 transmembrane domain, a B7-2 transmembrane domain, or a CD8a transmembrane domain. In certain embodiments, L is a B7-1 transmembrane domain. In some embodiments, L has the amino acid sequence of SEQ ID NO: 239.

[0089] In exemplary embodiments, the one or more membrane-anchored immunomodulatory fusion proteins independently conform, from N-terminus to C-terminus, to the formula: S1-IA1-L1-C1-L2-S2-IA2-L3-C2, where S1 and S2 are each independently a signal peptide, IA1 and IA2 are each independently an immunomodulatory agent, L1-L3 are each independently a linker, and C1 and C2 are each independently a cell membrane anchor moiety. In some embodiments, S1 and S2 are the same. In exemplary embodiments, C1 and C2 are the same. In some embodiments, L2 is a cleavable linker. In certain embodiments, L2 is a furin-cleavable linker.

[0090] In some embodiments, IA1 and IA2 are each independently a cytokine. In some embodiments, IA1 and IA2 are each independently selected from the group consisting of IL-2, IL-6, IL-7, IL-9, IL-12, IL-15, IL-18, IL-21, IL-23, IL-27, IFN-gamma, TNFα, IFN-alpha, IFN-beta, GM-CSF, GCSF, or variants thereof. In some embodiments, IA1 and IA2 are each independently selected from the group consisting of IL-2 and IL-12, with the proviso that one of IA1 and IA2 is IL-2 and the other is IL-12. In some embodiments, IA1 and IA2 are each independently selected from the group consisting of IL-15 and IL-21, with the proviso that one of IA1 and IA2 is IL-15 and the other is IL-21.

[0091] In exemplary embodiments, C1 and C2 are each independently a CD8a transmembrane intracellular domain, a B7-1 transmembrane domain, a B7-2 transmembrane domain, or a CD8a transmembrane domain. In some embodiments, C1 and C2 are each a B7-1 transmembrane domain. In some embodiments, C1 and C2 each have the amino acid sequence of SEQ ID NO: 239.

[0092] In certain embodiments, the modified TILs express one or more membrane-anchored immunomodulatory fusion proteins under the control of an NFAT promoter, an EF-1a promoter, an MND promoter, or an SSFV promoter. In some embodiments, the modified TILs are transduced with a retroviral vector to express one or more membrane-anchored immunomodulatory fusion proteins. In some embodiments, the modified TILs are transduced with a vector (e.g., an adenoviral vector, a retroviral vector, a retroviral vector, or an adeno-associated vector (AAV)) or a piggyBac transposon to express one or more membrane-anchored immunomodulatory fusion proteins.

[0093] Also provided are compositions comprising modified TILs produced by any of the methods described herein. [Brief explanation of the drawings]

[0094] [Figure 1] An exemplary Gen2 (Process 2A) chart providing an overview of steps A-F. [Figure 2A] 1 is a process flow diagram of an embodiment of Gen2 (Process 2A) for TIL fabrication. [Figure 2B] 1 is a process flow diagram of an embodiment of Gen2 (Process 2A) for TIL fabrication. [Figure 2C] 1 is a process flow diagram of an embodiment of Gen2 (Process 2A) for TIL fabrication. [Figure 3] FIG. 1 shows a diagram of an embodiment of an exemplary manufacturing process (approximately 22 days) for cryopreserved TILs. [Figure 4] FIG. 1 shows a diagram of an embodiment of Gen2 (Process 2A), a 22-day process for TIL fabrication. [Figure 5] 1 is a comparison table of steps A-F from exemplary embodiments of Process 1C and Gen2 (Process 2A) for TIL fabrication. [Figure 6] Detailed comparison of Process 1C and Gen2 (Process 2A) embodiments for TIL fabrication. [Figure 7] 1. Exemplary Gen3 TIL fabrication process. [Figure 8A] A comparison of embodiments of the 2A process (an approximately 22 day process) and the Gen3 process (an approximately 14-16 day process) for TIL fabrication is shown. [Figure 8B] An exemplary Process Gen3 chart providing an overview of steps A-F (approximately a 14-16 day process). [Figure 8C] A chart providing three exemplary Gen3 processes along with an overview of steps A-F (approximately 14- to 16-day processes) for each of the three process variations. [Figure 8D]An exemplary modified Gen2-like process (approximately a 22-day process) providing an overview of steps A-F. [Figure 9] 1 provides an experimental flow chart for the comparison between Gen2 (Process 2A) and Gen3 processes. [Figure 10] 1 shows a comparison of various Gen2 (Process 2A) and Gen3.1 process embodiments. [Figure 11] 1 is a table illustrating various features of embodiments of the Gen2, Gen2.1, and Gen3.0 processes. [Figure 12] Summary of media conditions for an embodiment of the Gen3 process, designated Gen3.1. [Figure 13] 1 is a table illustrating various features of embodiments of the Gen2, Gen2.1, and Gen3.0 processes. [Figure 14] 1 is a table comparing various features of embodiments of the Gen2 and Gen3.0 processes. [Figure 15] 1 is a table providing media use in various embodiments of the described expansion process. [Figure 16] Schematic of an exemplary embodiment of the Gen3 process (16-day process). [Figure 17] Schematic of an exemplary embodiment of a method for expanding T cells from hematopoietic malignancies using the Gen3 expansion platform. [Figure 18]Structures IA and IB are provided. The cylinders refer to individual polypeptide binding domains. Structures IA and IB comprise three linearly linked TNFRSF-binding domains derived from antibodies that bind, for example, to 4-1BBL or 4-1BB, that fold to form a trivalent protein, which is then linked to a second trivalent protein via IgG1-Fc (comprising the CH3 and CH2 domains), which is then used to link two of the trivalent proteins together via disulfide bonds (small oblong ellipses), stabilizing the structure and providing an agonist that can bring together the six receptor and intracellular signaling domains of the signaling protein to form a signaling complex. The TNFRSF-binding domains shown as cylinders can be, for example, scFv domains comprising VH and VL chains connected by a linker that may contain hydrophilic residues and Gly and Ser sequences for flexibility, and Glu and Lys for solubility. [Figure 19] Schematic of an exemplary embodiment of the Gen3 process (16-day process). [Figure 20] 1 provides a process overview of an exemplary embodiment of the Gen3.1 process (16-day process). [Figure 21] Schematic of an exemplary embodiment of the Gen3.1 testing process (16-17 day process). [Figure 22] Schematic of an exemplary embodiment of the Gen3 process (16-day process). [Figure 23A] 1 is a comparison table of an exemplary Gen2 process and an exemplary Gen3 process. [Figure 23B] 1 is a comparison table of an exemplary Gen2 process and an exemplary Gen3 process. [Figure 24] Schematic of an exemplary embodiment of the preparation timeline for the Gen3 process (16-17 day process). [Figure 25] Schematic of an exemplary embodiment of the Gen3 process (14-16 day process). [Figure 26A] Schematic of an exemplary embodiment of the Gen3 process (16-day process). [Figure 26B]Schematic of an exemplary embodiment of the Gen3 process (16-day process). [Figure 27] Schematic of an exemplary embodiment of the Gen3 process (16-day process). [Figure 28] Comparison of Gen2, Gen2.1, and Gen3 process (16-day process) embodiments. [Figure 29] Comparison of Gen2, Gen2.1, and Gen3 process (16-day process) embodiments. [Figure 30] Components of a Gen3 embodiment. [Figure 31] Flowchart comparison of Gen3 embodiments (Gen3.0, Gen3.1 control, Gen3.1 test). [Figure 32] Components of an exemplary embodiment of the Gen3 process (16-17 day process) are shown. [Figure 33] Approval criteria table [Figure 34] 1 depicts several embodiments of a TIL manufacturing process, including an electroporation step, for use in gene editing processes (including TALEN, zinc finger nuclease, and CRISPR methods described herein). [Figure 35] A depiction of an embodiment of a TIL manufacturing process including an electroporation step for use in gene editing processes (including TALEN, zinc finger nuclease, and CRISPR methods described herein). [Figure 36A] Exemplary membrane-anchored immunomodulatory fusion proteins that may be included in the TILs described herein. [Figure 36B] Exemplary membrane-anchored immunomodulatory fusion proteins that may be included in the TILs described herein. [Figure 36C] Exemplary membrane-anchored immunomodulatory fusion proteins that may be included in the TILs described herein. [Figure 36D] Exemplary membrane-anchored immunomodulatory fusion proteins that may be included in the TILs described herein. [Figure 36E]Exemplary membrane-anchored immunomodulatory fusion proteins that may be included in the TILs described herein. [Figure 36F] Exemplary membrane-anchored immunomodulatory fusion proteins that may be included in the TILs described herein. [Figure 36G] Exemplary membrane-anchored immunomodulatory fusion proteins that may be included in the TILs described herein. [Figure 36H] Exemplary membrane-anchored immunomodulatory fusion proteins that may be included in the TILs described herein. [Figure 36I] Exemplary membrane-anchored immunomodulatory fusion proteins that may be included in the TILs described herein. [Figure 36J] Exemplary membrane-anchored immunomodulatory fusion proteins that may be included in the TILs described herein. [Figure 37A] Exemplary membrane-anchored immunomodulatory fusion proteins that may be included in the TILs described herein. [Figure 37B] Exemplary membrane-anchored immunomodulatory fusion proteins that may be included in the TILs described herein. [Figure 37C] Exemplary membrane-anchored immunomodulatory fusion proteins that may be included in the TILs described herein. [Figure 37D] Exemplary membrane-anchored immunomodulatory fusion proteins that may be included in the TILs described herein. [Figure 38A] Summary of studies to evaluate the expression and signaling of pre-REP TILs transduced with membrane-bound IL-15 / IL-21. [Figure 38B] Summary of studies to evaluate the expression and signaling of pre-REP TILs transduced with membrane-bound IL-15 / IL-21. [Figure 38C] Summary of studies to evaluate the expression and signaling of pre-REP TILs transduced with membrane-bound IL-15 / IL-21. [Figure 39A] Summary of studies to evaluate the expression of mIL-15 / IL21 and CD8 and CD4 T cell subsets in mIL-15 / IL-21-transduced REP TILs. [Figure 39B]Summary of studies to evaluate the expression of mIL-15 / IL21 and CD8 and CD4 T cell subsets in mIL-15 / IL-21-transduced REP TILs. [Figure 40A] Summary of studies to evaluate the phenotype of mIL-15 / IL-21-transduced CD8+REP TILs. [Figure 40B] Summary of studies to evaluate the phenotype of mIL-15 / IL-21-transduced CD8+REP TILs. [Figure 40C] Summary of studies to evaluate the phenotype of mIL-15 / IL-21-transduced CD8+REP TILs. [Figure 41A] Summary of studies to evaluate the phenotype of mIL-15 / IL-21 transduced CD4+ cells. [Figure 41B] Summary of studies to evaluate the phenotype of mIL-15 / IL-21 transduced CD4+ cells. [Figure 41C] Summary of studies to evaluate the phenotype of mIL-15 / IL-21 transduced CD4+ cells. [Figure 42] Summary of studies to evaluate the fold expansion, cell viability, and transduction efficiency of TeIL-18 and TeDR-IL18 expressing TILs after gene transduction and the 11-day REP process described herein. [Figure 43A] Summary of studies to evaluate surface expression of IL-18 and DRIL-18 on TeIL-18- and TeDR-IL18-expressing TILs. [Figure 43B] Summary of studies to evaluate surface expression of IL-18 and DRIL-18 on TeIL-18- and TeDR-IL18-expressing TILs. [Figure 44] Summary of studies to evaluate IFN-γ production by TeIL-18 and TeDR-IL18 expressing TILs with and without TCR stimulation using the anti-CD3 antibody OKT3. [Figure 45]Summary of a study to evaluate the expression and IL-18 activity of TeIL-18 and TeDR-IL18 expressing REP TILs, where TeIL-18 and TeDRIL-18 are under the control of an inducible NFAT promoter. [Figure 46A] Summary of TeIL-IL18 and TeDRIL-18 REP TIL functional studies using KILR-THP-I cytotoxicity assays. Freshly thawed TILs. [Figure 46B] Summary of TeIL-IL18 and TeDRIL-18 REP TIL functional studies using KILR-THP-I cytotoxicity assays with freshly thawed TILs, including additional experiments with repeatedly stimulated TILs. [Figure 46C] Summary of TeIL-IL18 and TeDRIL-18 REP TIL functional studies using KILR-THP-I cytotoxicity assays with freshly thawed TILs, including additional experiments with repeatedly stimulated TILs. [Figure 47A] Summary of evaluation of IFN-γ production by TeIL-18 and TeDRIL-18 ub KILR-THP-I cytotoxicity assay. [Figure 47B] Summary of evaluation of IFN-γ production by TeIL-18 and TeDRIL-18 ub KILR-THP-I cytotoxicity assay. [Figure 48A] Summary of phenotypic and functional analyses of TeIL-18 and TeDRIL-18 REP TILs. (A) and (B): Assessment of differentiation in freshly thawed CD4+ (A) and CD8+ (B) TeIL-18 and TeDRIL-18 REP TILs. (C) and (D): Assessment of differentiation in repeatedly stimulated CD4+ (C) and CD8+ (D) TeIL-18 and TeDRIL-18 REP TILs. (E) and (F): Assessment of activation in freshly thawed (E) and repeatedly stimulated (F) TeIL-18 and TeDRIL-18 REP TILs. (F) and (G): Assessment of differentiation in repeatedly stimulated CD4+ and CD8+ TeIL-18 and TeDRIL-18 REP TILs. [Figure 48B]Summary of phenotypic and functional analyses of TeIL-18 and TeDRIL-18 REP TILs. (A) and (B): Assessment of differentiation in freshly thawed CD4+ (A) and CD8+ (B) TeIL-18 and TeDRIL-18 REP TILs. (C) and (D): Assessment of differentiation in repeatedly stimulated CD4+ (C) and CD8+ (D) TeIL-18 and TeDRIL-18 REP TILs. (E) and (F): Assessment of activation in freshly thawed (E) and repeatedly stimulated (F) TeIL-18 and TeDRIL-18 REP TILs. (F) and (G): Assessment of differentiation in repeatedly stimulated CD4+ and CD8+ TeIL-18 and TeDRIL-18 REP TILs. [Figure 48C] Summary of phenotypic and functional analyses of TeIL-18 and TeDRIL-18 REP TILs. (A) and (B): Assessment of differentiation in freshly thawed CD4+ (A) and CD8+ (B) TeIL-18 and TeDRIL-18 REP TILs. (C) and (D): Assessment of differentiation in repeatedly stimulated CD4+ (C) and CD8+ (D) TeIL-18 and TeDRIL-18 REP TILs. (E) and (F): Assessment of activation in freshly thawed (E) and repeatedly stimulated (F) TeIL-18 and TeDRIL-18 REP TILs. (F) and (G): Assessment of differentiation in repeatedly stimulated CD4+ and CD8+ TeIL-18 and TeDRIL-18 REP TILs. [Figure 48D] Summary of phenotypic and functional analyses of TeIL-18 and TeDRIL-18 REP TILs. (A) and (B): Assessment of differentiation in freshly thawed CD4+ (A) and CD8+ (B) TeIL-18 and TeDRIL-18 REP TILs. (C) and (D): Assessment of differentiation in repeatedly stimulated CD4+ (C) and CD8+ (D) TeIL-18 and TeDRIL-18 REP TILs. (E) and (F): Assessment of activation in freshly thawed (E) and repeatedly stimulated (F) TeIL-18 and TeDRIL-18 REP TILs. (F) and (G): Assessment of differentiation in repeatedly stimulated CD4+ and CD8+ TeIL-18 and TeDRIL-18 REP TILs. [Figure 48E]Summary of phenotypic and functional analyses of TeIL-18 and TeDRIL-18 REP TILs. (A) and (B): Assessment of differentiation in freshly thawed CD4+ (A) and CD8+ (B) TeIL-18 and TeDRIL-18 REP TILs. (C) and (D): Assessment of differentiation in repeatedly stimulated CD4+ (C) and CD8+ (D) TeIL-18 and TeDRIL-18 REP TILs. (E) and (F): Assessment of activation in freshly thawed (E) and repeatedly stimulated (F) TeIL-18 and TeDRIL-18 REP TILs. (F) and (G): Assessment of differentiation in repeatedly stimulated CD4+ and CD8+ TeIL-18 and TeDRIL-18 REP TILs. [Figure 48F] Summary of phenotypic and functional analyses of TeIL-18 and TeDRIL-18 REP TILs. (A) and (B): Assessment of differentiation in freshly thawed CD4+ (A) and CD8+ (B) TeIL-18 and TeDRIL-18 REP TILs. (C) and (D): Assessment of differentiation in repeatedly stimulated CD4+ (C) and CD8+ (D) TeIL-18 and TeDRIL-18 REP TILs. (E) and (F): Assessment of activation in freshly thawed (E) and repeatedly stimulated (F) TeIL-18 and TeDRIL-18 REP TILs. (F) and (G): Assessment of differentiation in repeatedly stimulated CD4+ and CD8+ TeIL-18 and TeDRIL-18 REP TILs. [Figure 48G] Summary of phenotypic and functional analyses of TeIL-18 and TeDRIL-18 REP TILs. (A) and (B): Assessment of differentiation in freshly thawed CD4+ (A) and CD8+ (B) TeIL-18 and TeDRIL-18 REP TILs. (C) and (D): Assessment of differentiation in repeatedly stimulated CD4+ (C) and CD8+ (D) TeIL-18 and TeDRIL-18 REP TILs. (E) and (F): Assessment of activation in freshly thawed (E) and repeatedly stimulated (F) TeIL-18 and TeDRIL-18 REP TILs. (F) and (G): Assessment of differentiation in repeatedly stimulated CD4+ and CD8+ TeIL-18 and TeDRIL-18 REP TILs. [Figure 48H]Summary of phenotypic and functional analyses of TeIL-18 and TeDRIL-18 REP TILs. (A) and (B): Assessment of differentiation in freshly thawed CD4+ (A) and CD8+ (B) TeIL-18 and TeDRIL-18 REP TILs. (C) and (D): Assessment of differentiation in repeatedly stimulated CD4+ (C) and CD8+ (D) TeIL-18 and TeDRIL-18 REP TILs. (E) and (F): Assessment of activation in freshly thawed (E) and repeatedly stimulated (F) TeIL-18 and TeDRIL-18 REP TILs. (F) and (G): Assessment of differentiation in repeatedly stimulated CD4+ and CD8+ TeIL-18 and TeDRIL-18 REP TILs. [Figure 49A] Summary of studies to evaluate the effects of TeIL-18 and TeDRIL-18 on THP-I MHC-I and -II expression. [Figure 49B] Summary of studies to evaluate the effects of TeIL-18 and TeDRIL-18 on THP-I MHC-I and -II expression. [Figure 50] In the subject TIL embodiments provided herein, exemplary nucleic acids that enable expression of member anchor IL-12 (TeIL-12) and PD-1 shRNA are shown. [Figure 51] 1 shows an exemplary workflow for the preparation of TILs expressing TeIL-12 and / or NFAT-TeIL-12 for administration to a subject. [Figure 52A] Summary of studies evaluating the expression of TeIL-12 and / or NFAT-TeIL-12 on REP TILs. (A) After REP harvest, surface expression of TeIL-12 on TeIL-12 TILs was examined by flow assay using IL-12P70 flow Ab (B). NFAT-TeIL-12-transduced REP-TILs were stimulated with TransACT at the indicated dilutions or PMA. 48 hours after stimulation, surface-expressed TeIL-12 expression was examined. [Figure 52B]Summary of studies evaluating the expression of TeIL-12 and / or NFAT-TeIL-12 on REP TILs. (A) After REP harvest, surface expression of TeIL-12 on TeIL-12 TILs was examined by flow assay using IL-12P70 flow Ab (B). NFAT-TeIL-12-transduced REP-TILs were stimulated with TransACT at the indicated dilutions or PMA. 48 hours after stimulation, surface-expressed TeIL-12 expression was examined. [Figure 53] Summary of studies to evaluate IL-12 activity in TeIL-12-expressing TILs. [Figure 54A] Summary of studies to assess (A) expansion and (B) survival of post-REP TILs transduced to express TeIL-12 or NFAT-TeIL-12. [Figure 54B] Summary of studies to assess (A) expansion and (B) survival of post-REP TILs transduced to express TeIL-12 or NFAT-TeIL-12. [Figure 55A] (A) Summary of studies to assess the frequency of TeIL-12 and / or NFAT-TeIL-12 in REP TIL populations from various tissues (including two lung, one head and neck, one breast, and one ovarian tumor samples) and (B) viral genome copy number per cell (VCN). [Figure 55B] (A) Summary of studies to assess the frequency of TeIL-12 and / or NFAT-TeIL-12 in REP TIL populations from various tissues (including two lung, one head and neck, one breast, and one ovarian tumor samples) and (B) viral genome copy number per cell (VCN). [Figure 56A] (A) and (B) Overview of studies to evaluate cytotoxicity in a THP-1-based allogeneic cytotoxicity assay, (C) and (D) IFN-γ production of TeIL-12 REP-TILs and NFAT-driven inducible TeIL-12 REP-TILs. [Figure 56B](A) and (B) Overview of studies to evaluate cytotoxicity in a THP-1-based allogeneic cytotoxicity assay, (C) and (D) IFN-γ production of TeIL-12 REP-TILs and NFAT-driven inducible TeIL-12 REP-TILs. [Figure 56C] (A) and (B) Overview of studies to evaluate cytotoxicity in a THP-1-based allogeneic cytotoxicity assay, (C) and (D) IFN-γ production of TeIL-12 REP-TILs and NFAT-driven inducible TeIL-12 REP-TILs. [Figure 56D] (A) and (B) Overview of studies to evaluate cytotoxicity in a THP-1-based allogeneic cytotoxicity assay, (C) and (D) IFN-γ production of TeIL-12 REP-TILs and NFAT-driven inducible TeIL-12 REP-TILs. [Figure 57A] Summary of studies to assess the cytotoxicity of TeIL-12-expressing TILs was also assessed by xCelligence RTCA assay using two target cell populations (A) and (B). [Figure 57B] Summary of studies to assess the cytotoxicity of TeIL-12-expressing TILs was also assessed by xCelligence RTCA assay using two target cell populations (A) and (B). [Figure 58A] Summary of studies to evaluate TIL killing efficacy. (A) Schematic of experimental design. (B) KILR® THP-1 cytotoxicity assay and IFN-g quantification, and (C) Xcellgene RTCA killing assay were performed. [Figure 58B] Summary of studies to evaluate TIL killing efficacy. (A) Schematic of experimental design. (B) KILR® THP-1 cytotoxicity assay and IFN-g quantification, and (C) Xcellgene RTCA killing assay were performed. [Figure 58C] Summary of studies to evaluate TIL killing efficacy. (A) Schematic of experimental design. (B) KILR® THP-1 cytotoxicity assay and IFN-g quantification, and (C) Xcellgene RTCA killing assay were performed. [Figure 59] A summary of studies to assess the distribution of CD8+, CD4+, and CD4+ / FoxP3- T cells within REP TIL populations transduced to express TeIL-12 or NFAT-TeIL-12 is shown. [Figure 60A] Summary of studies to assess T cell differentiation of (A) CD8+ and (B) CD4+ T cells, as measured by various cell markers, within REP TIL populations transduced to express TeIL-12 or NFAT-TeIL-12. [Figure 60B] Summary of studies to assess T cell differentiation of (A) CD8+ and (B) CD4+ T cells, as measured by various cell markers, within REP TIL populations transduced to express TeIL-12 or NFAT-TeIL-12. [Figure 61A] Summary of studies to assess T cell depletion of (A) CD8+ and (B) CD4+ T cells, as measured by various cellular markers, within REP TIL populations transduced to express TeIL-12 or NFAT-TeIL-12. [Figure 61B] Summary of studies to assess T cell depletion of (A) CD8+ and (B) CD4+ T cells, as measured by various cellular markers, within REP TIL populations transduced to express TeIL-12 or NFAT-TeIL-12. [Figure 62A] Summary of studies to assess T cell depletion of (A) CD8+ and (B) CD4+ T cells, as measured by various cellular markers, within REP TIL populations transduced to express TeIL-12 or NFAT-TeIL-12. [Figure 62B] Summary of studies to assess T cell depletion of (A) CD8+ and (B) CD4+ T cells, as measured by various cellular markers, within REP TIL populations transduced to express TeIL-12 or NFAT-TeIL-12. [Figure 63A]Summary of studies to assess T cell function of (A) CD8+ and (B) CD4+ T cells, as measured by various cellular markers, within REP TIL populations transduced to express TeIL-12 or NFAT-TeIL-12. [Figure 63B] Summary of studies to assess T cell function of (A) CD8+ and (B) CD4+ T cells, as measured by various cellular markers, within REP TIL populations transduced to express TeIL-12 or NFAT-TeIL-12. [Figure 64A] (A) Cell expansion and (B) surface expression of TeIL-15 after treatment are shown. After gene transduction with TeIL-15 lentivirus, pre-REP TILs were treated for REP expansion with feeder cells, 3000 IU / ml of IL-2, and aCD3 Ab OKT3 or HIT3a. OKT3 (30 ng / ml) or HIT3a (30 ng / ml) was added to the REP cell medium on different days (days 0, 2, and 4) after the REP process was set up. After 11 days of REP expansion, post-REP TILs were harvested and analyzed. [Figure 64B] (A) Cell expansion and (B) surface expression of TeIL-15 after treatment are shown. After gene transduction with TeIL-15 lentivirus, pre-REP TILs were treated for REP expansion with feeder cells, 3000 IU / ml of IL-2, and aCD3 Ab OKT3 or HIT3a. OKT3 (30 ng / ml) or HIT3a (30 ng / ml) was added to the REP cell medium on different days (days 0, 2, and 4) after the REP process was set up. After 11 days of REP expansion, post-REP TILs were harvested and analyzed. [Figure 65A](A) Cell expansion and (B) surface expression of TeIL-15 / TeIL-21 after treatment are shown. After gene transduction with TeIL-15 / TeIL-21 lentivirus, pre-REP TILs were treated for REP expansion with feeder cells, 3000 IU / ml of IL-2, and aCD3 Ab OKT3 or HIT3a. OKT3 (30 ng / ml) or HIT3a (30 ng / ml) was added to the REP cell medium on different days (days 0, 2, and 4) after the REP process was set up. After 11 days of REP expansion, post-REP TILs were harvested and analyzed. [Figure 65B] (A) Cell expansion and (B) surface expression of TeIL-15 / TeIL-21 after treatment are shown. After gene transduction with TeIL-15 / TeIL-21 lentivirus, pre-REP TILs were treated for REP expansion with feeder cells, 3000 IU / ml of IL-2, and aCD3 Ab OKT3 or HIT3a. OKT3 (30 ng / ml) or HIT3a (30 ng / ml) was added to the REP cell medium on different days (days 0, 2, and 4) after the REP process was set up. After 11 days of REP expansion, post-REP TILs were harvested and analyzed. [Figure 66A] (A) Cell expansion and (B) surface expression of TeIL-15 after treatment are shown. After transduction of TeIL-15 lentiviral gene, pre-REP TILs were treated with the indicated concentrations of feeder cells, 3000 IU / ml IL-2, and OKT3 for 11 days of REP expansion. After 11 days of REP expansion, post-REP TILs were harvested and analyzed. [Figure 66B] (A) Cell expansion and (B) surface expression of TeIL-15 after treatment are shown. After transduction of TeIL-15 lentiviral gene, pre-REP TILs were treated with the indicated concentrations of feeder cells, 3000 IU / ml IL-2, and OKT3 for 11 days of REP expansion. After 11 days of REP expansion, post-REP TILs were harvested and analyzed. [Figure 67A](A) Cell expansion and (B) surface expression of TeIL-15 / TeIL-21 after treatment are shown. After transduction of TeIL-15 / TeIL-21 lentiviral genes, pre-REP TILs were treated for REP expansion with the indicated concentrations of feeder cells, 3000 IU / ml IL-2, and OKT3. After 11 days of REP expansion, post-REP TILs were harvested and analyzed. [Figure 67B] (A) Cell expansion and (B) surface expression of TeIL-15 / TeIL-21 after treatment are shown. After transduction of TeIL-15 / TeIL-21 lentiviral genes, pre-REP TILs were treated for REP expansion with the indicated concentrations of feeder cells, 3000 IU / ml IL-2, and OKT3. After 11 days of REP expansion, post-REP TILs were harvested and analyzed.

[0095] Brief Description of Sequence Listing SEQ ID NO: 1 is the amino acid sequence of the heavy chain of muromonab.

[0096] SEQ ID NO: 2 is the amino acid sequence of the light chain of muromonab.

[0097] SEQ ID NO: 3 is the amino acid sequence of recombinant human IL-2 protein.

[0098] SEQ ID NO: 4 is the amino acid sequence of aldesleukin.

[0099] SEQ ID NO: 5 is an IL-2 form.

[0100] SEQ ID NO: 6 is the amino acid sequence of nemvaleukin alpha.

[0101] SEQ ID NO: 7 is an IL-2 form.

[0102] SEQ ID NO: 8 is a mucin domain polypeptide.

[0103] SEQ ID NO: 9 is the amino acid sequence of recombinant human IL-4 protein.

[0104] SEQ ID NO: 10 is the amino acid sequence of recombinant human IL-7 protein.

[0105] SEQ ID NO: 11 is the amino acid sequence of recombinant human IL-15 protein.

[0106] SEQ ID NO: 12 is the amino acid sequence of recombinant human IL-21 protein.

[0107] SEQ ID NO: 13 is the IL-2 sequence.

[0108] SEQ ID NO: 14 is the IL-2 mutein sequence.

[0109] SEQ ID NO: 15 is the sequence of an IL-2 mutein.

[0110] SEQ ID NO: 16 is HCDR1_IL-2 of IgG.IL2R67A.H1.

[0111] SEQ ID NO: 17 is HCDR2 of IgG.IL2R67A.H1.

[0112] SEQ ID NO: 18 is the HCDR3 of IgG.IL2R67A.H1.

[0113] SEQ ID NO: 19 is HCDR1_IL-2 Kabat of IgG.IL2R67A.H1.

[0114] SEQ ID NO: 20 is the HCDR2 Kabat of IgG.IL2R67A.H1.

[0115] SEQ ID NO: 21 is the HCDR3 Kabat of IgG.IL2R67A.H1.

[0116] SEQ ID NO: 22 is the HCDR1_IL-2 clotia of IgG.IL2R67A.H1.

[0117] SEQ ID NO: 23 is the HCDR2 clone of IgG.IL2R67A.H1.

[0118] SEQ ID NO: 24 is the HCDR3 clone of IgG.IL2R67A.H1.

[0119] SEQ ID NO: 25 is HCDR1_IL-2 IMGT of IgG.IL2R67A.H1.

[0120] SEQ ID NO: 26 is HCDR2 IMGT of IgG.IL2R67A.H1.

[0121] SEQ ID NO: 27 is the HCDR3 IMGT of IgG.IL2R67A.H1.

[0122] SEQ ID NO: 28 is the V H It's a chain.

[0123] SEQ ID NO: 29 is the heavy chain of IgG.IL2R67A.H1.

[0124] SEQ ID NO: 30 is the LCDR1 Kabat of IgG.IL2R67A.H1.

[0125] SEQ ID NO: 31 is the LCDR2 Kabat of IgG.IL2R67A.H1.

[0126] SEQ ID NO: 32 is the LCDR3 Kabat of IgG.IL2R67A.H1.

[0127] SEQ ID NO: 33 is the LCDR1 chothia of IgG.IL2R67A.H1.

[0128] SEQ ID NO: 34 is the LCDR2 chothia of IgG.IL2R67A.H1.

[0129] SEQ ID NO: 35 is the LCDR3 chothia of IgG.IL2R67A.H1.

[0130] SEQ ID NO: 36 is V L It's a chain.

[0131] SEQ ID NO: 37 is the light chain.

[0132] SEQ ID NO: 38 is the light chain.

[0133] SEQ ID NO: 39 is the light chain.

[0134] SEQ ID NO: 40 is the amino acid sequence of human 4-1BB.

[0135] SEQ ID NO: 41 is the amino acid sequence of mouse 4-1BB.

[0136] SEQ ID NO: 42 is the heavy chain of the 4-1BB agonist monoclonal antibody utomilumab (PF-05082566).

[0137] SEQ ID NO: 43 is the light chain of the 4-1BB agonist monoclonal antibody utomilumab (PF-05082566).

[0138] SEQ ID NO: 44 represents the heavy chain variable region (V) of the 4-1BB agonist monoclonal antibody utomilumab (PF-05082566). H )

[0139] SEQ ID NO: 45 is the light chain variable region (V) of the 4-1BB agonist monoclonal antibody utomilumab (PF-05082566). L )

[0140] SEQ ID NO: 46 is the heavy chain CDR1 of the 4-1BB agonist monoclonal antibody utomilumab (PF-05082566).

[0141] SEQ ID NO: 47 is the heavy chain CDR2 of the 4-1BB agonist monoclonal antibody utomilumab (PF-05082566).

[0142] SEQ ID NO: 48 is the heavy chain CDR3 of the 4-1BB agonist monoclonal antibody utomilumab (PF-05082566).

[0143] SEQ ID NO: 49 is the light chain CDR1 of the 4-1BB agonist monoclonal antibody utomilumab (PF-05082566).

[0144] SEQ ID NO: 50 is the light chain CDR2 of the 4-1BB agonist monoclonal antibody utomilumab (PF-05082566).

[0145] SEQ ID NO: 51 is the light chain CDR3 of the 4-1BB agonist monoclonal antibody utomilumab (PF-05082566).

[0146] SEQ ID NO: 52 is the heavy chain of the 4-1BB agonist monoclonal antibody urelumab (BMS-663513).

[0147] SEQ ID NO: 53 is the light chain of the 4-1BB agonist monoclonal antibody urelumab (BMS-663513).

[0148] SEQ ID NO: 54 is the heavy chain variable region (VH) of the 4-1BB agonist monoclonal antibody urelumab (BMS-663513).

[0149] SEQ ID NO: 55 is the light chain variable region (VL) of the 4-1BB agonist monoclonal antibody urelumab (BMS-663513).

[0150] SEQ ID NO: 56 is the heavy chain CDR1 of the 4-1BB agonist monoclonal antibody urelumab (BMS-663513).

[0151] SEQ ID NO: 57 is the heavy chain CDR2 of the 4-1BB agonist monoclonal antibody urelumab (BMS-663513).

[0152] SEQ ID NO: 58 is the heavy chain CDR3 of the 4-1BB agonist monoclonal antibody urelumab (BMS-663513).

[0153] SEQ ID NO: 59 is the light chain CDR1 of the 4-1BB agonist monoclonal antibody urelumab (BMS-663513).

[0154] SEQ ID NO: 60 is the light chain CDR2 of the 4-1BB agonist monoclonal antibody urelumab (BMS-663513).

[0155] SEQ ID NO: 61 is the light chain CDR3 of the 4-1BB agonist monoclonal antibody urelumab (BMS-663513).

[0156] SEQ ID NO: 62 is the Fc domain of the TNFRSF agonist fusion protein.

[0157] SEQ ID NO: 63 is the linker of the TNFRSF agonist fusion protein.

[0158] SEQ ID NO: 64 is the linker of the TNFRSF agonist fusion protein.

[0159] SEQ ID NO: 65 is the linker of the TNFRSF agonist fusion protein.

[0160] SEQ ID NO: 66 is the linker of the TNFRSF agonist fusion protein.

[0161] SEQ ID NO: 67 is the linker for the TNFRSF agonist fusion protein.

[0162] SEQ ID NO: 68 is the linker for the TNFRSF agonist fusion protein.

[0163] SEQ ID NO: 69 is the linker for the TNFRSF agonist fusion protein.

[0164] SEQ ID NO: 70 is the linker of the TNFRSF agonist fusion protein.

[0165] SEQ ID NO: 71 is the linker for the TNFRSF agonist fusion protein.

[0166] SEQ ID NO: 72 is the linker for the TNFRSF agonist fusion protein.

[0167] SEQ ID NO: 73 is the Fc domain of the TNFRSF agonist fusion protein.

[0168] SEQ ID NO: 74 is the linker for the TNFRSF agonist fusion protein.

[0169] SEQ ID NO: 75 is the linker for the TNFRSF agonist fusion protein.

[0170] SEQ ID NO: 76 is the linker for the TNFRSF agonist fusion protein.

[0171] SEQ ID NO: 77 is the 4-1BB ligand (4-1BBL) amino acid sequence.

[0172] SEQ ID NO: 78 is the soluble portion of the 4-1BBL polypeptide.

[0173] SEQ ID NO: 79 is the heavy chain variable region (V) of 4-1BB agonist antibody 4B4-1-1 version 1 H )

[0174] SEQ ID NO: 80 represents the light chain variable region (V) of 4-1BB agonist antibody 4B4-1-1 version 1 L )

[0175] SEQ ID NO: 81 is the heavy chain variable region (V H )

[0176] SEQ ID NO: 82 is the light chain variable region (V) of 4-1BB agonist antibody 4B4-1-1 version 2 L )

[0177] SEQ ID NO: 83 identifies the heavy chain variable region (V H )

[0178] SEQ ID NO: 84 identifies the light chain variable region (V L )

[0179] SEQ ID NO: 85 is the amino acid sequence of human OX40.

[0180] SEQ ID NO: 86 is the amino acid sequence of mouse OX40.

[0181] SEQ ID NO: 87 is the heavy chain of the OX40 agonist monoclonal antibody tabolixizumab (MEDI-0562).

[0182] SEQ ID NO: 88 is the light chain of the OX40 agonist monoclonal antibody tabolixizumab (MEDI-0562).

[0183] SEQ ID NO: 89 is the heavy chain variable region (V) of the OX40 agonist monoclonal antibody tabolixizumab (MEDI-0562). H )

[0184] SEQ ID NO: 90 is the light chain variable region (V) of the OX40 agonist monoclonal antibody tabolixizumab (MEDI-0562). L )

[0185] SEQ ID NO: 91 is the heavy chain CDR1 of the OX40 agonist monoclonal antibody tabolixizumab (MEDI-0562).

[0186] SEQ ID NO: 92 is the heavy chain CDR2 of the OX40 agonist monoclonal antibody tabolixizumab (MEDI-0562).

[0187] SEQ ID NO: 93 is the heavy chain CDR3 of the OX40 agonist monoclonal antibody tabolixizumab (MEDI-0562).

[0188] SEQ ID NO: 94 is the light chain CDR1 of the OX40 agonist monoclonal antibody tabolixizumab (MEDI-0562).

[0189] SEQ ID NO: 95 is the light chain CDR2 of the OX40 agonist monoclonal antibody tabolixizumab (MEDI-0562).

[0190] SEQ ID NO: 96 is the light chain CDR3 of the OX40 agonist monoclonal antibody tabolixizumab (MEDI-0562).

[0191] SEQ ID NO: 97 is the heavy chain of the OX40 agonist monoclonal antibody 11D4.

[0192] SEQ ID NO: 98 is the light chain of the OX40 agonist monoclonal antibody 11D4.

[0193] SEQ ID NO: 99 is the heavy chain variable region (V) of the OX40 agonist monoclonal antibody 11D4. H )

[0194] SEQ ID NO: 100 is the light chain variable region (V) of the OX40 agonist monoclonal antibody 11D4. L )

[0195] SEQ ID NO: 101 is the heavy chain CDR1 of the OX40 agonist monoclonal antibody 11D4.

[0196] SEQ ID NO: 102 is the heavy chain CDR2 of the OX40 agonist monoclonal antibody 11D4.

[0197] SEQ ID NO: 103 is the heavy chain CDR3 of the OX40 agonist monoclonal antibody 11D4.

[0198] SEQ ID NO: 104 is the light chain CDR1 of the OX40 agonist monoclonal antibody 11D4.

[0199] SEQ ID NO: 105 is the light chain CDR2 of the OX40 agonist monoclonal antibody 11D4.

[0200] SEQ ID NO: 106 is the light chain CDR3 of the OX40 agonist monoclonal antibody 11D4.

[0201] SEQ ID NO: 107 is the heavy chain of the OX40 agonist monoclonal antibody 18D8.

[0202] SEQ ID NO: 108 is the light chain of the OX40 agonist monoclonal antibody 18D8.

[0203] SEQ ID NO: 109 is the heavy chain variable region (V) of the OX40 agonist monoclonal antibody 18D8. H )

[0204] SEQ ID NO: 110 is the light chain variable region (V) of the OX40 agonist monoclonal antibody 18D8. L )

[0205] SEQ ID NO: 111 is the heavy chain CDR1 of the OX40 agonist monoclonal antibody 18D8.

[0206] SEQ ID NO: 112 is the heavy chain CDR2 of the OX40 agonist monoclonal antibody 18D8.

[0207] SEQ ID NO: 113 is the heavy chain CDR3 of the OX40 agonist monoclonal antibody 18D8.

[0208] SEQ ID NO: 114 is the light chain CDR1 of the OX40 agonist monoclonal antibody 18D8.

[0209] SEQ ID NO: 115 is the light chain CDR2 of the OX40 agonist monoclonal antibody 18D8.

[0210] SEQ ID NO: 116 is the light chain CDR3 of the OX40 agonist monoclonal antibody 18D8.

[0211] SEQ ID NO: 117 is the heavy chain variable region (V) of the OX40 agonist monoclonal antibody Hu119-122. H )

[0212] SEQ ID NO: 118 is the light chain variable region (V) of the OX40 agonist monoclonal antibody Hu119-122. L )

[0213] SEQ ID NO: 119 is the heavy chain CDR1 of the OX40 agonist monoclonal antibody Hu119-122.

[0214] SEQ ID NO: 120 is the heavy chain CDR2 of the OX40 agonist monoclonal antibody Hu119-122.

[0215] SEQ ID NO: 121 is the heavy chain CDR3 of the OX40 agonist monoclonal antibody Hu119-122.

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

[0217] SEQ ID NO: 123 is the light chain CDR2 of the OX40 agonist monoclonal antibody Hu119-122.

[0218] SEQ ID NO: 124 is the light chain CDR3 of the OX40 agonist monoclonal antibody Hu119-122.

[0219] SEQ ID NO: 125 is the heavy chain variable region (V) of the OX40 agonist monoclonal antibody Hu106-222. H )

[0220] SEQ ID NO: 126 is the light chain variable region (V) of the OX40 agonist monoclonal antibody Hu106-222. L )

[0221] SEQ ID NO: 127 is the heavy chain CDR1 of the OX40 agonist monoclonal antibody Hu106-222.

[0222] SEQ ID NO: 128 is the heavy chain CDR2 of the OX40 agonist monoclonal antibody Hu106-222.

[0223] SEQ ID NO: 129 is the heavy chain CDR3 of the OX40 agonist monoclonal antibody Hu106-222.

[0224] SEQ ID NO: 130 is the light chain CDR1 of the OX40 agonist monoclonal antibody Hu106-222.

[0225] SEQ ID NO: 131 is the light chain CDR2 of the OX40 agonist monoclonal antibody Hu106-222.

[0226] SEQ ID NO: 132 is the light chain CDR3 of the OX40 agonist monoclonal antibody Hu106-222.

[0227] SEQ ID NO: 133 is the OX40 ligand (OX40L) amino acid sequence.

[0228] SEQ ID NO: 134 is the soluble portion of the OX40L polypeptide.

[0229] SEQ ID NO: 135 is an alternative soluble portion of the OX40L polypeptide.

[0230] SEQ ID NO: 136 is the heavy chain variable region (V H )

[0231] SEQ ID NO: 137 is the light chain variable region (V) of OX40 agonist monoclonal antibody 008. L )

[0232] SEQ ID NO: 138 is the heavy chain variable region (V H )

[0233] SEQ ID NO: 139 is the light chain variable region (V) of the OX40 agonist monoclonal antibody 011. L )

[0234] SEQ ID NO: 140 is the heavy chain variable region (V) of the OX40 agonist monoclonal antibody 021. H )

[0235] SEQ ID NO: 141 is the light chain variable region (V) of the OX40 agonist monoclonal antibody 021. L )

[0236] SEQ ID NO: 142 is the heavy chain variable region (V) of the OX40 agonist monoclonal antibody 023. H )

[0237] SEQ ID NO: 143 is the light chain variable region (V) of the OX40 agonist monoclonal antibody 023. L )

[0238] SEQ ID NO: 144 is the heavy chain variable region (V H )

[0239] SEQ ID NO: 145 is the light chain variable region (V) of the OX40 agonist monoclonal antibody L )

[0240] SEQ ID NO: 146 is the heavy chain variable region (V H )

[0241] SEQ ID NO: 147 is the light chain variable region (V) of the OX40 agonist monoclonal antibody L )

[0242] SEQ ID NO: 148 is the heavy chain variable region (V H )

[0243] SEQ ID NO: 149 is the heavy chain variable region (V) of a humanized OX40 agonist monoclonal antibody. H )

[0244] SEQ ID NO: 150 is the light chain variable region (V) of a humanized OX40 agonist monoclonal antibody. L)

[0245] SEQ ID NO: 151 is the light chain variable region (V) of a humanized OX40 agonist monoclonal antibody. L )

[0246] SEQ ID NO: 152 is the heavy chain variable region (V H )

[0247] SEQ ID NO: 153 is the heavy chain variable region (V) of a humanized OX40 agonist monoclonal antibody. H )

[0248] SEQ ID NO: 154 is the light chain variable region (V) of a humanized OX40 agonist monoclonal antibody. L )

[0249] SEQ ID NO: 155 is the light chain variable region (V) of a humanized OX40 agonist monoclonal antibody. L )

[0250] SEQ ID NO: 156 is the heavy chain variable region (V H )

[0251] SEQ ID NO: 157 is the light chain variable region (V) of the OX40 agonist monoclonal antibody L )

[0252] SEQ ID NO: 158 is the heavy chain amino acid sequence of the PD-1 inhibitor nivolumab.

[0253] SEQ ID NO: 159 is the light chain amino acid sequence of the PD-1 inhibitor nivolumab.

[0254] SEQ ID NO: 160 represents the heavy chain variable region (V) of the PD-1 inhibitor nivolumab H ) amino acid sequence.

[0255] SEQ ID NO: 161 represents the light chain variable region (V) of the PD-1 inhibitor nivolumab L) amino acid sequence.

[0256] SEQ ID NO: 162 is the heavy chain CDR1 amino acid sequence of the PD-1 inhibitor nivolumab.

[0257] SEQ ID NO: 163 is the heavy chain CDR2 amino acid sequence of the PD-1 inhibitor nivolumab.

[0258] SEQ ID NO: 164 is the heavy chain CDR3 amino acid sequence of the PD-1 inhibitor nivolumab.

[0259] SEQ ID NO: 165 is the light chain CDR1 amino acid sequence of the PD-1 inhibitor nivolumab.

[0260] SEQ ID NO: 166 is the light chain CDR2 amino acid sequence of the PD-1 inhibitor nivolumab.

[0261] SEQ ID NO: 167 is the light chain CDR3 amino acid sequence of the PD-1 inhibitor nivolumab.

[0262] SEQ ID NO: 168 is the heavy chain amino acid sequence of the PD-1 inhibitor pembrolizumab.

[0263] SEQ ID NO: 169 is the light chain amino acid sequence of the PD-1 inhibitor pembrolizumab.

[0264] SEQ ID NO: 170 represents the heavy chain variable region (V) of the PD-1 inhibitor pembrolizumab H ) amino acid sequence.

[0265] SEQ ID NO: 171 is the light chain variable region (V) of the PD-1 inhibitor pembrolizumab L ) amino acid sequence.

[0266] SEQ ID NO: 172 is the heavy chain CDR1 amino acid sequence of the PD-1 inhibitor pembrolizumab.

[0267] SEQ ID NO: 173 is the heavy chain CDR2 amino acid sequence of the PD-1 inhibitor pembrolizumab.

[0268] SEQ ID NO: 174 is the heavy chain CDR3 amino acid sequence of the PD-1 inhibitor pembrolizumab.

[0269] SEQ ID NO: 175 is the light chain CDR1 amino acid sequence of the PD-1 inhibitor pembrolizumab.

[0270] SEQ ID NO: 176 is the light chain CDR2 amino acid sequence of the PD-1 inhibitor pembrolizumab.

[0271] SEQ ID NO: 177 is the light chain CDR3 amino acid sequence of the PD-1 inhibitor pembrolizumab.

[0272] SEQ ID NO: 178 is the heavy chain amino acid sequence of the PD-L1 inhibitor durvalumab.

[0273] SEQ ID NO: 179 is the light chain amino acid sequence of the PD-L1 inhibitor durvalumab.

[0274] SEQ ID NO: 180 represents the heavy chain variable region (V) of the PD-L1 inhibitor durvalumab H ) amino acid sequence.

[0275] SEQ ID NO: 181 represents the light chain variable region (V) of the PD-L1 inhibitor durvalumab L ) amino acid sequence.

[0276] SEQ ID NO: 182 is the heavy chain CDR1 amino acid sequence of the PD-L1 inhibitor durvalumab.

[0277] SEQ ID NO: 183 is the heavy chain CDR2 amino acid sequence of the PD-L1 inhibitor durvalumab.

[0278] SEQ ID NO: 184 is the heavy chain CDR3 amino acid sequence of the PD-L1 inhibitor durvalumab.

[0279] SEQ ID NO: 185 is the light chain CDR1 amino acid sequence of the PD-L1 inhibitor durvalumab.

[0280] SEQ ID NO: 186 is the light chain CDR2 amino acid sequence of the PD-L1 inhibitor durvalumab.

[0281] SEQ ID NO: 187 is the light chain CDR3 amino acid sequence of the PD-L1 inhibitor durvalumab.

[0282] SEQ ID NO: 188 is the heavy chain amino acid sequence of the PD-L1 inhibitor avelumab.

[0283] SEQ ID NO: 189 is the light chain amino acid sequence of the PD-L1 inhibitor avelumab.

[0284] SEQ ID NO: 190 represents the heavy chain variable region (V) of the PD-L1 inhibitor avelumab H ) amino acid sequence.

[0285] SEQ ID NO: 191 is the light chain variable region (V) of the PD-L1 inhibitor avelumab L ) amino acid sequence.

[0286] SEQ ID NO: 192 is the heavy chain CDR1 amino acid sequence of the PD-L1 inhibitor avelumab.

[0287] SEQ ID NO: 193 is the heavy chain CDR2 amino acid sequence of the PD-L1 inhibitor avelumab.

[0288] SEQ ID NO: 194 is the heavy chain CDR3 amino acid sequence of the PD-L1 inhibitor avelumab.

[0289] SEQ ID NO: 195 is the light chain CDR1 amino acid sequence of the PD-L1 inhibitor avelumab.

[0290] SEQ ID NO: 196 is the light chain CDR2 amino acid sequence of the PD-L1 inhibitor avelumab.

[0291] SEQ ID NO: 197 is the light chain CDR3 amino acid sequence of the PD-L1 inhibitor avelumab.

[0292] SEQ ID NO: 198 is the heavy chain amino acid sequence of the PD-L1 inhibitor atezolizumab.

[0293] SEQ ID NO: 199 is the light chain amino acid sequence of the PD-L1 inhibitor atezolizumab.

[0294] SEQ ID NO: 200 represents the heavy chain variable region (V) of the PD-L1 inhibitor atezolizumab H ) amino acid sequence.

[0295] SEQ ID NO: 201 is the light chain variable region (V) of the PD-L1 inhibitor atezolizumab L ) amino acid sequence.

[0296] SEQ ID NO: 202 is the heavy chain CDR1 amino acid sequence of the PD-L1 inhibitor atezolizumab.

[0297] SEQ ID NO: 203 is the heavy chain CDR2 amino acid sequence of the PD-L1 inhibitor atezolizumab.

[0298] SEQ ID NO: 204 is the heavy chain CDR3 amino acid sequence of the PD-L1 inhibitor atezolizumab.

[0299] SEQ ID NO: 205 is the light chain CDR1 amino acid sequence of the PD-L1 inhibitor atezolizumab.

[0300] SEQ ID NO: 206 is the light chain CDR2 amino acid sequence of the PD-L1 inhibitor atezolizumab.

[0301] SEQ ID NO: 207 is the light chain CDR3 amino acid sequence of the PD-L1 inhibitor atezolizumab.

[0302] SEQ ID NO: 208 is the heavy chain amino acid sequence of the CTLA-4 inhibitor ipilimumab.

[0303] SEQ ID NO: 209 is the light chain amino acid sequence of the CTLA-4 inhibitor ipilimumab.

[0304] SEQ ID NO: 210 represents the heavy chain variable region (V) of the CTLA-4 inhibitor ipilimumab H ) amino acid sequence.

[0305] SEQ ID NO: 211 represents the light chain variable region (V) of the CTLA-4 inhibitor ipilimumab L ) amino acid sequence.

[0306] SEQ ID NO: 212 is the heavy chain CDR1 amino acid sequence of the CTLA-4 inhibitor ipilimumab.

[0307] SEQ ID NO: 213 is the heavy chain CDR2 amino acid sequence of the CTLA-4 inhibitor ipilimumab.

[0308] SEQ ID NO: 214 is the heavy chain CDR3 amino acid sequence of the CTLA-4 inhibitor ipilimumab.

[0309] SEQ ID NO: 215 is the light chain CDR1 amino acid sequence of the CTLA-4 inhibitor ipilimumab.

[0310] SEQ ID NO: 216 is the light chain CDR2 amino acid sequence of the CTLA-4 inhibitor ipilimumab.

[0311] SEQ ID NO: 217 is the light chain CDR3 amino acid sequence of the CTLA-4 inhibitor ipilimumab.

[0312] SEQ ID NO: 218 is the heavy chain amino acid sequence of the CTLA-4 inhibitor tremelimumab.

[0313] SEQ ID NO: 219 is the light chain amino acid sequence of the CTLA-4 inhibitor tremelimumab.

[0314] SEQ ID NO: 220 represents the heavy chain variable region (V) of the CTLA-4 inhibitor tremelimumab H ) amino acid sequence.

[0315] SEQ ID NO: 221 represents the light chain variable region (V) of the CTLA-4 inhibitor tremelimumab L ) amino acid sequence.

[0316] SEQ ID NO: 222 is the heavy chain CDR1 amino acid sequence of the CTLA-4 inhibitor tremelimumab.

[0317] SEQ ID NO: 223 is the heavy chain CDR2 amino acid sequence of the CTLA-4 inhibitor tremelimumab.

[0318] SEQ ID NO: 224 is the heavy chain CDR3 amino acid sequence of the CTLA-4 inhibitor tremelimumab.

[0319] SEQ ID NO: 225 is the light chain CDR1 amino acid sequence of the CTLA-4 inhibitor tremelimumab.

[0320] SEQ ID NO: 226 is the light chain CDR2 amino acid sequence of the CTLA-4 inhibitor tremelimumab.

[0321] SEQ ID NO: 227 is the light chain CDR3 amino acid sequence of the CTLA-4 inhibitor tremelimumab.

[0322] SEQ ID NO: 228 is the heavy chain amino acid sequence of the CTLA-4 inhibitor zalifrelimab.

[0323] SEQ ID NO: 229 is the light chain amino acid sequence of the CTLA-4 inhibitor zalifrelimab.

[0324] SEQ ID NO: 230 is the heavy chain variable region (V) of the CTLA-4 inhibitor zalifrelimab H ) amino acid sequence.

[0325] SEQ ID NO: 231 is the light chain variable region (V) of the CTLA-4 inhibitor zalifrelimab L ) amino acid sequence.

[0326] SEQ ID NO: 232 is the heavy chain CDR1 amino acid sequence of the CTLA-4 inhibitor zalifrelimab.

[0327] SEQ ID NO: 233 is the heavy chain CDR2 amino acid sequence of the CTLA-4 inhibitor zalifrelimab.

[0328] SEQ ID NO: 234 is the heavy chain CDR3 amino acid sequence of the CTLA-4 inhibitor zalifrelimab.

[0329] SEQ ID NO: 235 is the light chain CDR1 amino acid sequence of the CTLA-4 inhibitor zalifrelimab.

[0330] SEQ ID NO: 236 is the light chain CDR2 amino acid sequence of the CTLA-4 inhibitor zalifrelimab.

[0331] SEQ ID NO: 237 is the light chain CDR3 amino acid sequence of the CTLA-4 inhibitor zalifrelimab.

[0332] SEQ ID NO: 238 is the CD8a transmembrane domain.

[0333] SEQ ID NO: 239 is the B7-1 transmembrane intracellular domain.

[0334] SEQ ID NOs:240-245 are exemplary glycine-serine linkers useful in the immunomodulatory fusion proteins described herein.

[0335] SEQ ID NO:246 is an exemplary linker that is useful in the immunomodulatory fusion proteins described herein.

[0336] SEQ ID NO: 247 is the 2A peptide C-terminal sequence.

[0337] SEQ ID NO: 248 is the porcine teschovirus-1 2A peptide.

[0338] SEQ ID NO: 249 is the equine rhinitis A virus 2A peptide.

[0339] SEQ ID NO: 250 is the foot and mouth disease virus 2A peptide.

[0340] SEQ ID NO: 251 is an exemplary furin-cleavable 2A peptide.

[0341] SEQ ID NOs: 252 and 253 are human IgE signal peptide sequences. SEQ ID NO: 254 is a human IL-2 signal peptide sequence.

[0342] SEQ ID NO: 255 is the 6xNFAT IL-2 minimal promoter.

[0343] SEQ ID NO: 256 is an NFAT responsive element.

[0344] SEQ ID NO: 257 is the human IL-2 promoter sequence.

[0345] SEQ ID NO: 258 is human IL-15 (N72D mutant).

[0346] SEQ ID NO: 259 is the human IL-15R-alpha-Su / Fc domain.

[0347] SEQ ID NO: 260 is human IL-15R-alpha-Su (65 aa truncated extracellular domain).

[0348] SEQ ID NO: 261 is human IL-15 isoform 2.

[0349] SEQ ID NO: 262 is human IL-15 isoform 1.

[0350] SEQ ID NO: 263 is human IL-15 (without the signal peptide).

[0351] SEQ ID NO: 264 is human IL-15R-alpha (85 aa truncated extracellular domain).

[0352] SEQ ID NO: 265 is human IL-15R-alpha (182 aa truncated extracellular domain).

[0353] SEQ ID NO: 266 is human IL-15R-alpha.

[0354] SEQ ID NO: 267 is the human IL-12 p35 subunit.

[0355] SEQ ID NO: 268 is the human IL-12 p40 subunit.

[0356] SEQ ID NO: 269 is human IL-18.

[0357] SEQ ID NO: 270 is a human IL-18 variant.

[0358] SEQ ID NO: 271 is human IL-21.

[0359] SEQ ID NO: 272 is human IL-2.

[0360] SEQ ID NO: 273 is human CD40L.

[0361] SEQ ID NO: 274 is an agonist anti-human CD40 VH (sotigalimb).

[0362] SEQ ID NO: 275 is an agonist anti-human CD40 VL (sotigalimb).

[0363] SEQ ID NO: 276 is an agonist anti-human CD40 scFv (sotigalimb).

[0364] SEQ ID NO: 277 is an agonist anti-human CD40 VH (dacetuzumab).

[0365] SEQ ID NO: 278 is an agonist anti-human CD40 VL (dacetuzumab).

[0366] SEQ ID NO: 279 is an agonist anti-human CD40 scFv (dacetuzumab).

[0367] SEQ ID NO: 280 is an agonist anti-human CD40 VH (lucatuzumab).

[0368] SEQ ID NO: 281 is an agonist anti-human CD40 VL (lucatuzumab).

[0369] SEQ ID NO: 282 is an agonist anti-human CD40 scFv (lucatuzumab).

[0370] SEQ ID NO: 283 is an agonist anti-human CD40 VH (celicrelumab).

[0371] SEQ ID NO: 284 is an agonist anti-human CD40 VL (celicrelumab).

[0372] SEQ ID NO: 285 is an agonist anti-human CD40 scFv (celicrelumab).

[0373] SEQ ID NO: 286 is the target PD-1 sequence.

[0374] SEQ ID NO: 287 is the target PD-1 sequence.

[0375] SEQ ID NO: 288 is a repeated PD-1 left repeat sequence.

[0376] SEQ ID NO: 289 is a repeated PD-1 right repeat sequence.

[0377] SEQ ID NO: 290 is a repeated PD-1 left repeat sequence.

[0378] SEQ ID NO: 291 is a repeated PD-1 right repeat sequence.

[0379] SEQ ID NO: 292 is the PD-1 left TALEN nuclease sequence.

[0380] SEQ ID NO: 293 is the PD-1 right TALEN nuclease sequence.

[0381] SEQ ID NO: 294 is the PD-1 left TALEN nuclease sequence.

[0382] SEQ ID NO: 295 is the PD-1 right TALEN nuclease sequence.

[0383] SEQ ID NO:296 is the nucleic acid sequence encoding the tethered IL-15 of SEQ ID NO:328.

[0384] SEQ ID NO:297 is a nucleic acid sequence encoding the tethered IL-21 fusion protein of SEQ ID NO:

[0385] SEQ ID NO:298 is a nucleic acid sequence encoding the tethered IL-15 fusion protein of SEQ ID NO:328 and the tethered IL-21 fusion protein of SEQ ID NO:331.

[0386] SEQ ID NO: 299 is a nucleic acid sequence encoding the tethered IL-12 fusion protein of SEQ ID NO: 303. The nucleic acid sequence includes the NFAT promoter.

[0387] SEQ ID NO: 300 is a nucleic acid sequence encoding the tethered IL-15 fusion protein of SEQ ID NO: 328. The nucleic acid sequence includes the NFAT promoter.

[0388] SEQ ID NO:301 is a nucleic acid sequence encoding the tethered IL-21 fusion protein of SEQ ID NO:XX. The nucleic acid sequence includes the NFAT promoter.

[0389] SEQ ID NO: 302 is a nucleic acid sequence encoding a tethered IL-15 fusion protein of SEQ ID NO: 328 and a tethered IL-21 fusion protein of SEQ ID NO: 331. The nucleic acid sequence comprises an NFAT promoter.

[0390] SEQ ID NO:303 is the amino acid sequence of an exemplary tethered IL-12 (tethered IL-12-Lr1-Ar2).

[0391] SEQ ID NO:304 is a nucleic acid sequence encoding the tethered IL-12 of SEQ ID NO:303.

[0392] SEQ ID NO:305 is the amino acid sequence of an exemplary tethered IL-18 (tethered IL-18-Lr1-Ar2).

[0393] SEQ ID NO:306 is a nucleic acid sequence encoding the tethered IL-18 of SEQ ID NO:305.

[0394] SEQ ID NO: 307 is the amino acid sequence of an exemplary tethered variant IL-18 (tethered DR-IL-18(6-27 variant)-Lr1-Ar2).

[0395] SEQ ID NO:308 is a nucleic acid sequence encoding the tethered variant IL-18 of SEQ ID NO:307.

[0396] SEQ ID NO:309 is the amino acid sequence of an exemplary tethered IL-12 / IL-15.

[0397] SEQ ID NO:310 is a nucleic acid sequence encoding the tethered IL-12 / IL-15 of SEQ ID NO:309.

[0398] SEQ ID NO:311 is the amino acid sequence of an exemplary tethered IL-18 / IL-15.

[0399] SEQ ID NO:312 is a nucleic acid sequence encoding the tethered IL-18 / IL-15 of SEQ ID NO:311.

[0400] SEQ ID NO: 313 is the amino acid sequence of an exemplary tethered anti-CD40 scFV (APX005M).

[0401] SEQ ID NO: 314 is the nucleic acid sequence encoding the tethered anti-CD40scFV (APX005M) of SEQ ID NO: 313.

[0402] SEQ ID NO: 315 is the amino acid sequence of an exemplary tethered anti-CD40scFV (dacetuzumab).

[0403] SEQ ID NO: 316 is the nucleic acid sequence encoding the tethered anti-CD40scFV (dacetuzumab) of SEQ ID NO: 315.

[0404] SEQ ID NO: 317 is the amino acid sequence of an exemplary tethered anti-CD40 scFV (lucatuzumab).

[0405] SEQ ID NO: 318 is the nucleic acid sequence encoding the tethered anti-CD40 scFV (lucatuzumab) of SEQ ID NO: 317.

[0406] SEQ ID NO: 319 is the amino acid sequence of an exemplary tethered anti-CD40 scFV (celicrelumab).

[0407] SEQ ID NO: 320 is the nucleic acid sequence encoding the tethered anti-CD40 scFV (celicrelumab) of SEQ ID NO: 319.

[0408] SEQ ID NO:321 is the nucleic acid sequence encoding the CD40L of SEQ ID NO:273.

[0409] SEQ ID NO:322 is the amino acid sequence of an exemplary tethered CD40L / IL-15.

[0410] SEQ ID NO:323 is the nucleic acid sequence encoding the tethered CD40L / IL-15 of SEQ ID NO:311.

[0411] SEQ ID NO:324 is the amino acid sequence of an exemplary tethered IL-2.

[0412] SEQ ID NO:325 is the nucleic acid sequence encoding the tethered IL-2 of SEQ ID NO:313.

[0413] SEQ ID NO:326 is the amino acid sequence of an exemplary tethered IL-12.

[0414] SEQ ID NO:327 is the nucleic acid sequence encoding the tethered IL-12 of SEQ ID NO:315.

[0415] SEQ ID NO: 328 is the amino acid sequence of an exemplary tethered IL-15.

[0416] SEQ ID NO:329 is the nucleic acid sequence encoding the tethered IL-15 of SEQ ID NO:317.

[0417] SEQ ID NO: 330 is a nucleic acid sequence encoding GFP.

[0418] SEQ ID NOs: 331-385 are additional variant IL-18 nucleic acids (eg, decoy-resistant IL-18 or "DR-IL18").

[0419] SEQ ID NO: 386 is the Clo051 nuclease domain amino acid sequence.

[0420] SEQ ID NO:387 is an exemplary piggyBac (PB) transposase enzyme amino acid sequence.

[0421] SEQ ID NO:388 is an exemplary Sleeping Beauty transposase enzyme amino acid sequence.

[0422] SEQ ID NO: 389 is an exemplary hyperactive Sleeping Beauty (SB100X) transposase amino acid sequence.

[0423] SEQ ID NO: 390 is an exemplary nucleic acid sequence of a 6XNFAT binding motif.

[0424] SEQ ID NO: 391 is an exemplary nucleic acid sequence of the IL-2 min promoter.

[0425] SEQ ID NO: 392 is tethered IL-12 (TeIL-12).

[0426] SEQ ID NO: 393 is an exemplary nucleic acid sequence of an IRES.

[0427] SEQ ID NO: 394 is an exemplary nucleic acid sequence of a U6 promoter.

[0428] SEQ ID NOs:395 to 401 are exemplary nucleic acid sequences of PD-1 shRNAs. DETAILED DESCRIPTION OF THE INVENTION

[0429] I. Introduction Adoptive cell therapy using TILs is an effective approach for inducing tumor regression in various cancers, including leukemia and melanoma. The use of adjuvants, including immunostimulatory agents, is being explored to enhance adoptive cell therapy and extend such therapy to other solid tumors. However, coadministration of immunomodulatory agents, such as cytokines (e.g., interleukins), can result in undesirable toxicity due to the high doses required. Therefore, providing such adjuvants at the appropriate time and site appears to be important to avoid such undesirable effects.

[0430] Provided herein are compositions and methods for treating cancer using modified TILs, where the modified TILs comprise one or more immunomodulatory agents (e.g., cytokines) associated with their cell surface. The immunomodulatory agents associated with the TILs provide a local immunostimulatory effect that can advantageously enhance TIL survival and / or antitumor activity in a patient recipient. Thus, the compositions and methods disclosed herein provide effective cancer therapies.

[0431] II. Definition Unless otherwise defined, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this invention belongs. All patents and publications referenced herein are incorporated by reference in their entirety.

[0432] As used herein, the terms "co-administration," "co-administering," "administered in combination," "administering in combination," "simultaneous," and "concurrent" encompass administration of two or more active pharmaceutical ingredients (e.g., multiple TILs in preferred embodiments of the present invention) to a subject such that both active pharmaceutical ingredients and / or their metabolites are present in the subject at the same time. Simultaneous administration includes simultaneous administration in separate compositions, administration at different times in separate compositions, or administration in a composition in which two or more active pharmaceutical ingredients are present. Concurrent administration in separate compositions and administration in a composition in which both agents are present are preferred.

[0433] The term "in vivo" refers to events that take place inside a subject's body.

[0434] The term "in vitro" refers to events that occur outside a subject's body. In vitro assays include cell-based assays, in which living or dead cells are used, and can also include cell-free assays, in which no intact cells are used.

[0435] The term "ex vivo" refers to events involving the administration of a therapy or treatment to cells, tissues, and / or organs that have been removed from a subject's body. Suitably, the cells, tissues, and / or organs may be returned to the subject's body in a surgical or therapeutic manner.

[0436] The term "rapid expansion" refers to an increase in the number of antigen-specific TILs by at least about 3-fold (or 4, 5, 6, 7, 8, or 9-fold) over a one-week period, more preferably by at least about 10-fold (or 20, 30, 40, 50, 60, 70, 80, or 90-fold) over a one-week period, or most preferably by at least about 100-fold over a one-week period. Several rapid expansion protocols are described herein.

[0437] As used herein, "tumor infiltrating lymphocytes" or "TILs" refers to a population of cells that are initially acquired as leukocytes that have left a subject's bloodstream and migrated to a tumor. TILs include CD8 + Cytotoxic T cells (lymphocytes), Th1 and Th17 CD4 + These include, but are not limited to, T cells, natural killer cells, dendritic cells, and M1 macrophages. TILs include both primary and secondary TILs. "Primary TILs" are those obtained from a patient tissue sample as outlined herein (sometimes referred to as "freshly harvested"), and "secondary TILs" are any expanded or propagated TIL cell populations discussed herein, including, but not limited to, bulk TILs and expanded TILs ("REP TILs" or "post-REP TILs"). TIL cell populations may include genetically modified TILs.

[0438] As used herein, a "population of cells" (including TILs) refers to several cells that share a common trait. Generally, a population is roughly 1 x 10 6 ~1×10 10 The number of TILs ranges from approximately 1 x 10 to 1 x 10, with different TIL populations containing different numbers. For example, the initial growth of primary TILs in the presence of IL-2 is approximately 1 x 10 8 REP expansion typically results in a bulk TIL population of 1.5 x 10 cells for injection. 9 ~1.5×10 10 This is done to provide a population of cells.

[0439] As used herein, "cryopreserved TILs" refers to TILs, either primary, bulk, or expanded (REP TILs), that are processed and stored at temperatures ranging from approximately -150°C to -60°C. General methods for cryopreservation are described elsewhere herein, including in the Examples. For clarity, "cryopreserved TILs" can be distinguished from frozen tissue samples that may be used as a source of primary TILs.

[0440] By "thawed cryopreserved TILs" herein is meant a population of TILs that have previously been cryopreserved and then processed to return to room temperature or above, including but not limited to, cell culture temperature or the temperature at which the TILs can be administered to a patient.

[0441] TILs can generally be defined either biochemically using cell surface markers or functionally by their ability to infiltrate tumors and achieve therapy. TILs can generally be classified by expression of one or more of the following biomarkers: CD4, CD8, TCRαβ, CD27, CD28, CD56, CCR7, CD45Ra, CD95, PD-1, and CD25. Additionally and alternatively, TILs can be functionally defined by their ability to infiltrate solid tumors upon reintroduction into patients.

[0442] The term "cryopreservation media" or "cryopreservation medium" refers to any medium that can be used for cryopreserving cells. Such media can include media containing 7% to 10% DMSO. Exemplary media include CryoStor CS10, Hyperthermasol, and combinations thereof. The term "CS10" refers to cryopreservation media obtained from Stemcell Technologies or Biolife Solutions. CS10 medium may be referred to by the trade name "CryoStor® CS10." CS10 medium is a serum-free, animal-component-free medium that contains DMSO.

[0443] The term "central memory T cells" refers to cells that are CD45R0+ and CCR7 (CCR7 高 ) and CD62L (CD62 高) is a subset of T cells that constitutively express the CD4 receptor. The surface phenotype of central memory T cells also includes TCR, CD3, CD127 (IL-7R), and IL-15R. Transcription factors of central memory T cells include BCL-6, BCL-6B, MBD2, and BMI1. After TCR triggering, central memory T cells primarily secrete IL-2 and CD40L as effector molecules. Central memory T cells predominate in the CD4 compartment in the blood and are proportionally enriched in lymph nodes and tonsils in humans.

[0444] The term "effector memory T cells" refers to T cells that are CD45R0+ like central memory T cells, but have lost constitutive expression of CCR7 (CCR7 低 ), heterogeneous or low CD62L expression (CD62L 低 ), refers to a subset of human or mammalian T cells. The surface phenotype of central memory T cells also includes TCR, CD3, CD127 (IL-7R), and IL-15R. Transcription factors of central memory T cells include BLIMP1. Effector memory T cells rapidly secrete high levels of inflammatory cytokines after antigen stimulation, including interferon gamma, IL-4, and IL-5. Effector memory T cells predominate in the CD8 compartment in the blood and are proportionally enriched in the lung, liver, and intestine in humans. CD8+ effector memory T cells carry large amounts of perforin.

[0445] The term "closed system" refers to a system that is closed to the external environment. Any closed system suitable for cell culture methods can be used in the methods of the present invention. Closed systems include, but are not limited to, sealed G containers. Once tumor segments are added to the closed system, the system is not opened to the external environment until the TILs are ready to be administered to a patient.

[0446] The terms "fragmenting," "fragments," and "fragmented" as used herein to describe processes for destroying tumors include mechanical fragmentation methods such as crushing, slicing, dividing, and mincing tumor tissue, as well as any other method for disrupting the physical structure of tumor tissue.

[0447] The terms "peripheral blood mononuclear cells" and "PBMCs" refer to peripheral blood cells with round nuclei, including lymphocytes (T cells, B cells, NK cells) and monocytes. When used as antigen-presenting cells (PBMCs are a type of antigen-presenting cell), the peripheral blood mononuclear cells are preferably irradiated allogeneic peripheral blood mononuclear cells.

[0448] The terms "peripheral blood lymphocytes" and "PBLs" refer to T cells expanded from peripheral blood. In some embodiments, PBLs are isolated from whole blood or apheresis products from a donor. In some embodiments, PBLs are isolated from whole blood or apheresis products from a donor by positive or negative selection of a T cell phenotype, such as a CD3+CD45+ T cell phenotype.

[0449] The term "anti-CD3 antibody" refers to an antibody or variant thereof, e.g., a monoclonal antibody, including a human, humanized, chimeric, or murine antibody, directed against the CD3 receptor in the T cell antigen receptor of mature T cells. Anti-CD3 antibodies include OKT-3, also known as muromonab. Anti-CD3 antibodies also include the UHCT1 clone, also known as T3 and CD3ε. Other anti-CD3 antibodies include, for example, otelixizumab, teplizumab, and visilizumab.

[0450] The term "OKT-3" (also referred to herein as "OKT3") refers to a monoclonal antibody, including a human, humanized, chimeric, or murine antibody against the CD3 receptor in the T cell antigen receptor of mature T cells, or a biosimilar or variant thereof, including commercially available forms such as OKT-3 (30 ng / mL, MACS GMP CD3 pure, Miltenyi Biotech, Inc., San Diego, CA, USA) and muromonab, or variants, conservative amino acid substitutions, glycoforms, or biosimilars thereof. The amino acid sequences of the heavy and light chains of muromonab are shown in Table 1 (SEQ ID NO: 1 and SEQ ID NO: 2). A hybridoma capable of producing OKT-3 has been deposited with the American Type Culture Collection and assigned ATCC accession number CRL8001. A hybridoma capable of producing OKT-3 has also been deposited with the European Collection of Authenticated Cell Cultures (ECACC) and assigned catalog number 86022706.

[0451] [Table 1]

[0452] The term "IL-2" (also referred to herein as "IL2") refers to the T cell growth factor known as interleukin-2 and includes all forms of IL-2, including human and mammalian forms, conservative amino acid substitutions, glycoforms, biosimilars, and variants. IL-2 is described, for example, in Nelson, J. Immunol. 2004, 172, 3983-88 and Malek, Annu. Rev. Immunol. 2008, 26, 453-79, the disclosures of which are incorporated herein by reference. The amino acid sequence of recombinant human IL-2 suitable for use in the present invention is shown in Table 2 (SEQ ID NO: 3). For example, the term IL-2 encompasses human recombinant IL-2 forms such as aldesleukin (PROLEUKIN, commercially available from multiple suppliers at 22 million IU per single-use vial), as well as the recombinant IL-2 form (catalog number CYT-209-b) marketed by CellGenix, Inc., Portsmouth, NH, USA (CELLGRO GMP) or ProSpec-Tany TechnoGene Ltd., East Brunswick, NJ, USA, and other commercial equivalents from other vendors. Aldesleukin (des-alanyl-1, serine-125 human IL-2) is a non-glycosylated human recombinant IL-2 form with a molecular weight of approximately 15 kDa. The amino acid sequence of aldesleukin suitable for use in the present invention is shown in Table 2 (SEQ ID NO: 4). The term IL-2 also refers to the pegylated IL-2 prodrug bempegaldesleukin (NKTR-214, an IL-2 prodrug in which an average of six lysine residues are replaced with [(2,7-bis{[methylpoly(oxyethylene)]carbamoyl}-9H-fluoren-9-yl)methoxy]carbonyl). 6The present invention also encompasses pegylated forms of IL-2 described herein, including pegylated human recombinant IL-2 such as SEQ ID NO: 4, which is available from Nektar Therapeutics (South San Francisco, CA, USA) or can be prepared by methods known in the art, such as the method described in Example 19 of International Patent Application Publication No. WO2018 / 132496 A1 or Example 1 of U.S. Patent Application Publication No. 2019 / 0275133 A1, the disclosures of which are incorporated herein by reference. Benpegaldesleukin (NKTR-214) and other pegylated IL-2 molecules suitable for use in the present invention are described in U.S. Patent Application Publication No. 2014 / 0328791 A1 and International Patent Application Publication No. WO2012 / 065086 A1, the disclosures of which are incorporated herein by reference. Alternative forms of conjugated IL-2 suitable for use in the present invention are described in U.S. Patent Nos. 4,766,106, 5,206,344, 5,089,261, and 4,902,502, the disclosures of which are incorporated herein by reference. Formulations of IL-2 suitable for use in the present invention are described in U.S. Patent No. 6,706,289, the disclosure of which is incorporated herein by reference.

[0453] In some embodiments, a suitable IL-2 form for use in the present invention is THOR-707, available from Synthorx, Inc. The preparation and properties of THOR-707 and additional alternative forms of IL-2 suitable for use in the present invention are described in U.S. Patent Application Publication Nos. 2020 / 0181220 A1 and 2020 / 0330601 A1, the disclosures of which are incorporated herein by reference. In some embodiments, a suitable IL-2 form for use in the present invention is an interleukin-2 (IL-2) complex comprising an isolated and purified IL-2 polypeptide and a conjugation moiety that binds to the isolated and purified IL-2 polypeptide at an amino acid position selected from K35, T37, R38, T41, F42, K43, F44, Y45, E61, E62, E68, K64, P65, V69, L72, and Y107, wherein the numbering of the amino acid residues corresponds to SEQ ID NO:5. In some embodiments, the amino acid position is selected from T37, R38, T41, F42, F44, Y45, E61, E62, E68, K64, P65, V69, L72, and Y107. In some embodiments, the amino acid position is selected from T37, R38, T41, F42, F44, Y45, E61, E62, E68, P65, V69, L72, and Y107. In some embodiments, the amino acid position is selected from T37, T41, F42, F44, Y45, P65, V69, L72, and Y107. In some embodiments, the amino acid position is selected from R38 and K64. In some embodiments, the amino acid position is selected from E61, E62, and E68. In some embodiments, the amino acid position is E62. In some embodiments, an amino acid residue selected from K35, T37, R38, T41, F42, K43, F44, Y45, E61, E62, E68, K64, P65, V69, L72, and Y107 is further mutated to lysine, cysteine, or histidine. In some embodiments, the amino acid residue is mutated to cysteine. In some embodiments, the amino acid residue is mutated to lysine. ...In some embodiments, the unnatural amino acid is N6-azidoethoxy-L-lysine (AzK), N6-propargylethoxy-L-lysine (PraK), BCN-L-lysine, norbornene lysine, TCO-lysine, methyltetrazine lysine, allyloxycarbonyl lysine, 2-amino-8-oxononanoic acid, 2-amino-8-oxooctanoic acid, p-acetyl-L-phenylalanine, p-azidomethyl-L-phenylalanine (pAMF), p-iodo-L-phenylalanine, m-acetylphenylalanine, 2-amino-8-oxononanoic acid, p-propargyloxyphenylalanine, p-propargyl-phenylalanine, 3-methyl-phenylalanine, L-dopa, fluorinated phenylalanine, isopropyl and selenocysteine, or selenocysteine. In some embodiments, the IL-2 conjugate has a reduced affinity for the IL-2 receptor alpha (IL-2Rα) subunit compared to a wild-type IL-2 polypeptide. In some embodiments, the reduced affinity is about a 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 95%, 99%, or greater than 99% reduction in binding affinity for IL-2Rα compared to a wild-type IL-2 polypeptide. In some embodiments, the reduced 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 greater, compared to a wild-type IL-2 polypeptide. In some embodiments, the conjugate moiety isimpairs or blocks the binding of IL-2 to IL-2Rα. In some embodiments, the conjugated moiety comprises a water-soluble polymer. In some embodiments, the additional conjugated moiety comprises a water-soluble polymer. In some embodiments, each of the water-soluble polymers independently comprises polyethylene glycol (PEG), poly(propylene glycol) (PPG), copolymers of ethylene glycol and propylene glycol, poly(oxyethylated polyol), poly(olefinic alcohol), poly(vinylpyrrolidone), poly(hydroxyalkyl methacrylamide), poly(hydroxyalkyl methacrylate), poly(saccharide), poly(α-hydroxy acid), poly(vinyl alcohol), polyphosphazene, polyoxazoline (POZ), poly(N-acryloylmorpholine), or a combination thereof. In some embodiments, each of the water-soluble polymers independently comprises PEG. In some embodiments, the PEG is linear PEG or branched PEG. In some embodiments, each of the water-soluble polymers independently comprises a polysaccharide. In some embodiments, the polysaccharide comprises dextran, polysialic acid (PSA), hyaluronic acid (HA), amylose, heparin, heparan sulfate (HS), dextrin, or hydroxyethyl starch (HES). In some embodiments, each of the water-soluble polymers independently comprises a glycan. In some embodiments, each of the water-soluble polymers independently comprises a polyamine. In some embodiments, the conjugation moiety comprises a protein. In some embodiments, the additional conjugation moieties comprise a protein. In some embodiments, each of the proteins independently comprises albumin, transferrin, or transthyretin. In some embodiments, each of the proteins independently comprises an Fc portion. In some embodiments, each of the proteins independently comprises an Fc portion of IgG. In some embodiments, the conjugation moiety comprises a polypeptide. In some embodiments, the additional conjugation moieties comprise a polypeptide. In some embodiments, each of the proteins independently comprises an XTEN peptide, a glycine-rich homoamino acid polymer (HAP), a PAS polypeptide, an elastin-like polypeptide (ELP),In some embodiments, the conjugation moiety is directly attached to the isolated and purified IL-2 polypeptide. In some embodiments, the conjugation moiety is indirectly attached to the isolated and purified IL-2 polypeptide via a linker. In some embodiments, the linker comprises a homobifunctional linker. In some embodiments, the homobifunctional linker is selected from the group consisting of the Romant reagents dithiobis(succinimidyl propionate) DSP, 3'3'-dithiobis(sulfosuccinimidyl propionate) (DTSSP), disuccinimidyl suberate (DSS), bis(sulfosuccinimidyl) suberate (BS), disuccinimidyl tartrate (DST), disulfosuccinimidyl tartrate (sulfoDS T), ethylene glycobis(succinimidyl succinate) (EGS), disuccinimidyl glutarate (DSG), N,N'-disuccinimidyl carbonate (DSC), dimethyl adipimidate (DMA), dimethyl pimelimidate (DMP), dimethyl suberimidate (DMS), dimethyl-3,3'-dithiobispropionimidate (DTBP), 1,4-di-(3'-(2'-pyridyldithio) (e) propionamido) butane (DPDPB), bismaleimidohexane (BMH), halogenated aryl-containing compounds (DFDNB), such as 1,5-difluoro-2,4-dinitrobenzene or 1,3-difluoro-4,6-dinitrobenzene, 4,4'-difluoro-3,3'-dinitrophenyl sulfone (DFDNPS), bis-[β-(4-azidosalicylamido)ethyl] disulfide (BASED), formaldehyde, glutaraldehyde, 1,4-butanediol diglycidyl ether, adipic acid dihydrazide, carbohydrazide, o-toluidine, 3,3'-dimethylbenzidine, benzidine, α,α'-p-diaminodiphenyl, diiodo-p-xylene sulfonic acid, N,N'-ethylene-bis(iodoacetamide), or N,N'-hexamethylenebis(iodoacetamide). In some embodiments, the linker comprises a heterobifunctional linker.Heterobifunctional linkers include 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), and sulfosuccinimidyl-4-(N-maleimidomethyl)cyclohexane-1-carboxylate. Silyl group (sulfo-sMCC), m-maleimidobenzoyl-N-hydroxysuccinimide ester (MB), m-maleimidobenzoyl-N-hydroxysulfosuccinimide ester (sulfo-MB), N-succinimidyl (4-iodoacetyl)aminobenzoate (sIAB), sulfosuccinimidyl (4-iodoacetyl)aminobenzoate (sulfo-sIAB), succinimidyl-4-(p-maleimidophenyl)butyrate (sMPB), sulfosuccinimidyl-4-(p-maleimidophenyl)butyrate (sulfo-sMPB), N-(γ-maleimidobutyryloxy)succinimide ester (GMB), N-(γ-maleimidobutyryloxy)sulfosuccinimide ester (sulfo-GMB), succinimidyl 6-((iodoacetyl)amino)hexanoate (sIAX), succinimidyl 6-[6-(((iodoacetyl)amino)hexanoyl)amino]hexanoate (slAXX), succinimidyl 4-(((iodoacetyl)amino)methyl)cyclohexane-1-carboxylate (sIAC), succinimidyl 6-(((((4-iodoacetyl)amino)methyl)cyclohexane-1-carbonyl)amino)hexanoate (sIACX), p-nitrophenyl iodoacetate (NPIA), carbonyl-reactive and sulfhydryl-reactive crosslinkers, such as 4-(4-N-maleimidophenyl)butyric acid hydrazide (MPBH),4-(N-maleimidomethyl)cyclohexane-1-carboxyl-hydrazide-8 (M2C2H), 3-(2-pyridyldithio)propionylhydrazide (PDPH), N-hydroxysuccinimidyl-4-azidosalicylate (NHs-AsA), N-hydroxysulfosuccinimidyl-4-azidosalicylate (sulfo-NHs-AsA), sulfosuccinimidyl-, (4-Azidosalicylamido)hexanoate (sulfo-NHs-LC-AsA), sulfosuccinimidyl-2-(p-azidosalicylamido)ethyl-1,3'-dithiopropionate (sAsD), N-hydroxysuccinimidyl-4-azidobenzoate (HsAB), N-hydroxysulfosuccinimidyl-4-azidobenzoate (sulfo-HsAB), N-succinimidyl-6-(4'-azido-2'-nitrophenylamino)hexanoate (sANPAH), sulfosuccinimidyl-6-(4'-azido-2'-nitrophenylamino)hexanoate Sulfosuccinimidyl 4-(p-azidophenyl)butyrate (sulfo-sANPAH), N-5-azido-2-nitrobenzoyloxysuccinimide (ANB-NO), sulfosuccinimidyl-2-(m-azido-o-nitrobenzamido)-ethyl-1,3'-dithiopropionate (sAND), N-succinimidyl-4(4-azidophenyl)1,3'-dithiopropionate (sADP), N-sulfosuccinimidyl (4-azidophenyl)-1,3'-dithiopropionate (sulfo-sADP), sulfosuccinimidyl 4-(p-azidophenyl)butyrate (sulfo-sAPB), sulfosuccinimidyl 2-(7-azido-4-methylcoumarin-3-acetamido)ethyl-1,3'-dithiopropionate (sAED), sulfosuccinimidyl 7-azido-4-methylcoumarin-3-acetate (sulfo-sAMCA), p-nitrophenyl diazopyruvate (pNPDP), p-nitrophenyl-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 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 optionally comprises a maleimide group, including maleimidocaproyl (mc), succinimidyl-4-(N-maleimidomethyl)cyclohexane-1-carboxylate (sMCC), or sulfosuccinimidyl-4-(N-maleimidomethyl)cyclohexane-1-carboxylate (sulfo-sMCC). In some embodiments, the linker further comprises a spacer. In some embodiments, the spacer comprises p-aminobenzyl alcohol (PAB), p-aminobenzyloxycarbonyl (PABC), a derivative, or an analog thereof. In some embodiments, the conjugated moiety is capable of extending the serum half-life of the IL-2 conjugate. In some embodiments, the additional conjugated moiety is capable of extending the serum half-life of the IL-2 conjugate. In some embodiments, the IL-2 form suitable for use in the present invention is a fragment of any of the IL-2 forms described herein. In some embodiments, IL-2 forms suitable for use in the present invention are pegylated as disclosed in U.S. Patent Application Publication Nos. 2020 / 0181220 A1 and 2020 / 0330601 A1. In some embodiments, IL-2 forms suitable for use in the present invention are IL-2 conjugates comprising an IL-2 polypeptide comprising N6-azidoethoxy-L-lysine (AzK) covalently attached to a conjugation moiety comprising polyethylene glycol (PEG), wherein the IL-2 polypeptide comprises an amino acid sequence having at least 80% sequence identity to SEQ ID NO:5, and wherein AzK substitutes an amino acid at position K35, F42, F44, K43, E62, P65, R38, T41, E68, Y45, V69, or L72 relative to amino acid position within SEQ ID NO:5. In some embodiments, the IL-2 polypeptide comprises an N-terminal deletion of one residue relative to SEQ ID NO:5. In some embodiments, forms of IL-2 suitable for use in the present invention lack IL-2R alpha chain association but retain normal binding to the intermediate affinity IL-2R beta-gamma signaling complex.In some embodiments, a suitable IL-2 form for use in the present invention is an IL-2 conjugate comprising an IL-2 polypeptide comprising N6-azidoethoxy-L-lysine (AzK) covalently attached to a conjugation moiety comprising polyethylene glycol (PEG), wherein the IL-2 polypeptide comprises an amino acid sequence having at least 90% sequence identity to SEQ ID NO:5, and wherein AzK substitutes an amino acid at position K35, F42, F44, K43, E62, P65, R38, T41, E68, Y45, V69, or L72 relative to amino acid position in SEQ ID NO:5. In some embodiments, a suitable IL-2 form for use in the present invention is an IL-2 conjugate comprising an IL-2 polypeptide comprising N6-azidoethoxy-L-lysine (AzK) covalently attached to a conjugation moiety comprising polyethylene glycol (PEG), wherein the IL-2 polypeptide comprises an amino acid sequence having at least 95% sequence identity to SEQ ID NO:5, and wherein AzK substitutes an amino acid at position K35, F42, F44, K43, E62, P65, R38, T41, E68, Y45, V69, or L72 relative to amino acid position in SEQ ID NO:5. In some embodiments, a suitable IL-2 form for use in the present invention is an IL-2 conjugate comprising an IL-2 polypeptide comprising N6-azidoethoxy-L-lysine (AzK) covalently attached to a conjugation moiety comprising polyethylene glycol (PEG), wherein the IL-2 polypeptide comprises an amino acid sequence having at least 98% sequence identity to SEQ ID NO:5, and wherein AzK substitutes an amino acid at position K35, F42, F44, K43, E62, P65, R38, T41, E68, Y45, V69, or L72 relative to amino acid position in SEQ ID NO:5.

[0454] In some embodiments, a form of IL-2 suitable for use in the present invention is nembareukin alfa, also known as ALKS-4230 (SEQ ID NO: 6), available from Alkermes, Inc. Nembareukin alfa is a nucleotide analogue of IL-2 that is linked via a peptidyl linker ( 60 GG 61 ) and fused to human interleukin-2 fragment (62-132) via a peptidyl linker ( 133 GSGGGS138 Human interleukin-2 receptor α-chain fragment (139-303) fused via a nucleotide sequence (Cys), produced in Chinese hamster ovary (CHO) cells, and glycosylated. 125 >Ser 51 ; human interleukin-2 (IL-2) (4-74)-peptide (62-132) fused via a G2 peptide linker (60-61) and human interleukin-2 receptor alpha chain (IL2R subunit alpha, IL2Rα, IL2RA) (1-165)-peptide (139-303) fused via a GSG3S peptide linker (133-138), produced in Chinese hamster ovary (CHO) cells, and alpha-glycosylated human interleukin-2 (IL-2) (75-133)-peptide [Cys 125(51)>Ser]-mutant (1-59). The amino acid sequence of nemvaleukin alfa is set forth in SEQ ID NO: 6. In some embodiments, nemvaleukin alfa exhibits the following post-translational modifications: disulfide bridges at the following positions: 31-116, 141-285, 184-242, 269-301, 166-197, or 166-199, 168-199, or 168-197 (using the numbering of SEQ ID NO: 6), and glycosylation sites at the following positions: N187, N206, T212, using the numbering of SEQ ID NO: 6. The preparation and properties of nemvaleukin alfa, as well as additional alternative forms of IL-2 suitable for use in the present invention, are described in U.S. Patent Application Publication No. 2021 / 0038684 A1 and U.S. Patent No. 10,183,979, the disclosures of which are incorporated herein by reference. In some embodiments, an IL-2 form suitable for use in the present invention is a protein having at least 80%, at least 90%, at least 95%, or at least 90% sequence identity to SEQ ID NO:6. In some embodiments, an IL-2 form suitable for use in the present invention has the amino acid sequence set forth in SEQ ID NO:6 or conservative amino acid substitutions thereof. In some embodiments, an IL-2 form suitable for use in the present invention is a fusion protein comprising amino acids 24-452 of SEQ ID NO:7, or a variant, fragment, or derivative thereof. In some embodiments, an IL-2 form suitable for use in the present invention is a fusion protein comprising an amino acid sequence having at least 80%, at least 90%, at least 95%, or at least 90% sequence identity to amino acids 24-452 of SEQ ID NO:7, or a variant, fragment, or derivative thereof. Other IL-2 forms suitable for use in the present invention are described in U.S. Pat. No. 10,183,979, the disclosure of which is incorporated herein by reference.Optionally, in some embodiments, a form of IL-2 suitable for use in the present invention is a fusion protein comprising a first fusion partner linked to a second fusion partner by a mucin domain polypeptide linker, wherein the first fusion partner is IL-1Rα or a protein having at least 98% amino acid sequence identity to IL-1Rα and having receptor antagonist activity for IL-Rα, the second fusion partner comprises all or a portion of an immunoglobulin comprising an Fc region, and the mucin domain polypeptide linker comprises SEQ ID NO:8 or an amino acid sequence having at least 90% sequence identity to SEQ ID NO:8, and wherein the half-life of the fusion protein is improved compared to the fusion of the first fusion partner with the second fusion partner in the absence of the mucin domain polypeptide linker.

[0455] [Table 2]

[0456] In some embodiments, IL-2 forms suitable for use in the present invention comprise a heavy chain variable region (V) comprising complementarity determining regions HCDR1, HCDR2, and HCDR3. H ) and a light chain variable region (V L ) and V H or V L and an IL-2 molecule or a fragment thereof grafted onto the CDR of a heavy chain variable region (V) comprising complementarity determining regions HCDR1, HCDR2, and HCDR3. H ) and a light chain variable region (V L ) and V H or V Land an IL-2 molecule or fragment thereof grafted onto the CDRs of, wherein the IL-2 molecule is a mutein, and the antibody cytokine grafted protein preferentially expands T effector cells over regulatory T cells. In some embodiments, the IL-2 regimen comprises administration of an antibody described in U.S. Patent Application Publication No. 2020 / 0270334 A1, the disclosure of which is incorporated herein by reference. In some embodiments, the antibody cytokine grafted protein comprises a heavy chain variable region (VH) comprising complementarity determining regions HCDR1, HCDR2, and HCDR3, a light chain variable region (VL) comprising LCDR1, LCDR2, and LCDR3, and a VL comprising complementarity determining regions HCDR1, HCDR2, and LCDR3. H or V L or a fragment thereof grafted onto the CDR of the antibody cytokine grafted protein, wherein the IL-2 molecule is a mutein, and the antibody cytokine grafted protein preferentially expands T effector cells over regulatory T cells, and the antibody further comprises an IgG class heavy chain and an IgG class light chain selected from the group consisting of an IgG class light chain comprising SEQ ID NO: 39 and an IgG class heavy chain comprising SEQ ID NO: 38, an IgG class light chain comprising SEQ ID NO: 37 and an IgG class heavy chain comprising SEQ ID NO: 29, an IgG class light chain comprising SEQ ID NO: 39 and an IgG class heavy chain comprising SEQ ID NO: 29, an IgG class light chain comprising SEQ ID NO: 37 and an IgG class heavy chain comprising SEQ ID NO: 38.

[0457] In some embodiments, the IL-2 molecule or fragment thereof is V H In some embodiments, the IL-2 molecule or fragment thereof is a mutein. H In some embodiments, the IL-2 molecule or fragment thereof is a mutein. H In some embodiments, the IL-2 molecule or fragment thereof is a mutein. L In some embodiments, the IL-2 molecule or a fragment thereof is grafted onto LCDR1 of V LIn some embodiments, the IL-2 molecule or fragment thereof is grafted onto LCDR2 of V L The LCDR3 of the IL-2 molecule is grafted onto the LCDR3 of the IL-2 molecule, and the IL-2 molecule is a mutein.

[0458] The insertion of the IL-2 molecule can be at or near the N-terminal region of the CDR, the middle region of the CDR, or at or near the C-terminal region of the CDR. In some embodiments, the antibody cytokine graft protein comprises an IL-2 molecule incorporated into the CDR, where the IL2 sequence does not frameshift the CDR sequence. In some embodiments, the antibody cytokine graft protein comprises an IL-2 molecule incorporated into the CDR, where the IL-2 sequence replaces all or part of the CDR sequence. The replacement with the IL-2 molecule can be at or near the N-terminal region of the CDR, the middle region of the CDR, or the C-terminal region of the CDR. The replacement with the IL-2 molecule can be as little as one or two amino acids of the CDR sequence, or the entire CDR sequence.

[0459] In some embodiments, the IL-2 molecule is directly grafted onto the CDR without a peptide linker and without additional amino acids between the CDR sequence and the IL-2 sequence. In some embodiments, the IL-2 molecule is indirectly grafted onto the CDR using a peptide linker with one or more additional amino acids between the CDR sequence and the IL-2 sequence.

[0460] In some embodiments, the IL-2 molecule described herein is an IL-2 mutein. In some cases, the IL-2 mutein comprises an R67A substitution. In some embodiments, the IL-2 mutein comprises the amino acid sequence of SEQ ID NO: 14 or SEQ ID NO: 15. In some embodiments, the IL-2 mutein comprises the amino acid sequence of Table 1 of U.S. Patent Application Publication No. 2020 / 0270334 A1, the disclosure of which is incorporated herein by reference.

[0461] In some embodiments, the antibody cytokine transplant protein comprises an HCDR1 selected from the group consisting of SEQ ID NO: 16, SEQ ID NO: 19, SEQ ID NO: 22, and SEQ ID NO: 25. In some embodiments, the antibody cytokine transplant protein comprises an HCDR1 selected from the group consisting of SEQ ID NO: 7, SEQ ID NO: 10, SEQ ID NO: 13, and SEQ ID NO: 16. In some embodiments, the antibody cytokine transplant protein comprises an HCDR1 selected from the group consisting of an HCDR2 selected from the group consisting of SEQ ID NO: 17, SEQ ID NO: 20, SEQ ID NO: 23, and SEQ ID NO: 26. In some embodiments, the antibody cytokine transplant protein comprises an HCDR3 selected from the group consisting of SEQ ID NO: 18, SEQ ID NO: 21, SEQ ID NO: 24, and SEQ ID NO: 27. In some embodiments, the antibody cytokine transplant protein comprises a V comprising the amino acid sequence of SEQ ID NO: 28. H In some embodiments, the antibody cytokine transplant protein comprises a heavy chain comprising the amino acid sequence of SEQ ID NO: 29. In some embodiments, the antibody cytokine transplant protein comprises a V region comprising the amino acid sequence of SEQ ID NO: 36. L In some embodiments, the antibody cytokine transplant protein comprises a light chain comprising the amino acid sequence of SEQ ID NO: 37. In some embodiments, the antibody cytokine transplant protein comprises a V region comprising the amino acid sequence of SEQ ID NO: 28. H V comprising the region and the amino acid sequence of SEQ ID NO: 36 LIn some embodiments, the antibody cytokine transplant protein comprises a heavy chain region comprising the amino acid sequence of SEQ ID NO:29 and a light chain region comprising the amino acid sequence of SEQ ID NO:37. In some embodiments, the antibody cytokine transplant protein comprises a heavy chain region comprising the amino acid sequence of SEQ ID NO:29 and a light chain region comprising the amino acid sequence of SEQ ID NO:39. In some embodiments, the antibody cytokine transplant protein comprises a heavy chain region comprising the amino acid sequence of SEQ ID NO:38 and a light chain region comprising the amino acid sequence of SEQ ID NO:37. In some embodiments, the antibody cytokine transplant protein comprises a heavy chain region comprising the amino acid sequence of SEQ ID NO:38 and a light chain region comprising the amino acid sequence of SEQ ID NO:39. In some embodiments, the antibody cytokine transplant protein comprises IgG.IL2F71A.H1 or IgG.IL2R67A.H1 of U.S. Patent Application Publication No. 2020 / 0270334 A1, or a variant, derivative, or fragment thereof, or conservative amino acid substitutions thereof, or a protein having at least 80%, at least 90%, at least 95%, or at least 98% sequence identity thereto. In some embodiments, the antibody component of the antibody cytokine transplant proteins described herein comprises immunoglobulin sequences, framework sequences, or CDR sequences of palivizumab. In some embodiments, the antibody cytokine transplant proteins described herein have a longer serum half-life than a wild-type IL-2 molecule, such as, but not limited to, aldesleukin or an equivalent molecule. In some embodiments, the antibody cytokine transplant proteins described herein have a sequence set forth in Table 3.

[0462] [Table 3-1] [Table 3-2]

[0463] The term "IL-4" (also referred to herein as "IL4") refers to the cytokine known as interleukin 4, which is produced by Th2 T cells, as well as eosinophils, basophils, and mast cells. IL-4 regulates the differentiation of naive helper T cells (Th0 cells) into Th2 T cells. Steinke and Borish, Respir. Res. 2001, 2, 66-70. Upon activation by IL-4, Th2 T cells subsequently produce additional IL-4 in a positive feedback loop. IL-4 also stimulates B cell proliferation and class II MHC expression, inducing class switching from B cells to IgE and IgG1 expression. Recombinant human IL-4 suitable for use in the present invention is commercially available from several suppliers, including ProSpec-Tany TechnoGene Ltd., East Brunswick, NJ, USA (catalog number CYT-211) and ThermoFisher Scientific, Inc., Waltham, MA, USA (human IL-15 recombinant protein, catalog number Gibco CTP0043). The amino acid sequence of recombinant human IL-4 suitable for use in the present invention is shown in Table 2 (SEQ ID NO: 9).

[0464] The term "IL-7" (also referred to herein as "IL7") refers to a glycosylated tissue-derived cytokine known as interleukin-7, which can be obtained from stromal and epithelial cells, as well as dendritic cells. Fry and Mackall, Blood 2002, 99, 3892-904. IL-7 can stimulate T cell development. IL-7 binds to the IL-7 receptor, a heterodimer consisting of the IL-7 receptor alpha and the common gamma chain receptor, which is a series of signals important for T cell development in the thymus and survival in the periphery. Recombinant human IL-7 suitable for use in the present invention is commercially available from several suppliers, including ProSpec-Tany TechnoGene Ltd., East Brunswick, NJ, USA (catalog number CYT-254) and ThermoFisher Scientific, Inc., Waltham, MA, USA (human IL-15 recombinant protein, catalog number Gibco PHC0071). The amino acid sequence of a recombinant human IL-7 suitable for use in the present invention is shown in Table 2 (SEQ ID NO: 10).

[0465] The term "IL-15" (also referred to herein as "IL15") refers to the T-cell growth factor known as interleukin-15 and includes all forms of IL-2, including human and mammalian forms, conservative amino acid substitutions, glycoforms, biosimilars, and variants. IL-15 is described, for example, in Fehniger and Caligiuri, Blood 2001, 97, 14-32, the disclosure of which is incorporated herein by reference. IL-15 shares β and γ signaling receptor subunits with IL-2. Recombinant human IL-15 is a single, non-glycosylated polypeptide chain containing 114 amino acids (and an N-terminal methionine) with a molecular weight of 12.8 kDa. Recombinant human IL-15 is commercially available from several suppliers, including ProSpec-Tany TechnoGene Ltd., East Brunswick, NJ, USA (catalog number CYT-230-b) and ThermoFisher Scientific, Inc., Waltham, MA, USA (human IL-15 recombinant protein, catalog number 34-8159-82). The amino acid sequence of recombinant human IL-15 suitable for use in the present invention is shown in Table 2 (SEQ ID NO: 11).

[0466] The term "IL-21" (also referred to herein as "IL21") refers to the pleiotropic cytokine protein known as interleukin-21, and includes all forms of IL-21, including human and mammalian forms, conservative amino acid substitutions, glycoforms, biosimilars, and variants. IL-21 is described, for example, in Spolski and Leonard, Nat. Rev. Drug. Disc. 2014, 13, 379-95, the disclosure of which is incorporated herein by reference. IL-21 primarily stimulates natural killer T cells and activated human CD4 +It is produced by T cells. Recombinant human IL-21 is a single, non-glycosylated polypeptide chain containing 132 amino acids with a molecular weight of 15.4 kDa. Recombinant human IL-21 is commercially available from several suppliers, including ProSpec-Tany TechnoGene Ltd., East Brunswick, NJ, USA (catalog number CYT-408-b) and ThermoFisher Scientific, Inc., Waltham, MA, USA (human IL-21 recombinant protein, catalog number 14-8219-80). The amino acid sequence of recombinant human IL-21 suitable for use in the present invention is shown in Table 2 (SEQ ID NO: 12).

[0467] When an "antitumor effective amount," "tumor inhibiting effective amount," or "therapeutic amount" is indicated, the exact amount of the composition of the present invention to be administered can be determined by a physician, taking into account individual differences in the age, weight, tumor size, extent of infection or metastasis, and health status of the patient (subject). Generally, the tumor-infiltrating lymphocytes (e.g., secondary TILs or genetically modified cytotoxic lymphocytes) described herein are administered at a dose of 10 per kg of body weight. 4 ~10 11 cells (e.g., 10 per kg body weight) 5 ~10 6 , 10 5 ~10 10 , 10 5 ~10 11 , 10 6 ~10 10 , 10 6 ~10 11 , 10 7 ~10 11 , 10 7 ~10 10 , 10 8 ~10 11 , 10 8 ~10 10 , 10 9 ~10 11 , or 10 9 ~10 10The TIL (optionally including genetically engineered TIL) compositions may be administered at doses of 1000-15000 cells (including all integer values within those ranges). TIL (optionally including genetically modified cytotoxic lymphocytes) compositions may also be administered multiple times at these doses. TIL (optionally including genetically engineered TIL) can be administered by using injection techniques commonly known in immunotherapy (see, e.g., Rosenberg et al., New Eng. J. of Med. 1988, 319, 1676). Optimal dosages and treatment regimes for a particular patient can be readily determined by one skilled in the medical arts by monitoring the patient for signs of disease and adjusting treatment accordingly.

[0468] The terms "hematological malignancies," "blood system malignancies," or terms of related meaning, refer to cancers and tumors of mammalian hematopoietic and lymphatic tissues, including, but not limited to, blood, bone marrow, lymph nodes, and lymphatic tissues. Hematological malignancies are also referred to as "liquid tumors." Hematological malignancies may include, but are not limited to, acute lymphoblastic leukemia (ALL), chronic lymphocytic lymphoma (CLL), small lymphocytic lymphoma (SLL), acute myeloid leukemia (AML), chronic myelogenous leukemia (CML), multiple myeloma, acute monocytic leukemia (AMoL), Hodgkin's lymphoma, and non-Hodgkin's lymphoma. The term "B-cell hematological malignancies" refers to hematological malignancies affecting B cells.

[0469] The term "liquid tumor" refers to an abnormal mass of cells that is fluid in nature. Liquid tumor cancers include, but are not limited to, leukemia, myeloma, and lymphoma, as well as other hematological malignancies. TILs obtained from liquid tumors may also be referred to herein as bone marrow-infiltrating lymphocytes (MILs). TILs obtained from liquid tumors, including those circulating in peripheral blood, may also be referred to herein as PBLs. The terms MILs, TILs, and PBLs are used interchangeably herein and differ only based on the tissue type from which the cells are derived.

[0470] The term "microenvironment" as used herein may refer to the solid or hematological tumor microenvironment as a whole, or to individual subsets of cells within the microenvironment. As used herein, the tumor microenvironment refers to a complex mixture of "cells, soluble factors, signaling molecules, extracellular matrix, and mechanical cues that promote neoplastic transformation, support tumor growth and invasion, protect tumors from host immunity, foster therapeutic resistance, and provide a niche for successful and dominant metastasis," as described in Swartz, et al., Cancer Res., 2012, 72, 2473. Tumors express antigens that are recognized by T cells, but tumor clearance by the immune system is rare due to immunosuppression by the microenvironment.

[0471] In some embodiments, the invention includes methods of treating cancer with a TIL population, wherein the patient is pretreated with non-myeloablative chemotherapy prior to infusion of TILs according to the invention. In some embodiments, a TIL population may be provided, wherein the patient is pretreated with non-myeloablative chemotherapy prior to infusion of TILs according to the invention. In some embodiments, the non-myeloablative chemotherapy is cyclophosphamide 60 mg / kg / day for 2 days (27 and 26 days before TIL infusion) and fludarabine 25 mg / m2 / day for 5 days (27-23 days before TIL infusion). In some embodiments, after non-myeloablative chemotherapy and TIL infusion according to the invention (day 0), the patient receives an intravenous infusion of IL-2 at 720,000 IU / kg every 8 hours to physiological tolerance.

[0472] Experimental findings indicate that lymphodepletion prior to adoptive transfer of tumor-specific T lymphocytes plays an important role in enhancing therapeutic efficacy by eliminating regulatory T cells and competing elements of the immune system ("cytokine sinks"). Accordingly, some embodiments of the present invention utilize a lymphodepletion step (sometimes referred to as "immunosuppressive conditioning") on patients prior to introducing the TILs of the present invention.

[0473] The term "effective amount" or "therapeutically effective amount" refers to an amount of a compound or combination of compounds described herein sufficient to achieve the intended use, including, but not limited to, disease treatment. A therapeutically effective amount may vary depending on the intended use (in vitro or in vivo), the subject and condition being treated (e.g., the subject's weight, age, and sex), the severity of the condition, or the method of administration. The term also applies to a dose that induces a specific response in target cells (e.g., reduced platelet adhesion and / or cell migration). The specific dose will vary depending on the particular compound selected, the dosing regimen to be followed, whether the compound is administered in combination with other compounds, the timing of administration, the tissue to which it is administered, and the physical delivery system by which the compound is delivered.

[0474] The terms "treatment," "treating," "treating," and the like refer to obtaining a desired pharmacological and / or physiological effect. The effect can be preventative, in terms of completely or partially preventing the disease or condition, and / or therapeutic, in terms of partially or completely curing the disease and / or side effects caused by the disease. "Treatment," as used herein, encompasses any treatment of disease in a mammal, particularly a human, and includes (a) preventing the disease from occurring in a subject who may be susceptible to the disease but has not yet been diagnosed with it; (b) suppressing the disease, i.e., arresting its onset or progression; and (c) palliating the disease, i.e., causing regression of the disease and / or alleviating one or more disease symptoms. "Treatment" is also intended to encompass the delivery of an agent to provide a pharmacological effect even in the absence of a disease or condition. For example, "treatment" encompasses the delivery of a composition capable of eliciting an immune response or conferring immunity in the absence of a pathology, e.g., in the case of a vaccine.

[0475] The term "heterologous," when used with reference to portions of a nucleic acid or protein, indicates that the nucleic acid or protein comprises two or more subsequences that are not found in the same relationship to each other in nature. For example, nucleic acids are typically produced recombinantly, with two or more sequences from unrelated genes arranged to create a new functional nucleic acid, e.g., a promoter from one source and a coding region from another source, or coding regions from different sources. Similarly, a heterologous protein indicates that the protein comprises two or more subsequences that are not found in the same relationship to each other in nature (e.g., a fusion protein).

[0476] The terms "sequence identity," "percent identity," and "percent sequence identity" (or their synonyms, e.g., "99% identical") in the context of two or more nucleic acids or polypeptides refer to two or more sequences or subsequences that are the same or have a specified percentage of the same nucleotide or amino acid residues when compared and aligned for maximum correspondence (introducing gaps, if necessary), without considering any conservative amino acid substitutions as part of the sequence identity. Percent identity can be measured using sequence comparison software or algorithms or by visual inspection. Various algorithms and software that can be used to obtain alignment of amino acid or nucleotide sequences are known in the art. Suitable programs for determining percent sequence identity include, for example, the BLAST suite of programs available from the BLAST website of the U.S. government's National Center for Biotechnology Information. Comparison between two sequences can be performed using either the BLASTN or BLASTP algorithm. BLASTN is used to compare nucleic acid sequences, and BLASTP is used to compare amino acid sequences. ALIGN, ALIGN-2 (Genentech, South San Francisco, California), or MegAlign, available from DNASTAR, are additional publicly available software programs that can be used to align sequences. Those skilled in the art can determine the appropriate parameters for maximum alignment depending on the particular alignment software. In certain embodiments, the default parameters of the alignment software are used.

[0477] As used herein, the term "variant" includes, but is not limited to, an antibody or fusion protein comprising an amino acid sequence that differs from the amino acid sequence of a reference antibody by one or more substitutions, deletions, and / or additions at specific positions within or adjacent to the amino acid sequence of the reference antibody. A variant may contain one or more conservative substitutions in its amino acid sequence compared to the amino acid sequence of the reference antibody. Conservative substitutions may include, for example, substitutions of similarly charged or uncharged amino acids. A variant retains the ability of the reference antibody to specifically bind to an antigen. The term variant also includes pegylated antibodies or proteins.

[0478] As used herein, "tumor infiltrating lymphocytes" or "TILs" refers to a population of cells that are initially acquired as leukocytes that have left a subject's bloodstream and migrated to a tumor. TILs include CD8 + Cytotoxic T cells (lymphocytes), Th1 and Th17 CD4 + These include, but are not limited to, T cells, natural killer cells, dendritic cells, and M1 macrophages. TILs include both primary and secondary TILs. "Primary TILs" are those obtained from a patient tissue sample as outlined herein (sometimes referred to as "freshly harvested"). "Secondary TILs" are any TIL cell populations that have been expanded or propagated as discussed herein, including, but not limited to, bulk TILs, expanded TILs ("REP TILs"), and "reREP TILs" as discussed herein. reREP TILs can include, for example, second expanded TILs or second additional expanded TILs (e.g., those described in step D of FIG. 8, including TILs designated as reREP TILs).

[0479] TILs can generally be defined either biochemically using cell surface markers or functionally by their ability to infiltrate tumors and achieve therapy. TILs can generally be classified by the expression of one or more of the following biomarkers: CD4, CD8, TCRαβ, CD27, CD28, CD56, CCR7, CD45Ra, CD95, PD-1, and CD25. Additionally or alternatively, TILs can be functionally defined by their ability to infiltrate solid tumors upon reintroduction into patients. TILs can be further characterized by potency; for example, TILs can be considered potent if their interferon (IFN) release is greater than about 50 pg / mL, greater than about 100 pg / mL, greater than about 150 pg / mL, or greater than about 200 pg / mL. For example, TILs can be considered potent if interferon (IFNγ) release is greater than about 50 pg / mL, greater than about 100 pg / mL, greater than about 150 pg / mL, or greater than about 200 pg / mL, greater than about 300 pg / mL, greater than about 400 pg / mL, greater than about 500 pg / mL, greater than about 600 pg / mL, greater than about 700 pg / mL, greater than about 800 pg / mL, greater than about 900 pg / mL, or greater than about 1000 pg / mL.

[0480] The term "deoxyribonucleotide" encompasses natural and synthetic, unmodified and modified deoxyribonucleotides. Modifications include changes to the sugar moiety, the base moiety, and / or the linkages between deoxyribonucleotides in an oligonucleotide.

[0481] The term "RNA" defines a molecule containing at least one ribonucleotide residue. The term "ribonucleotide" defines a nucleotide having a hydroxyl group at the 2' position of a bD-ribofuranose moiety. The term RNA includes double-stranded RNA, single-stranded RNA, isolated RNA such as partially purified RNA, essentially pure RNA, synthetic RNA, recombinantly produced RNA, and altered RNA that differs from naturally occurring RNA by the addition, deletion, substitution, and / or alteration of one or more nucleotides. The nucleotides of the RNA molecules described herein can also include non-standard nucleotides, such as non-naturally occurring nucleotides or chemically synthesized nucleotides or deoxynucleotides. These altered RNAs may be referred to as analogs or analogs of naturally occurring RNA.

[0482] The term "pharmaceutically acceptable carrier" or "pharmaceutically acceptable excipient" is intended to include any and all solvents, dispersion media, coatings, antibacterial and antifungal agents, isotonic and absorption delaying agents, and inactive ingredients. The use of such pharmaceutically acceptable carriers or pharmaceutically acceptable excipients for active pharmaceutical ingredients is well known in the art. Except insofar as any conventional pharmaceutically acceptable carrier or pharmaceutically acceptable excipient is incompatible with the active pharmaceutical ingredient, its use in the therapeutic compositions of the invention is contemplated. Additional active pharmaceutical ingredients, such as other drugs, can also be incorporated into the compositions and methods described.

[0483] The terms "about" and "approximately" mean within a statistically significant range of values. Such a range may be within one order of magnitude of a given value or range, preferably within 50%, more preferably within 20%, even more preferably within 10%, and even more preferably within 5%. The acceptable deviation encompassed by the term "about" or "approximately" depends on the particular system under study and can be readily understood by one of ordinary skill in the art. Furthermore, as used herein, the terms "about" and "approximately" mean that dimensions, sizes, formulations, parameters, shapes, and other quantities and characteristics are not, and need not be, exact, but may be approximate and / or larger or smaller, as appropriate, reflecting tolerances, conversion factors, rounding, measurement errors, etc., and other factors known to those of ordinary skill in the art. In general, a dimension, size, formulation, parameter, shape, or other quantity or characteristic is "about" or "approximately" whether or not expressly stated as such. It should be noted that embodiments of widely different sizes, shapes, and dimensions may employ the described configurations.

[0484] When used in the appended claims, the transitional terms "comprising," "consisting essentially of," and "consisting of" define the claim in its original and amended form, in terms of whether additional, unrecited claim elements or steps, if any, are excluded from the scope of the claim(s). The term "comprising" is intended to be inclusive or open-ended and does not exclude any additional, unrecited elements, methods, steps, or materials. The term "consisting of" excludes any element, step, or material other than those specified in the claim, and in the latter case, also excludes impurities normally associated with the specified material(s). The term "consisting essentially of" limits the claim to the specified element, step, or material(s) and does not materially affect the basic and novel feature(s) of the claimed invention. All compositions, methods, and kits described herein embodying the present invention may, in alternative embodiments, be more specifically defined by any of the transitional terms "comprising," "essentially consisting of," and "consisting of."

[0485] The terms "antibody" and its plural "antibodies" refer to whole immunoglobulins and any antigen-binding fragment ("antigen-binding portion") or single chains thereof. "Antibody" also refers to a glycoprotein comprising at least two heavy (H) chains and two light (L) chains interconnected by disulfide bonds, or antigen-binding portions thereof. Each heavy chain contains a heavy chain variable region (referred to herein as V H Each light chain is composed of a light chain variable region (abbreviated herein as V) and a heavy chain constant region. The heavy chain constant region is composed of three domains: CH1, CH2, and CH3. L The light chain constant region consists of one domain, C L The V of the antibody H and V L The regions can be further subdivided into regions of hypervariability, termed complementarity-determining regions (CDRs) or hypervariable regions (HVRs), which may be interspersed with more conserved regions called framework regions (FRs). H and VL is composed of three CDRs and four FRs arranged from the amino terminus to the carboxy terminus in the following order: FR1, CDR1, FR2, CDR2, FR3, CDR3, FR4. The variable regions of the heavy and light chains contain binding domains that interact with one or more antigen epitopes. The constant region of the antibody may mediate the binding of the immunoglobulin to host tissues or factors, including various cells of the immune system (e.g., effector cells) and the first component (Clq) of the classical complement system.

[0486] The term "antigen" refers to a substance that induces an immune response. In some embodiments, an antigen is a molecule that can be bound by an antibody or TCR when presented by a major histocompatibility complex (MHC) molecule. As used herein, the term "antigen" also encompasses T cell epitopes. An antigen can additionally be recognized by the immune system. In some embodiments, an antigen can induce a humoral or cellular immune response, leading to the activation of B and / or T lymphocytes. In some cases, this may require that the antigen contain or be bound by a Th cell epitope. An antigen may also have one or more epitopes (e.g., B- and T-epitopes). In some embodiments, an antigen preferably reacts with a corresponding antibody or TCR, typically in a highly specific and selective manner, and not with many other antibodies or TCRs that may be induced by other antigens.

[0487] The terms "monoclonal antibody," "mAb," "monoclonal antibody composition," or their plurals, refer to a preparation of antibody molecules of single molecular composition. A monoclonal antibody composition exhibits a single binding specificity and affinity for a particular epitope. Monoclonal antibodies specific for a particular receptor can be produced using knowledge and techniques in the art by injecting a test subject with an appropriate antigen and then isolating hybridomas expressing antibodies with the desired sequence or functional characteristics. DNA encoding the monoclonal antibody is readily isolated and sequenced using conventional procedures (e.g., by using oligonucleotide probes that can specifically bind to genes encoding the heavy and light chains of the monoclonal antibody). Hybridoma cells serve as a preferred source of such DNA. Once isolated, the DNA can be placed into an expression vector and then transfected into host cells such as E. coli cells, monkey COS cells, Chinese hamster ovary (CHO) cells, or myeloma cells that do not produce immunoglobulin protein to obtain the synthesis of the monoclonal antibody in the recombinant host cells. Recombinant production of antibodies is described in more detail below.

[0488] As used herein, the term "antigen-binding portion" or "antigen-binding fragment" of an antibody (or simply "antibody portion" or "fragment") refers to one or more fragments of an antibody that retain the ability to specifically bind to an antigen. It has been shown that the antigen-binding function of an antibody can be performed by fragments of a full-length antibody. Examples of binding fragments encompassed by the term "antigen-binding portion" of an antibody include: (i) V L , V H , C L (ii) a Fab fragment, which is a monovalent fragment consisting of two Fab fragments linked by a disulfide bridge at the hinge region; (iii) a V H and an Fd fragment consisting of the CH1 domain, (iv) a V of a single arm of an antibody L and V H Fv fragment consisting of domains, (v) V H or VL and (v) isolated complementarity-determining regions (CDRs). L and V H are encoded by separate genes, which can be synthesized using recombinant methods. L and V H The domains may be joined by a synthetic linker that allows them to pair as a single protein chain to form a monovalent molecule known as a single-chain Fv (scFv) (see, e.g., Bird, et al., Science 1988, 242, 423-426, and Huston, et al., Proc. Natl. Acad. Sci. USA 1988, 85, 5879-5883). Such scFv antibodies are also intended to be encompassed by the term "antigen-binding portion" or "antigen-binding fragment" of an antibody. These antibody fragments are obtained using conventional techniques known to those skilled in the art, and the fragments are screened for utility in the same manner as intact antibodies. In some embodiments, the scFv protein domain comprises a V H Part and V L The scFv molecule contains a V L If the domain is the N-terminal portion of the scFv molecule, V L -LV H , or V H If the domain is the N-terminal portion of the scFv molecule, V H -LV L Methods for producing scFv molecules and designing suitable peptide linkers are described in U.S. Pat. Nos. 4,704,692, 4,946,778, R. Raag and M. Whitlow, "Single Chain Fvs," FASEB Vol. 9:73-80 (1995), and RE Bird and BW Walker, Single Chain Antibody Variable Regions, TIBTECH, Vol. 9:132-137 (1991), the disclosures of which are incorporated herein by reference.

[0489] As used herein, the term "human antibody" is intended to include antibodies having variable regions in which both the framework and CDR regions are derived from human germline immunoglobulin sequences. Furthermore, if the antibody contains a constant region, the constant region also is derived from human germline immunoglobulin sequences. The human antibodies of the invention may include amino acid residues not encoded by human germline immunoglobulin sequences (e.g., mutations introduced by random or site-specific mutagenesis in vitro or by somatic mutation in vivo). As used herein, the term "human antibody" is not intended to include antibodies in which CDR sequences derived from the germline of another mammalian species, such as a mouse, have been grafted onto human framework sequences.

[0490] The term "human monoclonal antibody" refers to antibodies displaying a single binding specificity which have variable regions in which both the framework and CDR regions are derived from human germline immunoglobulin sequences. In some embodiments, human monoclonal antibodies are produced by hybridomas comprising B cells obtained from a transgenic non-human animal, e.g., a transgenic mouse, whose genome comprises human heavy chain and light chain transgenes fused to an immortalized cell.

[0491] As used herein, the term "recombinant human antibody" includes all human antibodies prepared, expressed, created, or isolated by recombinant means, such as (a) antibodies isolated from animals (such as mice) that are transgenic or transchromosomal for human immunoglobulin genes or hybridomas prepared therefrom (described further below); (b) antibodies isolated from host cells transformed to express human antibodies, e.g., from transfectomas; (c) antibodies isolated from recombinant combinatorial human antibody libraries; and (d) antibodies prepared, expressed, created, or isolated by any other means, including splicing human immunoglobulin gene sequences to other DNA sequences. Such recombinant human antibodies have variable regions in which the framework and CDR regions are derived from human germline immunoglobulin sequences. However, in certain embodiments, such recombinant human antibodies may be subjected to in vitro mutagenesis (or, when animals transgenic for human Ig sequences are used, in vivo somatic mutagenesis) to thereby modify the V sequences of the recombinant antibodies. H and V L The amino acid sequence of the region is human germline V H and V L These are sequences that are derived from and related to sequences, but may not naturally occur within the human antibody germline repertoire in vivo.

[0492] As used herein, "isotype" refers to the antibody class (e.g., IgM or IgG1) that is encoded by heavy chain constant region genes.

[0493] The phrases "an antibody that recognizes an antigen" and "an antibody that is specific for an antigen" are used interchangeably herein with the term "an antibody that specifically binds to an antigen."

[0494] The term "human antibody derivative" refers to any modified form of a human antibody, including a conjugate of the antibody with another active pharmaceutical ingredient or antibody. The terms "conjugate," "antibody drug conjugate," "ADC," or "immunoconjugate" refer to an antibody or fragment thereof conjugated to another therapeutic moiety, which can be conjugated to the antibodies described herein using methods available in the art.

[0495] The terms "humanized antibody," "humanized antibodies," and "humanization" are intended to refer to antibodies in which CDR sequences derived from the germline of another mammalian species, such as mouse, have been grafted onto human framework sequences. Additional framework region modifications can be made within the human framework sequences. Humanized forms of non-human (e.g., murine) antibodies are chimeric antibodies that contain minimal sequence derived from non-human immunoglobulin. In most cases, humanized antibodies are human immunoglobulins (recipient antibodies) in which residues from a hypervariable region of the recipient are replaced by residues from a hypervariable region of a non-human species (donor antibody) such as mouse, rat, rabbit, or non-human primate having the desired specificity, affinity, and capacity. In some cases, Fv framework (FR) residues of the human immunoglobulin are replaced by corresponding non-human residues. Furthermore, humanized antibodies may contain residues that are not found in the recipient or donor antibody. These modifications are made to further refine antibody performance. Generally, a humanized antibody will comprise substantially all of at least one, and typically two, variable domains, in which all or substantially all of the hypervariable loops correspond to those of a non-human immunoglobulin and all or substantially all of the FR regions are those of a human immunoglobulin sequence. The humanized antibody will also optionally comprise at least a portion of an immunoglobulin constant region (Fc), typically that of a human immunoglobulin. For further details, see Jones, et al., Nature 1986, 321, 522-525; Riechmann, et al., Nature 1988, 332, 323-329; and Presta, Curr. Op. Struct. Biol. 1992, 2, 593-596. The antibodies described herein can also be modified to use any Fc variant known to confer improved (e.g., reduced) effector function and / or FcR binding.Fc variants are described in, for example, International Patent Application Publication Nos. WO1988 / 07089A1, WO1996 / 14339A1, WO1998 / 05787A1, WO1998 / 23289A1, WO1999 / 51642A1, WO99 / 58572A1, WO2000 / 09560A2, WO2000 / 32767A1, WO2000 / 42072A2, and WO2002 / 4 4215A2, WO2002 / 060919A2, WO2003 / 074569A2, WO2004 / 016750A2, WO2004 / 029207A2, WO2004 / 03 5752A2, WO2004 / 063351A2, WO2004 / 074455A2, WO2004 / 099249A2, WO2005 / 040217A2, WO2005 / 07 0963A1, WO2005 / 077981A2, WO2005 / 092925A2, WO2005 / 123780A2, WO2006 / 019447A1, WO2006 / 047350A2, and WO2006 / 085967A2, as well as U.S. Pat. Nos. 5,648,260, 5,739,277, 5,834,250, 5,869,046, 6,096 ,871, 6,121,022, 6,194,551, 6,242,195, 6,277,375, 6,528,624, 6,538,124, 6,737,056, 6,821,505, 6,998,253, and 7,083,784 (the disclosures of which are incorporated herein by reference).

[0496] The term "chimeric antibody" is intended to refer to an antibody in which the variable region sequences are derived from one species and the constant region sequences are derived from another species, e.g., an antibody in which the variable region sequences are derived from a murine antibody and the constant region sequences are derived from a human antibody.

[0497] A "diabody" is a small antibody fragment that has two antigen-binding sites. The fragments bind to the same polypeptide chain (VH -V L or V L -V H ) in the light chain variable domain (V L ) connected to the heavy chain variable domain (V H ). When a linker that is too short to pair the two domains on the same chain is used, the domains are forced to pair with complementary domains on another chain and create two antigen-binding sites. Bispecific antibodies are more fully described in, for example, European Patent No. 404,097, International Patent Publication No. WO 93 / 11161, and Bolliger, et al., Proc. Natl. Acad. Sci. USA 1993, 90, 6444-6448.

[0498] The term "glycosylation" refers to modified derivatives of antibodies. An aglycosylated antibody lacks glycosylation. Glycosylation can be altered to, for example, increase the affinity of an antibody for an antigen. Such carbohydrate modifications can be achieved, for example, by altering one or more glycosylation sites within the antibody sequence. For example, one or more amino acid substitutions can be made that result in the elimination of one or more variable region framework glycosylation sites, thereby eliminating glycosylation at those sites. As described in U.S. Pat. Nos. 5,714,350 and 6,350,861, aglycosylation can increase the affinity of an antibody for an antigen. Additionally or alternatively, antibodies can be generated with altered types of glycosylation, such as hypofucosylated antibodies with reduced amounts of fucosyl residues or antibodies with increased bisecting GlcNac structures. Such altered glycosylation patterns have been demonstrated to increase antibody potency. Such carbohydrate modifications can be achieved, for example, by expressing the antibody in a host cell with altered glycosylation machinery. Cells with altered glycosylation machinery have been described in the art and can be used as host cells to express the recombinant antibodies of the present invention, thereby producing antibodies with altered glycosylation. For example, the cell lines Ms704, Ms705, and Ms709 lack the fucosyltransferase gene FUT8 (alpha(1,6) fucosyltransferase), such that antibodies expressed in these cell lines lack fucose on their carbohydrates. The Ms704, Ms705, and Ms709 FUT8- / - cell lines were generated by targeted disruption of the FUT8 gene in CHO / DG44 cells using two replacement vectors (see, e.g., U.S. Patent Publication No. 2004 / 0110704 or Yamane-Ohnuki, et al., Biotechnol. Bioeng., 2004, 87, 614-622).As another example, European Patent No. 1,176,195 describes cell lines with a functionally disrupted FUT8 gene encoding a fucosyltransferase, thereby resulting in antibodies expressed in such cell lines exhibiting hypofucosylation by reducing or eliminating alpha-1,6 bond-associated enzymes. It also describes cell lines with reduced or no enzymatic activity for adding fucose to N-acetylglucosamine linked to the Fc region of antibodies, such as the rat myeloma cell line YB2 / 0 (ATCC CRL 1662). International Patent Publication No. WO 03 / 035835 describes a variant CHO cell line, Lec13 cells, that has a reduced ability to attach fucose to Asn(297)-linked carbohydrates, which also results in hypofucosylation of antibodies expressed in the host cells (see also Shields, et al., J. Biol. Chem. 2002, 277, 26733-26740). International Patent Publication No. WO 99 / 54342 describes cell lines engineered to express glycoprotein-modifying glycosyltransferases (e.g., beta(1,4)-N-acetylglucosaminyltransferase III (GnTIII)), such that antibodies expressed in the engineered cell lines exhibit increased bisecting GlcNac structures, resulting in increased ADCC activity of the antibodies (see also Umana, et al., Nat. Biotech. 1999, 17, 176-180). Alternatively, fucosidase enzymes can be used to cleave fucose residues from antibodies. For example, the fucosidase alpha-L-fucosidase removes fucosyl residues from antibodies, as described in Tarentino, et al., Biochem. 1975, 14, 5516-5523.

[0499] "PEGylation" refers to a modified antibody or fragment thereof that has been reacted with polyethylene glycol (PEG), such as a reactive ester or aldehyde derivative of PEG, under conditions that result in one or more PEG groups being attached to the antibody or antibody fragment. PEGylation can, for example, increase the biological (e.g., serum) half-life of the antibody. Preferably, PEGylation is carried out via an acylation reaction or an alkylation reaction with a reactive PEG molecule (or an analogous reactive water-soluble polymer). As used herein, the term "polyethylene glycol" refers to a mono (C1-C 10 PEG is intended to encompass any of the forms of PEG that have been used to derivatize other proteins, such as alkoxy- or aryloxy-polyethylene glycol, or polyethylene glycol-maleimide. The antibody to be pegylated may be an aglycosylated antibody. Methods for pegylation are known in the art and can be applied to the antibodies of the invention, for example, as described in European Patent Nos. 0154316 and 0401384, and U.S. Patent No. 5,824,778 (the disclosures of each of which are incorporated herein by reference).

[0500] The term "biosimilar" refers to a biological product that is highly similar to a reference biological product approved in the United States, despite minor differences in clinically inactive components, including monoclonal antibodies or proteins, and that has no clinically meaningful differences between the biological product and the reference product in terms of product safety, purity, and potency. Furthermore, a similar biological or "biosimilar" drug is a biological product similar to another biological product already approved for use by the European Medicines Agency. The term "biosimilar" is also used interchangeably by regulatory agencies in other countries and regions. Biological products or biological products are medicines made by or derived from biological sources, such as bacteria or yeast. They can consist of relatively small molecules, such as human insulin or erythropoietin, or complex molecules, such as monoclonal antibodies. For example, if the reference IL-2 protein is aldesleukin (proleukin), a protein approved by a drug regulatory agency for aldesleukin is a "biosimilar" of aldesleukin or a "biosimilar of" aldesleukin. In Europe, a similar biological or "biosimilar" medicinal product is a biological product similar to another biological product already authorized for use by the European Medicines Agency (EMA). The legal basis for similar biological uses in Europe is Article 6 of Regulation (EC) No. 726 / 2004, as amended, and Article 10(4) of Directive 2001 / 83 / EC. Therefore, in Europe, biosimilars may be authorized or approved for authorization or licensing purposes under Article 6 of Regulation (EC) No. 726 / 2004 and Article 10(4) of Directive 2001 / 83 / EC. The original biological product already authorized is sometimes referred to as the "reference medicinal product" in Europe. Some of the requirements for a product to be considered a biosimilar are outlined in the CHMP guideline on biosimilar medicinal products. Additionally, product-specific guidelines, including those related to monoclonal antibody biosimilars, are provided by the EMA on a product-by-product basis and are available on its website.Biosimilars described herein may be similar to the reference medicinal product in terms of quality characteristics, biological activity, mechanism of action, safety profile, and / or efficacy. Furthermore, biosimilars may be used or intended for use to treat the same condition as the reference medicinal product. Thus, biosimilars described herein may be considered to have similar or very similar quality characteristics to the reference medicinal product. Alternatively, or in addition, biosimilars described herein may be considered to have similar or very similar biological activity to the reference medicinal product. Alternatively, or in addition, biosimilars described herein may be considered to have a similar or very similar safety profile to the reference medicinal product. Alternatively, or in addition, biosimilars described herein may be considered to have similar or very similar efficacy to the reference medicinal product. As described herein, biosimilars in Europe are compared to reference medicinal products authorized by the EMA. However, in some cases, biosimilars may be compared in specific studies to biopharmaceuticals authorized outside the European Economic Area (non-EEA-authorized "comparators"). Such studies include, for example, specific clinical studies and in vivo nonclinical studies. As used herein, the term "biosimilar" also refers to a biopharmaceutical that has been or can be compared to a non-EEA-approved comparator. Particular biosimilars are proteins, such as antibodies, antibody fragments (e.g., antigen-binding portions), and fusion proteins. Protein biosimilars may have amino acid sequences with minor modifications to the amino acid structure (e.g., including amino acid deletions, additions, and / or substitutions) that do not significantly affect the function of the polypeptide. A biosimilar may have an amino acid sequence that has 97% or more sequence identity, e.g., 97%, 98%, 99%, or 100%, to the amino acid sequence of its reference pharmaceutical. A biosimilar may include one or more post-translational modifications, such as, but not limited to, glycosylation, oxidation, deamidation, and / or cleavage, that differ from the post-translational modifications of the reference pharmaceutical, provided that the differences do not result in a change in the safety and / or efficacy of the pharmaceutical. A biosimilar may have the same or a different glycosylation pattern as the reference pharmaceutical, provided that the differences do not result in a change in the safety and / or efficacy of the pharmaceutical.In particular, but not exclusively, biosimilars may have different glycosylation patterns if the differences address or are intended to address safety concerns associated with the reference drug. Additionally, biosimilars may deviate from the reference drug, for example, in its strength, dosage form, formulation, excipients, and / or presentation, provided that the drug's safety and efficacy are not compromised. Biosimilars may contain differences, for example, in their pharmacokinetic (PK) and / or pharmacodynamic (PD) profiles compared to the reference drug, but are still considered sufficiently similar to the reference drug to be approved or deemed suitable for approval. In certain circumstances, biosimilars exhibit different binding characteristics compared to the reference drug, and these different binding characteristics are not considered by regulatory authorities, such as the EMA, to be a barrier to approval as a similar biological product. The term "biosimilar" is also used interchangeably by regulatory agencies in other countries and regions.

[0501] III. Immunomodulator-associated tumor-infiltrating lymphocytes Provided herein are modified tumor-infiltrating lymphocytes (TILs) comprising one or more immunomodulatory agents associated with the TIL cell surface. In some embodiments, the subject modified TILs exhibit improved in vivo survival, proliferation, and / or anti-tumor efficacy in a patient recipient.

[0502] Immunomodulatory agents can be attached to the TILs disclosed herein (e.g., therapeutic TILs provided herein) using any suitable method. In some embodiments, one or more immunomodulatory agents are part of an immunomodulatory fusion protein that is attached to the TIL cell surface. In some embodiments, one or more immunomodulatory agents are included as part of a nanoparticle that associates with the TIL cell surface. The immunomodulatory agent can be any immunomodulatory agent that promotes TIL survival, proliferation, and / or anti-tumor effects in a patient recipient. In some embodiments, the immunomodulatory agent is a cytokine (e.g., an interleukin). In an exemplary embodiment, the TILs comprise IL-12, IL-15, and / or IL-21.

[0503] Any suitable TIL population can be modified to produce a composition of interest, including TILs produced using the manufacturing processes described herein. In some embodiments, the modified TILs are derived from TILs produced during any of the steps of the Process 2A method disclosed herein (see, e.g., Figures 2-6). In exemplary embodiments, the modified TILs are derived from TILs produced during any of the steps of the GEN3 method disclosed herein (see, e.g., Figure 7). In some embodiments, the TILs are PD-1-positive TILs derived from the methods disclosed herein.

[0504] In some embodiments, the TILs are further modified by a gene editing process disclosed herein, such as CRISPR, TALE, ZFN, tCas-CLOVER, shRNA, or a combination thereof, to alter the expression of one or more immune checkpoint genes in the TIL population. Non-limiting examples of immune checkpoint genes that can be silenced or inhibited by the gene editing methods 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, TET2, BAFF (BR3), CD96, CRTAM, LAIR1, SIGLEC7, SIGLEC9, CD244, TNFRSF10B, TNFRSF10C, TNFRSF10D, TNFRSF10E, TNFRSF10F, TNFRSF10G, TNFRSF10H ... These include FRSF10A, 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.

[0505] Aspects of the subject modified TILs are further detailed herein.

[0506] A. Immunomodulatory Fusion Proteins In some embodiments, the modified TILs provided herein comprise an immunomodulatory fusion protein comprising an immunomodulatory agent (e.g., a cytokine) linked to a moiety that facilitates tethering of the immunomodulatory agent to the surface of the TIL. In some embodiments, the fusion protein comprises a cell membrane anchor portion (transmembrane domain). In certain embodiments, the fusion protein comprises a TIL surface antigen-binding portion that binds to a TIL surface antigen. Aspects of these fusion proteins are discussed in further detail below.

[0507] Any suitable genetic modification method can be used to genetically modify the subject's TILs to include such immunomodulatory fusion proteins, including, for example, any of the gene editing methods described herein. In some embodiments, the genetic modification method is the CRISPR, TALE, zinc finger, or Cas-CLOVER method described herein. In some embodiments, such modified TILs are produced using any one of the retroviral methods (e.g., lentiviral methods) provided herein. In some embodiments, such modified TILs are produced using any of the transposon / transposase systems described herein, such as the piggyBac method (e.g., piggyBac transposon and transposase or piggyBac-like transposon and transposase), the Sleeping Beauty method (e.g., Sleeping Beauty or Sleeping Beauty-like transposon and transposase), the Helraiser method (e.g., Helraiser and Helraiser-like transposon and transposase), and the Tol2 method (e.g., Tol2 and Tol2-like transposon and transposase).

[0508] 1. Membrane-anchored immunomodulatory fusion proteins In some embodiments, the modified TILs provided herein comprise a membrane-anchored immunomodulatory fusion protein. The membrane-anchored immunomodulatory fusion protein comprises one or more immunomodulatory agents (e.g., cytokines) linked to a cell membrane anchor moiety. In such embodiments, the membrane-anchored immunomodulatory agent is tethered to the TIL surface membrane via the cell membrane anchor moiety, thus allowing the immunomodulatory agent to exert its effect in a targeted manner.

[0509] The immunomodulatory agent can be any suitable immunomodulatory agent, including, for example, any of the immunomodulatory agents provided herein. In some embodiments, the immunomodulatory agent is an interleukin that promotes an anti-tumor response. In some embodiments, the immunomodulatory agent is a cytokine. In specific embodiments, the immunomodulatory agent is IL-2, IL-12, IL-15, IL-18, IL-21, or a CD40 agonist (e.g., CD40L or an agonistic anti-CD40 binding domain (e.g., anti-CD40scFv)) or a bioactive variant thereof. In certain embodiments, two or more different membrane-anchored immunomodulatory fusion proteins are expressed on the TIL surface. In exemplary embodiments, the TIL comprises 2, 3, 4, 5, 6, 7, 8, 9, or 10 different membrane-anchored immunomodulatory fusion proteins.

[0510] The immunomodulatory agent is linked to a membrane anchor moiety that enables tethering of the immunomodulatory agent to the TIL cell surface. Suitable membrane anchor moieties include, for example, transmembrane domains of endogenous TIL cell surface proteins and fragments thereof. Exemplary transmembrane domains that can be used in the subject fusion proteins include, for example, the B7-1, B7-2, and CD8a transmembrane domains and fragments thereof. In some embodiments, the membrane anchor moiety further comprises the transmembrane domain and intracellular domain of an endogenous TIL cell surface protein, or a fragment thereof. In some embodiments, the membrane anchor moiety is a B7-1, B7-2, or CD8a transmembrane intracellular domain, or a fragment thereof. In certain embodiments, the membrane anchor moiety is a CD8a transmembrane domain having the amino acid sequence IYIWAPLAGTCGVLLLSLVIT (SEQ ID NO: 238). In certain embodiments, the membrane anchor moiety is a B7-1 transmembrane intracellular domain having the amino acid sequence LLPSWAITLISVNGIFVICCLTYCFAPRCRERRRNERLRRESVRPV (SEQ ID NO: 239). In certain embodiments, the cell membrane anchor moiety is a non-peptide cell membrane anchor moiety. In an exemplary embodiment, the non-peptide cell membrane anchor moiety is a glycophosphatidylinositol (GPI) anchor. A GPI anchor has a structure including a phosphoethanolamine linker, a carbohydrate core, and a phospholipid tail. In some embodiments, the carbohydrate core is modified with one or more side chains. In some embodiments, the carbohydrate core is modified with one or more of the following side chains: a phosphoethanolamine group, mannose, galactose, sialic acid, or other sugar.

[0511] The membrane-anchored immunomodulatory fusion protein comprises a linker that allows for the attachment of components of the membrane-anchored immunomodulatory fusion protein (e.g., the immunomodulatory agent to the cell membrane anchor moiety). Suitable linkers include those that are at least about 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, or 30 amino acid residues in length. In some embodiments, the linker is 5-10, 10-15, 15-20, 20-25, 25-30, 30-35, 35-40, 45-50, or 50-60 amino acids in length. Suitable linkers include, but are not limited to, cleavable linkers, non-cleavable linkers, peptide linkers, flexible linkers, rigid linkers, helical linkers, or non-helical linkers. In some embodiments, the linker is a peptide linker that optionally comprises Gly and Ser. In certain embodiments, the peptide linker utilizes a glycine-serine polymer, including, for example, (GS)n (SEQ ID NO:240), (GSGGS)n (SEQ ID NO:241), (GGGS)n (SEQ ID NO:242), (GGGGS)n (SEQ ID NO:243), (GGGGGS)n (SEQ ID NO:244), and (GGGGGGS)n (SEQ ID NO:245), where n is at least one integer (and generally, 3 to 10). Additional linkers that can be used in the present compositions and methods are described in U.S. Patent Publication Nos. 2006 / 0074008, 2005 / 0238649, and 2006 / 0024317, each of which is incorporated by reference in its entirety, particularly in relevant portions relating to linkers. In some embodiments, the peptide linker is SGGGGSGGGGSGGGGSGGGGSGGGSLQ (SEQ ID NO: 246).

[0512] In some embodiments, the linker is a non-cleavable linker. In exemplary embodiments, a cleavable linker allows for release of the immunomodulatory agent into the tumor microenvironment. Cleavable linkers are also useful in embodiments in which two membrane-anchored immunomodulatory fusion proteins are co-expressed in the same TIL (see, e.g., Figure 36 and Tables 58 and 59). In exemplary embodiments, the linker is a self-cleaving 2A peptide. See, e.g., Liu et al., Sci. Rep. 7(1):2193 (2017) (incorporated by reference in relevant portions relating to the 2A peptide). 2A peptides are viral oligopeptides that mediate cleavage of polypeptides during translation in eukaryotic cells. In some embodiments, the 2A peptide comprises a C-terminus having the amino acid sequence GDVEXiNPGP (SEQ ID NO: 247), where Xi is any naturally occurring amino acid residue. In certain embodiments, the 2A peptide is the porcine teschovirus-1 2A peptide (GSGATNFSLLKQAGDVEENPGP, SEQ ID NO: 248). In some embodiments, the 2A peptide is the equine rhinitis A virus 2A peptide (GSGQCTNYALLKLAGDVESNPGP, SEQ ID NO: 249). In certain embodiments, the 2A peptide is the foot-and-mouth disease virus 2A peptide: (GSGEGRGSLLTCGDVEENPGP, SEQ ID NO: 250). In some embodiments, the cleavable linker comprises a furin-cleavable sequence. Exemplary furin-cleavable sequences are described, for example, in Duckert et al., Protein Engineering, Design & Selection 17(1):107-112 (2004), and U.S. Patent No. 8,871,906, each of which is incorporated herein by reference, particularly in relevant portions relating to furin-cleavable sequences. In some embodiments, the linker comprises a 2A peptide and a furin-cleavable sequence. In an exemplary embodiment, the furin-cleavable 2A peptide comprises the amino acid sequence RAKRSGSGATNFSLLKQAGDVEENPGP (SEQ ID NO: 251).

[0513] In some embodiments, the immunomodulatory agent is attached to the membrane-anchored immunomodulatory fusion protein by a degradable linker (e.g., a disulfide linker) such that under physiological conditions, the linker degrades, thereby releasing the immunomodulatory agent. In some embodiments, the immunomodulatory agent is reversibly linked to a functional group via the degradable linker such that under physiological conditions, the linker degrades, thereby releasing the immunomodulatory agent. Suitable degradable linkers include, but are not limited to, protease-sensitive linkers that are sensitive to one or more enzymes present in a biological medium, such as proteases in the tumor microenvironment, such as matrix metalloproteinases (e.g., matrix metalloproteinase 2 (MMP2) or matrix metalloproteinase 9 (MMP9)) present in the tumor microenvironment or inflamed tissue.

[0514] In other embodiments, the components of the membrane-anchored immunomodulatory fusion protein are linked by an enzyme-sensitive linker. Exemplary cleavable linkers include those recognized by one of the following enzymes: metalloproteases MMP-1, MMP-2, MMP-3, MMP-8, MMP-9, MMP-14, plasmin, PSA, PSMA, cathepsin D, cathepsin K, cathepsin S, ADAM10, ADAM12, ADAMTS, caspase-1, caspase-2, caspase-3, caspase-4, caspase-5, caspase-6, caspase-7, caspase-8, caspase-9, caspase-10, caspase-11, caspase-12, caspase-13, caspase-14, and TACE. See, for example, US Pat. Nos. 8,541,203 and 8,580,244, each of which is incorporated by reference in its entirety and in relevant portions relating to cleavable linkers.

[0515] In certain embodiments, the membrane-anchored immunomodulatory fusion protein comprises a signal peptide that facilitates translocation of the fusion protein to the TIL cell membrane. Any suitable signal peptide that facilitates localization of the fusion protein to the TIL cell membrane can be used. In some embodiments, the signal peptide does not interfere with the biological activity of the immunomodulator. Exemplary signal peptide sequences include, but are not limited to, the human granulocyte-macrophage colony-stimulating factor (GM-CSF) receptor signal sequence, the human prolactin signal sequence, and the human IgE signal sequence. In certain embodiments, the fusion protein comprises a human IgE signal sequence. In an exemplary embodiment, the human IgE signal sequence has the amino acid sequence MDWTWILFLVAAATRVHS (SEQ ID NO: 252). In some embodiments, the human IgE signal sequence comprises the amino acid sequence NIKGSPWKGSLLLLLVSNLLLCQSVAP (SEQ ID NO: 253). In some embodiments, the signal peptide sequence is the IL-2 signal sequence having the amino acid sequence MYRMQLLSCIALSLALVTNS (SEQ ID NO: 254).

[0516] In some embodiments, the membrane-anchored immunomodulatory fusion protein has, from N-terminus to C-terminus, the formula: It is in accordance with S-IA-LC, wherein S is a signal peptide, IA is an immunomodulatory agent, L is a linker, and C is a cell membrane anchor moiety.

[0517] In some embodiments, the signal peptide S is any one of SEQ ID NOs: 252-254. In some embodiments, the cell membrane anchor portion is SEQ ID NO: 277. In exemplary embodiments, the immunomodulatory agent is IL-2, IL-12, IL-15, IL-18, IL-21, or a CD40 agonist (e.g., CD40L or an anti-CD40 scFv described herein). In some embodiments, C is the B7-1 transmembrane-intracellular domain (e.g., SEQ ID NO: 239). Exemplary membrane-anchored immunomodulatory fusion proteins according to the above formulas are shown in Figures 36 and 37.

[0518] In some embodiments, the TIL comprises, from N-terminus to C-terminus, two or more different membrane-anchored immunomodulatory fusion proteins according to the formula: S-IA-LC, wherein each of the different membrane-anchored immunomodulatory fusion proteins comprises a different immunomodulatory agent. In some embodiments, the two or more different immunomodulatory agents are selected from the group consisting of IL-12 and IL-15, IL-15 and IL-18, CD40L and IL-15, IL-15 and IL-21, and IL-2 and IL-12.

[0519] In some embodiments comprising two membrane-anchored immunomodulatory fusion proteins, the membrane-anchored immunomodulatory fusion protein has, from N-terminus to C-terminus, the formula: Arranged according to S1-IA1-L1-C1-L2-S2-IA2-L3-C2, wherein S1 and S2 are each a signal peptide, IA1 and IA2 are each an immunomodulatory agent, L1-L3 are each a linker, and C1 and C2 are each a cell membrane anchor moiety. In some embodiments, IA1 and IA2 are the same immunomodulatory agent. In certain embodiments, IA1 and IA2 are different immunomodulatory agents. Suitable immunomodulatory agents include any of those described herein. In some embodiments, IA1 and IA2 are independently selected from IL-2, IL-12, IL-15, IL-18, IL-21, a CD40 agonist (e.g., CD40L or an agonistic anti-CD40 binding domain (e.g., an anti-CD40 scFv)), or a bioactive variant thereof. In some further embodiments, IA1 and IA2 are selected from the group consisting of IL-12 and IL-15, IL-15 and IL-18, CD40L and IL-15, IL-15 and IL-21, and IL-2 and IL-12. In some embodiments, one or more of L1-L3 are cleavable linkers. In some embodiments, two or more of L1-L3 are different linkers. In an exemplary embodiment, L2 is a cleavable linker. In some embodiments, L2 is a furin-cleavable P2A linker (e.g., SEQ ID NO: 251). In some embodiments, C1 and C2 are independently a transmembrane domain and / or a transmembrane-intracellular domain. In certain embodiments, C1 and C2 are the same. In an exemplary embodiment, C1 and C2 are each a B7-1 transmembrane-intracellular domain (e.g., SEQ ID NO: 239). In an exemplary embodiment, C1 and C2 are different. Exemplary constructs containing two membrane-anchored immunomodulatory fusion proteins according to the above formula are shown in FIG. 36 and Tables 58 and 59.

[0520] Modified TILs comprising a cell membrane-anchored immunomodulatory fusion protein associated with their surface can be generated by genetically modifying a population of TILs to include a nucleic acid encoding the fusion protein. Any suitable genetic modification method can be used to produce such modified TILs, including, for example, the CRISPR, TALE, zinc finger, and Cas-CLOVER methods described herein.

[0521] Any suitable TIL population can be genetically modified to produce the subject modified TIL composition. In some embodiments, a TIL population produced during any of the steps of the Process 2A method disclosed herein (see, e.g., Figures 2-6) is genetically modified to produce the subject modified TIL. In an exemplary embodiment, a TIL population produced during any of the steps of the GEN3 method disclosed herein (see, e.g., Figure 7) is genetically modified to produce the subject modified TIL. In an exemplary embodiment, TILs produced from the second step of the Process 2A method and / or the rapid expansion step of the GEN3 method provided herein are genetically modified to produce the subject modified TIL. In some embodiments, PD-1-positive TILs preselected using the methods described herein are genetically modified to produce the subject modified TIL.

[0522] Any suitable TIL population can be transiently genetically modified to produce a subject's transiently modified TIL composition. In some embodiments, a TIL population produced during any of the steps of the Process 2A method disclosed herein (see, e.g., Figures 2-6) is transfected with a nucleic acid encoding a cell membrane-anchored immunomodulatory fusion protein to transiently express the cell membrane-anchored immunomodulatory fusion protein in the subject's transiently modified TILs. In an exemplary embodiment, a TIL population produced during any of the steps of the GEN3 method disclosed herein (see, e.g., Figure 7) is transfected with a nucleic acid encoding a cell membrane-anchored immunomodulatory fusion protein to transiently express the cell membrane-anchored immunomodulatory fusion protein in the subject's transiently modified TILs. In an exemplary embodiment, TILs produced from the first expansion step of the Process 2A method and / or the priming expansion step of the GEN3 method provided herein are transfected with a nucleic acid encoding a cell membrane-anchored immunomodulatory fusion protein to transiently express the cell membrane-anchored immunomodulatory fusion protein in the subject's transiently modified TILs. In exemplary embodiments, TILs produced from the second expansion step of the Process 2A method and / or the rapid expansion step of the GEN3 method provided herein are transfected with a nucleic acid encoding a cell membrane-anchored immunomodulatory fusion protein to transiently express the cell membrane-anchored immunomodulatory fusion protein in the subject's transiently modified TILs. In some embodiments, PD-1-positive TILs preselected using the methods described herein are transfected with a nucleic acid encoding a cell membrane-anchored immunomodulatory fusion protein to transiently express the cell membrane-anchored immunomodulatory fusion protein in the subject's transiently modified TILs.

[0523] Also provided herein are nucleic acids encoding membrane-anchored immunomodulatory fusion proteins, expression vectors containing such nucleic acids, and host cells containing the nucleic acids or expression vectors. Any suitable promoter can be used to express the membrane-anchored immunomodulatory fusion protein. In exemplary embodiments, the promoter is an inducible promoter. Vectors for expressing the subject membrane-anchored immunomodulatory fusion proteins include, but are not limited to, adenoviral vectors, retroviral vectors, lentiviral vectors, and adeno-associated vectors (AAV). In some embodiments, the piggyBac transposon is used to express the subject membrane-anchored immunomodulatory fusion proteins. Exemplary nucleic acids encoding exemplary membrane-anchored immunomodulatory fusion proteins and components of such fusion proteins are shown in Figures 36 and 37 and Tables 58 and 59.

[0524] In some embodiments, the nucleic acid encoding the membrane-anchored immunomodulatory fusion protein is an mRNA. In exemplary embodiments, the mRNA comprises one or more modifications that improve the intracellular stability and / or translation efficiency of the mRNA. In some embodiments, the mRNA comprises a 5' cap or cap analog that improves the half-life of the mRNA. Exemplary cap structures include ARCA, mCAP, m 7 GpppN(cap 0), m 7 GpppNm (cap 1), and m 7 In some embodiments, the 5' cap has the formula: m7 Gppp[N 2’Ome ] n [N] m in which: m7G is N7-methylated guanosine or any guanosine analog, N is any natural, modified, or unnatural nucleoside, "n" can be any integer from 0 to 4, and "m" can be an integer from 1 to 9. Exemplary 5' caps are disclosed in U.S. Pat. No. 10,703,789 and WO2017 / 053297, which are incorporated by reference in their entireties, particularly for their disclosure regarding 5' caps and cap analogs.

[0525] In some embodiments, the nucleic acid encoding the membrane-anchored immunomodulatory fusion protein is an mRNA and further comprises a 3' untranslated region (UTR) or a modified UTR. 3'UTRs are known to have adenosine and uridine stretches. These AU-rich signatures are particularly common in genes with high turnover rates. Based on their sequence characteristics and functional properties, AU-rich elements (AREs) can be divided into three classes (Chen et al., 1995): Class I AREs contain several dispersed copies of the AUUUA motif within the U-rich region. C-Myc and MyoD contain Class I AREs. Class II AREs have two or more overlapping UUAUUUA(U / A)(U / A) nonamers. Molecules containing this type of ARE include GM-CSF and TNF-α. Class III AREs are less well-defined. These U-rich regions do not contain the AUUUA motif. c-Jun and Myogenin are two well-studied examples of this class. While most proteins that bind to AREs are known to destabilize messengers, members of the ELAV family, particularly HuR, have been reported to increase mRNA stability. HuR binds to all three classes of AREs. Engineering a HuR-specific binding site into the 3'UTR of a nucleic acid molecule leads to HuR binding and therefore to message stabilization in vivo.

[0526] The introduction, removal, or modification of 3'UTR AU-rich elements (AREs) can be used to modulate the stability of the nucleic acids described herein. When engineering a particular nucleic acid, one or more copies of an ARE can be introduced to destabilize the polynucleotide of the invention, thereby suppressing translation and reducing production of the resulting protein. Similarly, AREs can be identified and removed or mutated to increase intracellular stability, thus increasing translation and production of the resulting protein. Transfection experiments can be performed using nucleic acids in relevant cell lines, and protein production can be assayed at various time points after transfection. For example, cells can be transfected with different ARE-engineered molecules and the relevant protein using an ELISA kit, and protein production can be assayed 6 hours, 12 hours, 24 hours, 48 hours, and 7 days after transfection.

[0527] In some embodiments, the nucleic acid encoding the membrane-anchored immunomodulatory fusion protein is operably linked to the nuclear factor of activated T cells (NFAT) promoter, or a functional portion or variant thereof. As used herein, "NFAT promoter" refers to one or more NFAT-responsive elements linked to a minimal promoter of any gene expressed by a T cell. Preferably, the minimal promoter of a gene expressed by a T cell is a minimal human IL-2 promoter. The NFAT-responsive elements can include, for example, NFAT1, NFAT2, NFAT3, and / or NFAT4-responsive elements. The NFAT promoter (or functional portion or variant thereof) can include any number of binding motifs, e.g., at least two, at least three, at least four, at least five, or at least six, at least seven, at least eight, at least nine, at least ten, at least eleven, or up to twelve binding motifs.

[0528] [Table 4]

[0529] In preferred embodiments, the NFAT promoter comprises six NFAT binding motifs. See, e.g., U.S. Patent No. 8,556,882 (incorporated by reference in its entirety, particularly with respect to relevant portions relating to the NFAT promoter). In some embodiments, the NFAT promoter system controls expression of an immunomodulatory fusion protein comprising any of the immunomodulatory agents described herein. In certain embodiments, the immunomodulatory agent is selected from IL-2, IL-12, IL-15, IL-18, IL-21, and a CD40 agonist (e.g., CD40L or an agonistic anti-CD40 binding domain (e.g., anti-CD40scFv)) or a bioactive variant thereof. Exemplary nucleic acids encoding exemplary membrane-anchored immunomodulatory fusion proteins of interest operably linked to the NFAT promoter are shown in Table 59. In some embodiments, the NFAT promoter system controls expression of an immunomodulatory fusion protein comprising IL-15. In some embodiments, the NFAT promoter system controls expression of an immunomodulatory fusion protein comprising IL-21. In some embodiments, the NFAT promoter system controls the expression of an immunomodulatory fusion protein comprising IL-15 and IL-21.

[0530] In some embodiments, the invention provides TILs genetically modified to include DNA encoding an immunomodulatory fusion protein operably linked to an NFAT promoter. In some embodiments, the NFAT promoter controls expression of DNA encoding an immunomodulatory fusion protein comprising any of the immunomodulatory agents described herein. In certain embodiments, the immunomodulatory agent is selected from IL-2, IL-12, IL-15, IL-18, IL-21, and a CD40 agonist (e.g., CD40L or an agonistic anti-CD40 binding domain (e.g., anti-CD40 scFv)) or a bioactive variant thereof. In some embodiments, the NFAT promoter controls expression of DNA encoding an immunomodulatory fusion protein comprising IL-15. In some embodiments, the NFAT promoter controls expression of DNA encoding an immunomodulatory fusion protein comprising IL-21. In some embodiments, the NFAT promoter controls expression of DNA encoding an immunomodulatory fusion protein comprising IL-15 and IL-21.

[0531] In some embodiments, the invention provides TILs that have been genetically modified to comprise DNA encoding an immunomodulatory fusion protein operably linked to an NFAT promoter, the immunomodulatory fusion protein comprising, from N-terminus to C-terminus, a sequence of the formula: Arranged according to S1-IA1-L1-C1-L2-S2-IA2-L3-C2, wherein S1 and S2 are each a signal peptide, IA1 and IA2 are each an immunomodulatory agent, L1-L3 are each a linker, and C1 and C2 are each a cell membrane anchor moiety. In some embodiments, IA1 and IA2 are the same immunomodulatory agent. In certain embodiments, IA1 and IA2 are different immunomodulatory agents. Suitable immunomodulatory agents include any of those described herein. In some embodiments, IA1 and IA2 are independently selected from IL-2, IL-12, IL-15, IL-18, IL-21, a CD40 agonist (e.g., CD40L or an agonistic anti-CD40 binding domain (e.g., an anti-CD40 scFv)), or a bioactive variant thereof. In some further embodiments, IA1 and IA2 are selected from the group consisting of IL-12 and IL-15, IL-15 and IL-18, CD40L and IL-15, IL-15 and IL-21, and IL-2 and IL-12. In some embodiments, IA1 and IA2 are independently selected from IL-15 and IL-21. In some embodiments, IA1 is IL-15 and IA2 is IL-21. In some embodiments, IA1 is IL-21 and IA2 is IL-15. In some embodiments, one or more of L1-L3 are cleavable linkers. In some embodiments, two or more of L1-L3 are different linkers. In an exemplary embodiment, L2 is a cleavable linker. In some embodiments, L2 is a furin-cleavable P2A linker (e.g., SEQ ID NO: 251). In some embodiments, C1 and C2 are independently transmembrane domains and / or transmembrane-intracellular domains. In certain embodiments, C1 and C2 are the same. In an exemplary embodiment, C1 and C2 are each the B7-1 transmembrane-intracellular domain (e.g., SEQ ID NO: 239). In an exemplary embodiment, C1 and C2 are different. An exemplary construct comprising two membrane-anchored immunomodulatory fusion proteins according to the above formula is shown in Figure 36.

[0532] A nucleic acid encoding a membrane-anchored immunomodulatory fusion protein of interest can be introduced into a TIL population to produce transiently modified or genetically modified TILs expressing the membrane-anchored immunomodulatory fusion protein using any suitable method. In some embodiments, the nucleic acid encoding the membrane-anchored immunomodulatory fusion protein is introduced into a TIL population using a microfluidic platform. In some embodiments, the microfluidic platform is a microfluidic platform that does not contain an SQZ vector. See, for example, International Patent Application Publication Nos. WO2013 / 059343A1, WO2017 / 008063A1, or WO2017 / 123663A1, or U.S. Patent Application Publication Nos. 2014 / 0287509A1, 2018 / 0201889A1, or 2018 / 0245089A1 (all of which are incorporated herein by reference in their entirety, particularly for disclosure of microfluidic platforms for nucleic acid delivery). In the SQZ platform, the cell membrane of cells for modification (e.g., TILs) is temporarily disrupted by microfluidic contraction, thereby allowing nucleic acids encoding membrane-anchored immunomodulatory fusion proteins to be delivered into the cells.

[0533] In some embodiments, the nucleic acid encoding the membrane-anchored immunomodulatory fusion protein is mRNA, and a microfluidic platform (e.g., a microfluidic platform without an SQZ vector) is used to deliver the mRNA to TILs to produce transiently modified TILs. In some embodiments, the nucleic acid encoding the membrane-anchored immunomodulatory fusion protein is DNA, and a microfluidic platform (e.g., a microfluidic platform without an SQZ vector) is used to deliver the DNA to TILs to produce stable genetically modified TILs. A microfluidic platform (e.g., a microfluidic platform without an SQZ vector) may also be used to deliver the nucleic acid to any TIL population produced during any step of the Process 2A method disclosure herein (see, e.g., Figures 2-6) or the GEN3 method disclosure herein (see, e.g., Figure 7) to produce modified TILs. In some embodiments, the membrane-anchored immunomodulatory fusion protein comprises IL-2, IL-12, IL-15, IL-18, IL-21, a CD40 agonist (e.g., CD40L or an agonist anti-CD40 binding domain (e.g., anti-CD40scFv)), or any combination thereof.

[0534] In exemplary embodiments, the modified TILs provided herein comprise two membrane-anchored immunomodulatory fusion proteins, each comprising a different immunomodulatory agent (i.e., a first and a second immunomodulatory agent), where the first immunomodulatory agent is IL-15. In some embodiments, the second immunomodulatory agent is IL-2, IL-12, IL-18, IL-21, CD40L, or an anti-CD40 binding domain (e.g., an anti-CD40 scFv).

[0535] In exemplary embodiments, the modified TILs provided herein comprise two membrane-anchored immunomodulatory fusion proteins, each comprising a different immunomodulatory agent (i.e., a first and a second immunomodulatory agent), where the first immunomodulatory agent is CD40L. In some embodiments, the second immunomodulatory agent is IL-2, IL-12, IL-15, IL-18, IL-21, or a CD40 agonist (e.g., CD40L or an agonistic anti-CD40 binding domain (e.g., anti-CD40scFv)) or a bioactive variant thereof.

[0536] In exemplary embodiments, the modified TILs provided herein comprise two membrane-anchored immunomodulatory fusion proteins, each comprising a different immunomodulatory agent (i.e., a first and a second immunomodulatory agent), where the first immunomodulatory agent is IL-12. In some embodiments, the second immunomodulatory agent is IL-2, IL-15, IL-18, IL-21, CD40L, or an anti-CD40 binding domain (e.g., an anti-CD40 scFv).

[0537] In exemplary embodiments, the modified TILs provided herein comprise two membrane-anchored immunomodulatory fusion proteins, each comprising a different immunomodulatory agent (i.e., a first and a second immunomodulatory agent), where the first immunomodulatory agent is IL-18. In some embodiments, the second immunomodulatory agent is IL-2, IL-12, IL-15, IL-21, CD40L, or an anti-CD40 binding domain (e.g., an anti-CD40 scFv).

[0538] In exemplary embodiments, the modified TILs provided herein comprise two membrane-anchored immunomodulatory fusion proteins, each comprising a different immunomodulatory agent (i.e., a first and a second immunomodulatory agent), where the first immunomodulatory agent is IL-21. In some embodiments, the second immunomodulatory agent is IL-2, IL-12, IL-15, IL-18, CD40L, or an anti-CD40 binding domain (e.g., an anti-CD40 scFv).

[0539] In exemplary embodiments, the modified TILs provided herein comprise two membrane-anchored immunomodulatory fusion proteins, each comprising a different immunomodulatory agent (i.e., a first and a second immunomodulatory agent), where the first immunomodulatory agent is IL-2. In some embodiments, the second immunomodulatory agent is IL-2, IL-12, IL-15, IL-18, IL-21, CD40L, or an anti-CD40 binding domain (e.g., an anti-CD40 scFv).

[0540] In an exemplary embodiment, the modified TILs provided herein comprise two membrane-anchored immunomodulatory fusion proteins, each comprising a different immunomodulatory agent (i.e., a first and a second immunomodulatory agent), wherein the first immunomodulatory agent is IL-2 and the second immunomodulatory agent is IL-12.

[0541] In an exemplary embodiment, the modified TILs provided herein comprise two membrane-anchored immunomodulatory fusion proteins, each comprising a different immunomodulatory agent (i.e., a first and a second immunomodulatory agent), wherein the first immunomodulatory agent is IL-2 and the second immunomodulatory agent is IL-15.

[0542] In an exemplary embodiment, the modified TILs provided herein comprise two membrane-anchored immunomodulatory fusion proteins, each comprising a different immunomodulatory agent (i.e., a first and a second immunomodulatory agent), wherein the first immunomodulatory agent is IL-2 and the second immunomodulatory agent is IL-18.

[0543] In an exemplary embodiment, the modified TILs provided herein comprise two membrane-anchored immunomodulatory fusion proteins, each comprising a different immunomodulatory agent (i.e., a first and a second immunomodulatory agent), wherein the first immunomodulatory agent is IL-2 and the second immunomodulatory agent is IL-21.

[0544] In an exemplary embodiment, the modified TILs provided herein comprise two membrane-anchored immunomodulatory fusion proteins, each comprising a different immunomodulatory agent (i.e., a first and a second immunomodulatory agent), wherein the first immunomodulatory agent is IL-2 and the second immunomodulatory agent is a CD40L or anti-CD40 binding domain (e.g., an anti-CD40 scFv).

[0545] In an exemplary embodiment, the modified TILs provided herein comprise two membrane-anchored immunomodulatory fusion proteins, each comprising a different immunomodulatory agent (i.e., a first and a second immunomodulatory agent), wherein the first immunomodulatory agent is IL-12 and the second immunomodulatory agent is IL-15.

[0546] In an exemplary embodiment, the modified TILs provided herein comprise two membrane-anchored immunomodulatory fusion proteins, each comprising a different immunomodulatory agent (i.e., a first and a second immunomodulatory agent), wherein the first immunomodulatory agent is IL-12 and the second immunomodulatory agent is IL-18.

[0547] In an exemplary embodiment, the modified TILs provided herein comprise two membrane-anchored immunomodulatory fusion proteins, each comprising a different immunomodulatory agent (i.e., a first and a second immunomodulatory agent), wherein the first immunomodulatory agent is IL-12 and the second immunomodulatory agent is IL-21.

[0548] In an exemplary embodiment, the modified TILs provided herein comprise two membrane-anchored immunomodulatory fusion proteins, each comprising a different immunomodulatory agent (i.e., a first and a second immunomodulatory agent), wherein the first immunomodulatory agent is IL-12 and the second immunomodulatory agent is a CD40L or anti-CD40 binding domain (e.g., an anti-CD40 scFv).

[0549] In an exemplary embodiment, the modified TILs provided herein comprise two membrane-anchored immunomodulatory fusion proteins, each comprising a different immunomodulatory agent (i.e., a first and a second immunomodulatory agent), wherein the first immunomodulatory agent is IL-15 and the second immunomodulatory agent is IL-18.

[0550] In an exemplary embodiment, the modified TILs provided herein comprise two membrane-anchored immunomodulatory fusion proteins, each comprising a different immunomodulatory agent (i.e., a first and a second immunomodulatory agent), wherein the first immunomodulatory agent is IL-15 and the second immunomodulatory agent is IL-21.

[0551] In an exemplary embodiment, the modified TILs provided herein comprise two membrane-anchored immunomodulatory fusion proteins, each comprising a different immunomodulatory agent (i.e., a first and a second immunomodulatory agent), wherein the first immunomodulatory agent is IL-15 and the second immunomodulatory agent is a CD40L or anti-CD40 binding domain (e.g., an anti-CD40 scFv).

[0552] In an exemplary embodiment, the modified TILs provided herein comprise two membrane-anchored immunomodulatory fusion proteins, each comprising a different immunomodulatory agent (i.e., a first and a second immunomodulatory agent), wherein the first immunomodulatory agent is IL-18 and the second immunomodulatory agent is IL-21.

[0553] In an exemplary embodiment, the modified TILs provided herein comprise two membrane-anchored immunomodulatory fusion proteins, each comprising a different immunomodulatory agent (i.e., a first and a second immunomodulatory agent), wherein the first immunomodulatory agent is IL-18 and the second immunomodulatory agent is a CD40L or anti-CD40 binding domain (e.g., an anti-CD40 scFv).

[0554] In an exemplary embodiment, the modified TILs provided herein comprise two membrane-anchored immunomodulatory fusion proteins, each comprising a different immunomodulatory agent (i.e., a first and a second immunomodulatory agent), wherein the first immunomodulatory agent is IL-21 and the second immunomodulatory agent is a CD40L or anti-CD40 binding domain (e.g., an anti-CD40 scFv).

[0555] Additional membrane-anchored immunomodulatory fusion proteins that can be included in the modified TILs provided herein are described in WO2019 / 157130A1 (which is incorporated by reference in its entirety, particularly the relevant portions relating to membrane-anchored immunomodulatory fusion proteins).

[0556] Exemplary membrane-anchored immunomodulatory fusion proteins included in the modified TILs provided herein are shown in Figures 36 and 37 and Tables 58 and 59.

[0557] In some embodiments, a nucleic acid encoding any of the above membrane-anchored immunomodulatory fusion proteins is operably linked to an NFAT promoter or a functional portion or variant thereof.

[0558] 2. Immunomodulator-TIL antigen-binding domain fusion proteins In some embodiments, the modified TILs provided herein comprise immunomodulatory fusion proteins, such fusion proteins comprising one or more immunomodulatory agents linked to a TIL antigen binding domain (ABD). In some embodiments, the one or more immunomodulatory agents are tethered to the TIL surface membrane upon binding of the TIL ABD to a TIL surface antigen.

[0559] The TIL antigen-binding domain comprises an antibody variable heavy domain (VH) and variable light domain (VL). In some embodiments, the TIL antigen-binding domain is a full-length antibody comprising a heavy chain according to the formula: VH-CH1-hinge-CH2-CH3 and a light chain according to the formula: VL-CL, where VH is a variable heavy domain, CH1, CH2, CH3 are heavy chain constant domains, VL is a variable light domain, and CL is a light chain constant domain. In some embodiments, the TIL antigen-binding domain is an antibody fragment. In certain embodiments, the TIL antigen-binding domain is a Fab, Fab', F(ab')2, F(ab)2, variable fragment (Fv), domain antibody (dAb), or single-chain variable fragment (scFv).

[0560] The TIL antigen-binding domain can bind to any suitable TIL antigen that enables attachment of the immunomodulator-TIL ABD fusion protein to the surface of TILs. In exemplary embodiments, the TIL antigen-binding domain can bind to a TIL surface antigen, including, but not limited to, D16, CD45, CD4, CD8, CD3, CD11a, CD11b, CD11c, CD18, LFA-1, CD25, CD127, CD56, CD19, CD20, CD22, HLA-DR, CD197, CD38, CD27, CD137, OX40, GITR, CD56, CD196, CXCR3, CXCR4, CXCR5, CD84, CD229, CCR1, CCR5, CCR4, CCR6, CCR8, and / or CCR10. In some embodiments, the ABD binds to CD45. In certain embodiments, the ABD binds to a CD45 isoform selected from CD45RA, CD45RB, CD45RC, or CD45Rβ. In certain embodiments, the ABD binds to CD45 that is primarily expressed on T cells.

[0561] In certain embodiments, the ABD binds to a checkpoint inhibitor. Exemplary checkpoint inhibitors include, but are not limited to, PD-1, PD-L1, LAG-3, TIM-3, and CTLA-4 (see, e.g., Qin et al., Molecular Cancer 18:155 (2019)). In some embodiments, the ABD binds to a checkpoint inhibitor expressed on an immune effector cell (e.g., a T cell or an NK cell). Exemplary anti-PD-1 antibodies are disclosed, for example, in U.S. Pat. Nos. 7,695,715, 7,332,582, 9,205,148, 8,686,119, 8,735,553, 7,488,802, 8,927,697, 8,993,731, and 9,102,727, which are incorporated by reference in their entirety, particularly the relevant portions relating to anti-PD-1 antibodies. Exemplary anti-PD-L1 antibodies are disclosed in U.S. Patent Nos. 8,217,149, 8,779,108, 8,168,179, 8,552,154, 8,460,927, and 9,175,082, which are incorporated by reference in their entireties, particularly the relevant portions relating to anti-PD-L1 antibodies. Exemplary anti-LAG-3 antibodies are disclosed in U.S. Patent Nos. 9,244,059, 9,244,059, and 9,505,839, which are incorporated by reference in their entireties, particularly the relevant portions relating to anti-LAG-3 antibodies. Exemplary TIM-3 antibodies are disclosed in WO2016 / 161270, US8,841,418, and US9,163,087, which are incorporated by reference in their entirety, particularly relevant portions relating to anti-TIM-3 antibodies. Exemplary CTLA-4 antibodies are disclosed in US6,984,720 and US7,411,057, which are incorporated by reference in their entirety, particularly relevant portions relating to anti-CTLA-4 antibodies.

[0562] In some embodiments, the ABD is an anti-CD45 antibody or a fragment thereof. In certain embodiments, the anti-CD45 antibody is a human anti-CD45 antibody, a humanized anti-CD45 antibody, or a chimeric anti-CD45 antibody. In exemplary embodiments, the ABD comprises vhCDR1-3 and vlCDR1-3 of the anti-CD45 antibody BC8 (see US2017 / 0326259, particularly the relevant portions relating to the anti-CD45 antibody sequences, which are incorporated herein by reference). In some embodiments, the ABD comprises the variable heavy domain and variable domain of the anti-CD45 antibody BC8. In some embodiments, the ABD comprises the vhCDR1-3 and vlCDR1-3 or the VH and VL of one of the following anti-CD45 antibodies: 10G10, UCHL1, 9.4, 4B2, or GAP8.3 (see Spertini et al., Immunology 113(4):441-452 (2004); Buzzi et al. Cancer Research 52:4027-4035 (1992)).

[0563] The immunomodulatory fusion protein can be any suitable immunomodulatory agent, including, for example, any of the immunomodulatory agents provided herein. In some embodiments, the immunomodulatory agent is an interleukin that promotes an anti-tumor response. In some embodiments, the immunomodulatory agent is a cytokine. In specific embodiments, the immunomodulatory agent is IL-2, IL-12, IL-15, IL-21, or a bioactive variant thereof. In certain embodiments, the fusion protein comprises two or more immunomodulatory agents. In exemplary embodiments, the fusion protein comprises 2, 3, 4, 5, 6, 7, 8, 9, or 10 different immunomodulatory agents.

[0564] The TIL antigen-binding domain is attached to the immunomodulatory agent using any suitable linker. Suitable linkers include, but are not limited to, cleavable linkers, non-cleavable linkers, peptide linkers, flexible linkers, rigid linkers, helical linkers, or non-helical linkers. In some embodiments, the linker is a peptide linker optionally comprising Gly and Ser. Suitable linkers include linkers that are at least about 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, or 30 amino acid residues in length. In some embodiments, the linker is 5-10, 10-15, 15-20, 20-25, 25-30, 30-35, 35-40, 45-50, or 50-60 amino acids in length. In certain embodiments, the peptide linker is (GGGS) n or (GGGGS) n and a linker, where n represents the number of repeats of the motif and is an integer selected from 1 to 10. In some embodiments, the linker is an antibody hinge domain or a fragment thereof. In certain embodiments, the linker is a human immunoglobulin (Ig) hinge domain (e.g., an IgG1, IgG2, IgG3, IgG4, IgD, IgE, IgM, or IgA hinge) or a fragment thereof. In some embodiments, the immunomodulatory agent is directly linked to the TIL without a linker.

[0565] The immunomodulatory agent can be attached to the TIL antigen-binding domain at any suitable position that does not interfere with binding of the fusion protein to TIL. In some embodiments where the antigen-binding domain is a full-length antibody, the immunomodulatory agent is attached to the C- or N-terminus of either the heavy or light chain. In some embodiments where the antigen-binding domain is an scFv, the immunomodulatory agent is attached to the C- or N-terminus of the variable heavy or variable light domain. In some embodiments where the antigen-binding domain is a Fab, the immunomodulatory agent is attached to the C- or N-terminus of the variable heavy or variable light domain. In some embodiments where the antigen-binding domain is a Fab', the immunomodulatory agent is attached to the C- or N-terminus of the variable heavy or variable light domain. In some embodiments where the antigen-binding domain is a Fab'2, the immunomodulatory agent is attached to the C- or N-terminus of the variable heavy or variable light domain.

[0566] In some embodiments where the fusion protein comprises two or more immunomodulatory agents, the immunomodulatory agents are attached to each other using any of the linkers described herein. In some embodiments, the two or more immunomodulatory agents are attached to different positions on the antigen-binding domain. For example, in some embodiments where the TIL antigen-binding domain is a full-length antibody, the two or more immunomodulatory agents are attached to (i) different positions on the heavy chain, (ii) different positions on the light chain, or (iii) different positions on the heavy and / or light chain.

[0567] The subject immunomodulator-TIL antigen-binding domain fusion proteins can be produced using any suitable method. In one aspect, provided herein are nucleic acids encoding the subject fusion proteins, expression vectors containing such nucleic acids, and host cells containing the expression vectors. The host cells containing the expression vectors encoding the subject fusion proteins are cultured under conditions for expression of the fusion protein, after which the fusion protein is isolated and purified. In some embodiments, the purified fusion protein is then incubated with a population of TILs under conditions that allow binding of the fusion protein to the TILs.

[0568] In some embodiments, a subject immunomodulatory agent-TIL antigen binding domain fusion protein is attached to TILs produced during any of the steps of the Process 2A method disclosed herein (see, e.g., Figures 2-6). In exemplary embodiments, the fusion protein is attached to TILs produced during any of the steps of the GEN3 method disclosed herein (see, e.g., Figure 7). In exemplary embodiments, the fusion protein is attached to TILs produced from the first expansion step of the Process 2A method and / or the primed expansion step of the GEN3 method provided herein. In exemplary embodiments, the fusion protein is attached to TILs produced from the second expansion step of the Process 2A method and / or the rapid expansion step of the GEN3 method provided herein. In some embodiments, the TILs are PD-1-positive TILs preselected using the methods described herein.

[0569] A nucleic acid encoding a subject immunomodulator-TIL antigen binding domain fusion protein can be introduced into a TIL population to produce transiently modified or genetically modified TILs expressing the subject immunomodulator-TIL antigen binding domain fusion protein using any suitable method. In some embodiments, the nucleic acid encoding the subject immunomodulator-TIL antigen binding domain fusion protein is introduced into a TIL population using a microfluidic platform. In some embodiments, the microfluidic platform is a microfluidic platform that does not contain an SQZ vector. See, e.g., International Patent Application Publication Nos. WO2013 / 059343A1, WO2017 / 008063A1, or WO2017 / 123663A1, or U.S. Patent Application Publication Nos. 2014 / 0287509A1, 2018 / 0201889A1, or 2018 / 0245089A1 (all of which are incorporated by reference in their entireties, particularly for their disclosure of microfluidic platforms for nucleic acid delivery). In the SQZ platform, the cell membrane of cells for modification (e.g., TILs) is transiently disrupted by microfluidic contraction, thereby allowing delivery of a nucleic acid encoding an immunomodulator-TIL antigen-binding domain fusion protein into the cells.

[0570] In some embodiments, the nucleic acid encoding the subject immunomodulatory agent-TIL antigen-binding domain fusion protein is mRNA, and a microfluidic platform (e.g., a microfluidic platform without an SQZ vector) is used to deliver the mRNA to TILs to transiently produce modified TILs. In some embodiments, the nucleic acid encoding the subject immunomodulatory agent-TIL antigen-binding domain fusion protein is DNA, and a microfluidic platform (e.g., a microfluidic platform without an SQZ vector) is used to deliver the nucleic acid to TILs to produce stable genetically modified TILs. A microfluidic platform (e.g., a microfluidic platform without an SQZ vector) may also be used to deliver the nucleic acid to any TIL population produced during any step of the Process 2A method disclosure herein (see, e.g., Figures 2-6) or the GEN3 method disclosure herein (see, e.g., Figure 7) to produce modified TILs. In some embodiments, the membrane-anchored immunomodulatory fusion protein comprises IL-2, IL-12, IL-15, IL-21, or a combination thereof (e.g., IL-15 and IL-21).

[0571] Exemplary immunomodulatory agent-TIL antigen binding domain fusion proteins useful in the compositions and methods provided herein are further described, e.g., in U.S. Patent Application Publication No. 2020 / 0330514, which is incorporated by reference in its entirety, and relevant portions relating to immunomodulatory agent-TIL antigen binding domain fusion proteins.

[0572] B. Nanoparticle Composition In some embodiments, the subject modified TILs provided herein comprise one or more nanoparticles, which comprise one or more immunomodulatory agents. In some embodiments, the nanoparticles provided herein comprise a plurality of two or more proteins coupled to each other and / or a second component of the particle (e.g., reversibly linked via a degradable linker). In some embodiments, the protein of the nanoparticle is present in a polymer or silica. In certain embodiments, the nanoparticle comprises a nanoshell. The nanoparticles provided herein comprise one or more immunomodulatory agents. In some embodiments, the immunomodulatory agent is IL-2, IL-12, IL-15, IL-18, IL-21, or a CD40 agonist (e.g., CD40L or an agonistic anti-CD40 binding domain (e.g., anti-CD40 scFv)) or a bioactive variant thereof. The nanoparticles are attached to the surface of the TILs using any suitable technique described herein.

[0573] Exemplary nanoparticles for use in the subject modified TILs provided herein include, but are not limited to, liposomes, protein nanogels, nucleotide nanogels, polymeric nanoparticles, or solid nanoparticles. In some embodiments, the nanoparticle comprises a liposome. In exemplary embodiments, the nanoparticle comprises an immunomodulatory agent nanogel. In certain embodiments, the nanoparticle is an immunomodulatory agent nanogel having multiple immunomodulatory agents (e.g., cytokines) covalently bonded to each other. In some embodiments, the nanoparticle comprises at least one polymer, cationic polymer, or cationic block copolymer on the nanoparticle surface. Exemplary nanoparticles that can be used in the compositions provided herein are disclosed, for example, in U.S. Pat. Nos. 9,283,184 and 9,603,944, each of which is incorporated by reference in its entirety and incorporated by reference in relevant portions relating to nanoparticles.

[0574] The immunomodulatory agent can be any suitable immunomodulatory agent, including, for example, any of the immunomodulatory agents provided herein. In some embodiments, the immunomodulatory agent is an interleukin that promotes an anti-tumor response. In some embodiments, the immunomodulatory agent is a cytokine. In specific embodiments, the immunomodulatory agent is IL-2, IL-12, IL-15, IL-21, or a bioactive variant thereof. In certain embodiments, the fusion protein comprises two or more immunomodulatory agents. In exemplary embodiments, the fusion protein comprises 2, 3, 4, 5, 6, 7, 8, 9, or 10 different immunomodulatory agents.

[0575] In some embodiments, the nanoparticles comprise a protein and / or a second moiety (e.g., a degradable linker) covalently crosslinked to one another. In some embodiments, the nanoparticles comprise an immunomodulatory agent that is reversibly linked to a functional group or polymer via a degradable linker, or is "reversibly modified." In some embodiments, the nanoparticles are nanogels comprising multiple immunomodulatory agents crosslinked to one another via degradable linkers (see U.S. Pat. No. 9,603,944). In exemplary embodiments, the protein of the nanogel is crosslinked to a polymer (e.g., polyethylene glycol (PEG)). In some embodiments, the polymer is crosslinked to the nanogel surface.

[0576] In some embodiments, the immunomodulatory agents of the nanoparticles are reversibly linked to each other via a degradable linker (e.g., a disulfide linker) such that the linker degrades under physiological conditions, thereby releasing the immunomodulatory agent. In some embodiments, the immunomodulatory agents of the nanoparticles are reversibly linked to a functional group via a degradable linker such that the linker degrades under physiological conditions, thereby releasing the immunomodulatory agent. Suitable degradable linkers include, but are not limited to, two N-hydroxysuccinimide (NHS) ester groups joined together by a flexible disulfide-containing linker that is sensitive to reducing physiological environments; hydrolyzable linkers that are sensitive to acidic physiological environments (pH<7, e.g., pH 4-5, 5-6, or less than 6-7, e.g., 6.9); or protease-sensitive linkers that are sensitive to one or more enzymes present in biological media, such as matrix metalloproteinases present in tumor microenvironments or inflamed tissues (e.g., matrix metalloproteinase 2 (MMP2) or matrix metalloproteinase 9 (MMP9)). Crosslinkers sensitive to reducing physiological environments are, for example, crosslinkers with disulfide-containing linkers that react with amine groups on proteins due to the presence of NHS groups to crosslink the proteins into dense protein nanogels. In some embodiments, degradable crosslinkers include bis[2-(N-succinimidyl-oxycarbonyloxy)ethyl]disulfide.

[0577] In some embodiments, the degradable linker comprises at least one N-hydroxysuccinimide ester. In some embodiments, the degradable linker is a redox-responsive linker. In some embodiments, the redox-responsive linker comprises a disulfide bond. In some embodiments, the degradable linkers provided herein comprise at least one N-hydroxysuccinimide ester that can react with a protein at a neutral pH (e.g., about 6 to about 8 or about 7) without substantially denaturing the protein. In some embodiments, the degradable linker is a "redox-responsive" linker, meaning that it degrades in the presence of a reducing agent (e.g., glutathione, GSH) under physiological conditions (e.g., 20-40°C and / or pH 4-8), thereby reversibly releasing the intact protein from the compound to which it is linked. In some embodiments, the protein of the nanoparticle is linked to the degradable linker via a terminal or internal NH2 functional group (e.g., the side chain of lysine).

[0578] In other embodiments, the proteins of the nanoparticles are linked by enzyme-sensitive linkers. Exemplary cleavable linkers include those recognized by one of the following enzymes: metalloproteases MMP-1, MMP-2, MMP-3, MMP-8, MMP-9, MMP-14, plasmin, PSA, PSMA, cathepsin D, cathepsin K, cathepsin S, ADAM10, ADAM12, ADAMTS, caspase-1, caspase-2, caspase-3, caspase-4, caspase-5, caspase-6, caspase-7, caspase-8, caspase-9, caspase-10, caspase-11, caspase-12, caspase-13, caspase-14, and TACE. See, for example, US Pat. Nos. 8,541,203 and 8,580,244, each of which is incorporated by reference in its entirety and in relevant portions relating to cleavable linkers.

[0579] In some embodiments, the nanoparticles are nanogels comprising monodisperse immunomodulatory agents (e.g., cytokines). In some embodiments, the immunomodulatory agents of the nanogel are crosslinked to a polymer. In certain embodiments, the polymer is crosslinked to the surface of the nanogel. In certain embodiments, the nanogel comprises a) one or more immunomodulatory agents reversibly and covalently crosslinked to each other via a degradable linker, and b) a polymer crosslinked to a surface-exposed protein of the nanogel. Such nanogels can be made by contacting one or more immunomodulatory agents with a degradable linker under conditions that allow the immunomodulatory agents to be reversibly covalently linked to each other via the degradable linker to form multiple immunomodulatory agent nanogels. The immunomodulatory agent nanogel is then contacted with a polymer (e.g., polyethylene glycol) under conditions that allow crosslinking of the polymer to the immunomodulatory agent nanogel, thereby producing multiple immunomodulatory agent polymer nanogels.

[0580] In some embodiments, the nanoparticles comprise one or more polymers. Exemplary polymers include, but are not limited to, aliphatic polyesters, poly(lactic acid) (PLA), poly(glycolic acid) (PGA), copolymers of lactic and glycolic acid (PLGA), polycaprolactone (PCL), polyanhydrides, poly(ortho)esters, polyurethanes, poly(butyric acid), poly(valeric acid), and poly(lactide-co-caprolactone), as well as natural polymers such as alginate and other polysaccharides including dextran and cellulose, collagen, their chemical derivatives (e.g., including substitutions, additions of chemical groups such as alkyl, alkylene, hydroxylation, oxidation, and other modifications routinely performed by those skilled in the art), albumin and other hydrophilic proteins, zein and other prolamines, and hydrophilic proteins, copolymers, and mixtures thereof. In some embodiments, the immunomodulatory agent of the nanoparticles is linked to a hydrophilic polymer. Exemplary hydrophilic polymers include, but are not limited to, polyethylene glycol (PEG), polyethylene glycol-b-polylysine (PEG-PLL), and / or polyethylene glycol-b-polyarginine (PEG-PArg).

[0581] In some embodiments, nanoparticles (e.g., nanogels) comprise one or more polycations on their surface. Exemplary polycations for use in the subject nanoparticles include, but are not limited to, polylysine (poly-L-lysine and / or poly-D-lysine), poly(arginine acid glyceryl succinate) (PAGS, arginine-based polymers), polyethyleneimine, polyhistidine, polyarginine, protamine sulfate, polyethylene glycol-b-polylysine (PEG-PLL), and polyethylene glycol-g-polylysine.

[0582] In some embodiments, the nanoparticles associate with the TIL surface by electrostatic attraction to the TIL. In certain embodiments, the nanoparticles comprise ligands that have affinity for surface molecules (e.g., surface proteins, carbohydrates, and / or lipids) of the TIL.

[0583] In certain embodiments, the nanoparticles comprise an antigen-binding domain that binds to a TIL surface antigen described herein. In some embodiments, the antigen-binding domain is an antibody or a fragment thereof. In exemplary embodiments, the TIL surface antigen is CD45, LFA-1, CD11a (integrin alpha-L), CD18 (integrin beta-2), CD11b, CD11c, CD25, CD8, or CD4. In exemplary embodiments, the antigen-binding domain (ABD) is an anti-CD45 antibody or a fragment thereof. In certain embodiments, the anti-CD45 antibody is a human anti-CD45 antibody, a humanized anti-CD45 antibody, or a chimeric anti-CD45 antibody. In exemplary embodiments, the ABD comprises vhCDR1-3 and vlCDR1-3 of the anti-CD45 antibody BC8 (see US 2017 / 0326259, particularly the relevant portions relating to the anti-CD45 antibody sequence, which are incorporated herein by reference). In some embodiments, the ABD comprises the variable heavy domain and variable domain of the anti-CD45 antibody BC8. In some embodiments, the ABD comprises the vhCDR1-3 and vlCDR1-3 or VH and VL of one of the following anti-CD45 antibodies: 10G10, UCHL1, 9.4, 4B2, or GAP8.3 (see Spertini et al., Immunology 113(4):441-452 (2004); Buzzi et al. Cancer Research 52:4027-4035 (1992)). In such embodiments, the nanoparticles are attached to the surface of the TIL population by incubating the TILs in the presence of the nanoparticles under conditions in which the nanoparticles bind to the surface of the TILs.

[0584] In some embodiments, the nanoparticles associate with the TIL cell surface by electrostatic attraction. In some embodiments, the nanoparticles are covalently bound to the TIL. In other embodiments, the nanoparticles are not covalently bound to the TIL.

[0585] In some embodiments, the subject nanoparticles are attached to TILs produced during any of the steps of the Process 2A method disclosed herein (see, e.g., Figures 2-6). In an exemplary embodiment, the subject nanoparticles are attached to TILs produced during any of the steps of the GEN3 method disclosed herein (see, e.g., Figure 7). In an exemplary embodiment, the subject nanoparticles are attached to TILs produced from the first expansion step of the Process 2A method and / or the primed expansion step of the GEN3 method provided herein. In an exemplary embodiment, the subject nanoparticles are attached to TILs produced from the second expansion step of the Process 2A method and / or the rapid expansion step of the GEN3 method provided herein. In some embodiments, the TILs are PD-1-positive TILs preselected using the methods described herein.

[0586] Additional suitable nanoparticles for use in the modified TILs provided herein are disclosed in U.S. Patent Application Publication Nos. 2020 / 0131239 and WO2020 / 205808 (each of which is incorporated by reference in its entirety), and relevant portions relating to nanoparticles.

[0587] C. Immunomodulators The modified TILs provided herein can include one or more immunomodulatory agents attached to their surface. The immunomodulatory agent can be incorporated into any of the immunomodulatory fusion proteins described herein, including, for example, the membrane-anchored immunomodulatory fusion proteins described herein. Any suitable immunomodulatory agent can be included in the subject modified TILs. In some embodiments, the immunomodulatory agent enhances TIL survival and / or anti-tumor activity once transferred to a patient. Exemplary immunomodulatory agents include, for example, cytokines. In some embodiments, the modified TILs include one or more of the following cytokines: IL-2, IL-7, IL-12, IL-15, IL-18, IL-21, IL-23, IL-27, IL-4, IL-1α, IL-1β, IL-5, IFNγ, TNFα (TNFa), IFNα, IFNβ, GM-CSF, or GCSF, or a biologically active variant thereof. In some embodiments, the immunomodulatory agent is a costimulatory molecule. In certain embodiments, the costimulatory molecule is one of the following: an agonist of OX40, CD28, GITR, VISTA, CD40, CD3, or CD137. In some embodiments, the immunomodulatory agent is a CD40 agonist (e.g., CD40L or an agonist CD40 binding domain). Exemplary immunomodulatory agents are discussed in further detail below.

[0588] 1.IL-15 In some embodiments, the modified TILs provided herein comprise IL-15. In an exemplary embodiment, IL-15 is included as part of an immunomodulatory fusion protein (e.g., a membrane-anchored immunomodulatory fusion protein) described herein.

[0589] As used herein, "interleukin-15," "IL-15," and "IL15" all refer to an interleukin that binds to and signals through a complex composed of the IL-15-specific receptor alpha chain (IL-15Rα), the IL-2 / IL-15 receptor beta chain (CD122), and the common gamma chain (gamma-C, CD132) (e.g., Genbank Accession Nos. NM_00000585, NP_000576, and NP_751915 (human); and NM_001254747 and NP_001241676 (mouse)). IL-15 has been shown to stimulate intratumor T cell proliferation. IL-15 can also extend the viability of effector memory CD8+ T cells and is important for NK cell development. Therefore, without being bound by any particular theory of operation, it is believed that modified TILs that associate with IL-15 as described herein exhibit improved survival and / or antitumor efficacy.

[0590] IL-15 has a short half-life of less than 40 minutes in vivo. Modifications to the IL-15 monomer can improve its in vivo pharmacokinetics in cancer treatment. These modifications generally focus on improving IL-15 transpresentation by the alpha subunit of the IL-15 receptor, IL-15Rα. Such modifications include: 1) pre-association of IL-15 with its soluble receptor α-subunit-Fc fusion to form an IL-15:IL-15Rα-Fc complex (see, e.g., Rubinstein et al., Proc Natl Acad Sci USA 103:9166-71 (2006)); 2) expression of the superagonist IL-15-sIL-15Rα-sushi protein (see, e.g., Bessard et al., Molecular cancer therapeutics 8:2736-45 (2009)); and 3) pre-association of the human IL-15 variant IL-15N72D with an IL-15Rα-Fc sushi-Fc fusion complex (see, e.g., Zhu et al., Journal of Immunology 183:3598-6007 (2009)).

[0591] In some embodiments, the IL-15 associated with the modified TIL is full-length IL-15, a fragment, or a variant of IL-15. In some embodiments, the IL-15 is human IL-15 or a variant human IL-15. In an exemplary embodiment, the IL-15 is a biologically active human IL-15 variant. In some embodiments, the IL-15 comprises 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 mutations compared to wild-type IL-15. In certain embodiments, the IL-15 comprises an N72D mutation relative to wild-type human IL-15. In some embodiments, the variant IL-15 exhibits IL-15Rα binding activity.

[0592] In some embodiments, the immunomodulatory agent comprises the extracellular domains of IL-15 and IL-15Rα, hi certain embodiments, the immunomodulatory agent comprises IL-15 and IL-15Rα fused to an Fc domain (IL-15Rα-Fc).

[0593] [Table 5-1] [Table 5-2]

[0594] In some embodiments, the immunostimulatory protein is superagonist IL-15 (IL-15SA), which comprises a complex of human IL-15 and soluble human IL-15Rα. The combination of human IL-15 and soluble human IL-15Rα forms an IL-15SA complex with higher biological activity than human IL-15 alone. Soluble human IL-15Rα, as well as truncated forms of the extracellular domain, have been described in the art (Wei et al., 2001 J of Immunol. 167:277-282). The amino acid sequence of human IL-15Rα is set forth in SEQ ID NO: 266. In some embodiments, IL-15SA comprises a complex of human IL-15 and soluble human IL-15Rα, which comprises all or part of the extracellular domain, without the transmembrane or cytoplasmic domain. In some embodiments, IL-15SA comprises a complex of human IL-15 and soluble human IL-15Rα, comprising the intact extracellular domain or a truncated form of the extracellular domain that retains IL-15 binding activity.

[0595] In some embodiments, IL-15SA comprises a complex of human IL-15 and soluble human IL-15Rα, which comprises a truncated form of the extracellular domain that retains IL-15 binding activity. In some embodiments, the soluble human IL-15Rα comprises amino acids 1-60, 1-61, 1-62, 1-63, 1-64, or 1-65 of human IL-15Rα. In some embodiments, the soluble human IL-15Rα comprises amino acids 1-80, 1-81, 1-82, 1-83, 1-84, or 1-85 of human IL-15Rα. In some embodiments, the soluble human IL-15Rα comprises amino acids 1-180, 1-181, or 1-182 of human IL-15Rα.

[0596] In some embodiments, the immunomodulatory agent is IL-15SA, which comprises a complex of human IL-15 and soluble human IL-15Rα, which retains IL-15 binding activity and contains a truncated form of the extracellular domain containing the Sushi domain. The Sushi domain of IL-15Rα has been described in the art as being approximately 60 amino acids long and containing four cysteines (Wei et al., 2001). Truncated forms of soluble human IL-15Rα that retain IL-15 activity and contain the Sushi domain are useful in the IL-15SA of the present disclosure.

[0597] In some embodiments, the immunomodulatory agent comprises a complex comprising soluble human IL-15Rα and IL-15 expressed as a fusion protein, such as an Fc fusion (e.g., human IgG1 Fc) described herein. In some embodiments, IL-15SA comprises a dimeric human IL-15RαFc fusion protein (e.g., human IgG1 Fc) complexed with two human IL-15 molecules.

[0598] In some embodiments, the immunomodulatory agent is an IL-15SA cytokine complex comprising an IL-15 molecule comprising the amino acid sequence set forth in SEQ ID NO: 258, SEQ ID NO: 261, SEQ ID NO: 262, or SEQ ID NO: 263. In some embodiments, the IL-15SA cytokine complex comprises a soluble IL-15Rα molecule comprising the sequence of SEQ ID NO: 260, SEQ ID NO: 264, or SEQ ID NO: 265.

[0599] In some embodiments, the immunomodulatory agent is an IL-15SA cytokine complex comprising a dimeric IL-15RαFc fusion protein complexed with two IL-15 molecules. In some embodiments, the IL-15-SA comprises a dimeric IL-15RαSu (Sushi domain) / Fc (SEQ ID NO: 259) and two IL-15N72D (SEQ ID NO: 258) molecules (also known as ALT-803) described in 2014 / 0134128 (incorporated herein by reference). In some embodiments, the IL-15SA comprises a dimeric IL-15RαSu / Fc molecule (SEQ ID NO: 259) and two IL-15 molecules (SEQ ID NO: 261). In some embodiments, the IL-15SA comprises a dimeric IL-15RαSu / Fc molecule (SEQ ID NO: 259) and two IL-15 molecules (SEQ ID NO: 262). In some embodiments, IL-15SA comprises a dimeric IL-15RαSu / Fc molecule (SEQ ID NO: 259) and two IL-15 molecules (SEQ ID NO: 263).

[0600] In some embodiments, the IL-15SA comprises a dimeric IL-15RαSu / Fc molecule (SEQ ID NO: 259) and two IL-15 molecules having an amino acid sequence selected from SEQ ID NOs: 258, 258, 262, and 263.

[0601] In some embodiments, the IL-15SA comprises a soluble IL-15Rα molecule (SEQ ID NO: 260) and two IL-15 molecules (SEQ ID NO: 258). In some embodiments, the IL-15SA comprises a soluble IL-15Rα molecule (SEQ ID NO: 260) and two IL-15 molecules (SEQ ID NO: 261). In some embodiments, the IL-15SA comprises a soluble IL-15Rα molecule (SEQ ID NO: 260) and two IL-15 molecules (SEQ ID NO: 262). In some embodiments, the IL-15SA comprises a soluble IL-15Rα molecule (SEQ ID NO: 260) and two IL-15 molecules (SEQ ID NO: 263).

[0602] In some embodiments, the IL-15SA comprises a soluble IL-15Rα molecule (SEQ ID NO: 264) and two IL-15 molecules (SEQ ID NO: 258). In some embodiments, the IL-15SA comprises a soluble IL-15Rα molecule (SEQ ID NO: 264) and two IL-15 molecules (SEQ ID NO: 261). In some embodiments, the IL-15SA comprises a soluble IL-15Rα molecule (SEQ ID NO: 264) and two IL-15 molecules (SEQ ID NO: 262). In some embodiments, the IL-15SA comprises a soluble IL-15Rα molecule (SEQ ID NO: 264) and two IL-15 molecules (SEQ ID NO: 261).

[0603] In some embodiments, the IL-15SA comprises a soluble IL-15Rα molecule (SEQ ID NO: 265) and two IL-15 molecules (SEQ ID NO: 258). In some embodiments, the IL-15SA comprises a soluble IL-15Rα molecule (SEQ ID NO: 265) and two IL-15 molecules (SEQ ID NO: 261). In some embodiments, the IL-15SA comprises a soluble IL-15Rα molecule (SEQ ID NO: 265) and two IL-15 molecules (SEQ ID NO: 262). In some embodiments, the IL-15SA comprises a soluble IL-15Rα molecule (SEQ ID NO: 265) and two IL-15 molecules (SEQ ID NO: 263).

[0604] In some embodiments, IL-15SA comprises a dimeric IL-15RαSu / Fc (SEQ ID NO: 269) molecule and two IL-15 molecules (SEQ ID NO: 262). In some embodiments, IL-15SA comprises a dimeric IL-15RαSu / Fc (SEQ ID NO: 259) molecule and two IL-15 molecules (SEQ ID NO: 263).

[0605] In some embodiments, IL-15SA comprises SEQ ID NO:259 and SEQ ID NO:260. In some embodiments, IL-15SA comprises SEQ ID NO:261 or SEQ ID NO:262. In some embodiments, IL-15SA comprises SEQ ID NO:261 and SEQ ID NO:259. In some embodiments, IL-15SA comprises SEQ ID NO:262 and SEQ ID NO:259. In some embodiments, IL-15SA comprises SEQ ID NO:263 and SEQ ID NO:259. In some embodiments, IL-15SA comprises SEQ ID NO:261 and SEQ ID NO:260. In some embodiments, IL-15SA comprises SEQ ID NO:262 and SEQ ID NO:260.

[0606] In some embodiments, the TIL composition comprises an immunomodulatory fusion protein or nanoparticle composition comprising IL-15 or a bioactive variant thereof. Exemplary fusion proteins comprising IL-15 are shown in Figures 36 and 37 and Tables 58 and 59.

[0607] In exemplary embodiments, the TIL compositions provided herein comprise a nucleic acid encoding an immunomodulatory fusion protein comprising IL-15, wherein the nucleic acid is operably linked to an NFAT promoter, an EF-1a promoter, an MND promoter, or an SSFV promoter, as described herein. Exemplary NFAT promoter-driven constructs for expression of immunomodulatory fusion proteins comprising IL-15 are shown in Table 59.

[0608] 2.IL-12 In some embodiments, the modified TILs associate with IL-12 or a variant thereof. In an exemplary embodiment, IL-12 is included as part of an immunomodulatory fusion protein (e.g., a membrane-anchored immunomodulatory fusion protein) described herein.

[0609] As used herein, "interleukin 12," "IL-12," and "IL12" all refer to interleukin, a heterodimeric cytokine encoded by the IL-12A and IL-12B genes (Genbank accession numbers: NM_000882 (IL-12A) and NM_002187 (IL-12B)). IL-12 is composed of a bundle of four alpha helices and is involved in the differentiation of natural T cells into TH1 cells. It is encoded by two separate genes, IL-12A (p35) and IL-12B (p40). The active heterodimer (termed "p70") and a homodimer of p40 are formed after protein synthesis. IL-12 binds to the IL-12 receptor, a heterodimeric receptor formed by IL-12R-β1 and IL-12R-β2. IL-12 is known as a T cell stimulatory factor, capable of stimulating the growth and function of T cells. In particular, IL-12 can stimulate the production of interferon gamma (IFN-γ) and tumor necrosis factor-alpha (TNF-α) from T cells and natural killer (NK) cells and reduce IL-4-mediated suppression of IFN-γ. IL-12 can further mediate the enhancement of the cytotoxic activity of NK cells and CD8+ cytotoxic T lymphocytes. Furthermore, IL-12 can also have anti-angiogenic activity by increasing the production of interferon gamma, which in turn increases the production of chemokine-inducible protein-10 (IP-10 or CXCL10). IP-10 then mediates this anti-angiogenic effect. Therefore, without being bound by any particular theory of operation, it is believed that IL-12 can increase the viability and / or anti-tumor effect of the TIL compositions provided herein.

[0610] In some embodiments, the IL-12 associated with the modified TIL is full-length IL-12, a fragment, or a variant of IL-12. In some embodiments, the IL-12 is human IL-12 or a variant human IL-12. In exemplary embodiments, the IL-12 is a biologically active human IL-12 variant. In some embodiments, the IL-12 contains 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 mutations compared to wild-type IL-12.

[0611] In some embodiments, the IL-12 included in the modified TIL composition comprises the IL-12 p35 subunit or a variant thereof. In some embodiments, the IL-12 p35 subunit is a human IL-12 p35 subunit. In some embodiments, the IL-12 p35 subunit has the amino acid sequence: In certain embodiments, the IL-12 included in the modified TIL composition comprises the IL-12 p40 subunit or a variant thereof. In certain embodiments, the IL-12 is a single-chain IL-12 polypeptide comprising the IL-12 p35 subunit attached to the IL-12 p40 subunit. Such an IL-12 single-chain polypeptide advantageously retains one or more of the biological activities of wild-type IL-12. In some embodiments, the single-chain IL-12 polypeptides described herein conform, from N- to C-terminus, to the formula (p40)-(L)-(p35), where "p40" is the IL-12 p40 subunit, "p35" is the IL-12 p35 subunit, and L is a linker. In other embodiments, the single-chain IL-12 conforms, from N- to C-terminus, to the formula (p35)-(L)-(p40). Any suitable linker can be used in the single-chain IL-12 polypeptides, including those described herein. Suitable linkers include, for example, a linker having the amino acid sequence (GGGGS) xwhere x is an integer from 1 to 10. Other suitable linkers include, for example, the amino acid sequence GGGGGGS. Exemplary single-chain IL-12 linkers that can be used with the subject single-chain IL-12 polypeptides are also described in Lieschke et al., Nature Biotechnology 15:35-40 (1997), which is incorporated herein by reference in its entirety, particularly for its teaching of IL-12 polypeptide linkers. In an exemplary embodiment, the single-chain IL-12 polypeptide is a single-chain human IL-12 polypeptide (i.e., it comprises human p35 and p40 IL-12 subunits).

[0612] [Table 6]

[0613] In some embodiments, the TIL composition comprises an immunomodulatory fusion protein or nanoparticle composition comprising IL-12 or a bioactive variant thereof.

[0614] In exemplary embodiments, the TIL compositions provided herein comprise a nucleic acid encoding an immunomodulatory fusion protein comprising IL-12, wherein the nucleic acid is operably linked to an NFAT promoter, an EF-1a promoter, an MND promoter, or an SSFV promoter, as described herein. See, e.g., U.S. Patent No. 8,556,882, which is incorporated by reference in its entirety, particularly for relevant portions relating to the NFAT promoter for IL-12 expression. Exemplary fusion proteins comprising IL-12 are shown in Figures 36 and 37 and in Table 58.

[0615] 3.IL-18 In some embodiments, the modified TILs associate with IL-18 or a variant thereof. In an exemplary embodiment, IL-18 is included as part of an immunomodulatory fusion protein (e.g., a membrane-anchored immunomodulatory fusion protein) described herein.

[0616] As used herein, "interleukin-18," "IL-18," "IL18," "IGIF," "IL-1g," "interferon-gamma-inducing factor," and "IL1F4" all refer to interleukin, a heterodimeric cytokine encoded by the IL-18 gene (e.g., GenBank accession numbers: NM_001243211, NM_001562, and NM_001386420). Structurally similar to IL-1β, IL-18 is a member of the IL-1 superfamily of cytokines. This cytokine, expressed by many human lymphoid and non-lymphoid cells, plays an important role in inflammatory processes. IL-18 in combination with IL-12 can activate cytotoxic T cells (CTLs) and natural killer (NK) cells to produce IFN-γ, thus contributing to tumor immunity. Therefore, without being bound by any particular theory of operation, it is believed that IL-18 can enhance the antitumor effect of the TIL compositions provided herein.

[0617] In some embodiments, the IL-18 associated with the modified TIL is full-length IL-18, a fragment, or a variant of IL-18. In some embodiments, the IL-18 is human IL-18 or variant human IL-18. In exemplary embodiments, the IL-18 is a biologically active human IL-18 variant. In some embodiments, the IL-18 comprises 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, or 20 mutations compared to wild-type IL-18 (SEQ ID NO: 269). In some embodiments, the bioactive variant is a decoy-resistant IL-18 variant ("DR-IL18" or "DR-IL-18") that provides IL-18 signaling activity even in the presence of inhibitory molecules such as IL-18 binding protein (IL-18BP). Exemplary IL-18 variants that may be included in the subject modified TILs described herein are set forth below in Table 7. Additional IL-18 variants that may be included in the subject modified TILs are described in WO2022 / 094473, which is incorporated by reference in its entirety, particularly with respect to disclosures related to variant DR-IL-18.

[0618] In some embodiments, the variant IL-18 comprises a stabilizing mutation pair selection from C38S / C68S, C38S / C68G, C38S / C68A, C38S / C68D, and C38S / C68N [relative to human wild-type IL-18—SEQ ID NO: 269]. In some embodiments, the variant IL-18 comprises a stabilizing mutation pair selection from amino acid positions M51 (e.g., M51E, M51R, M51K, M51T, M51D, or M51N), K53 (e.g., K53G, K53S, K53T, or K53R), Q56 (e.g., Q56G, Q56R, Q56L, Q56E, Q56A, Q56V, or Q56K), D110 (e.g., D110S, D110N, D110G, D110R ... In addition to the amino acids D110K, D110H, D110Q, or D110E), and N111 (e.g., N111G, N111R, NilIS, NilID, N111H, or N111Y), stabilizing mutation pairs selected from the following: C38S / C68S, C38S / C68G, C38S / C68A, C38S / C68D, and C38S / C68N [compared to human wild-type IL-18 - SEQ ID NO: 269]. In some such cases, the stabilized IL-18 variant polypeptide additionally comprises a mutation at amino acid position S105 (e.g., S105D, S105A, S105N, S105R, S105D, or S105K), and optionally further comprises a mutation at amino acid position P57 (e.g., P57A, P57L, P57G, or P57K) and M60 (e.g., M60L, M60R, M60K, or M60Q).

[0619] [Table 7-1] [Table 7-2] [Table 7-3] [Table 7-4] [Table 7-5] [Table 7-6] [Table 7-7] [Table 7-8] [Table 7-9] [Table 7-10]

[0620] In some embodiments, the TIL composition comprises an immunomodulatory fusion protein or nanoparticle composition comprising IL-18 or a bioactive variant thereof (e.g., any one of the IL-18 variants included in Table 7). An exemplary fusion protein comprising IL-18 is shown in Figure 36.

[0621] In exemplary embodiments, the TIL compositions provided herein comprise a nucleic acid encoding an immunomodulatory fusion protein comprising IL-18, wherein the nucleic acid is operably linked to an NFAT promoter, an EF-1a promoter, an MND promoter, or an SSFV promoter, as described herein. Exemplary NFAT promoter-driven constructs for expression of immunomodulatory fusion proteins comprising IL-21 are shown in Table 59.

[0622] 4.IL-21 In some embodiments, the modified TILs associate with IL-21 or a variant thereof. In an exemplary embodiment, IL-21 is included as part of an immunomodulatory fusion protein (e.g., a membrane-anchored immunomodulatory fusion protein) described herein.

[0623] In certain embodiments, the cytokine-ABD comprises an IL-21 molecule or a fragment thereof. As used herein, "interleukin-21," "IL-21," and "IL21" (e.g., Genbank Accession Nos. NM_001207006 and NP_001193935 (human), and NM_0001291041 and NP_001277970 (mouse)) all refer to a member of the cytokine family that binds to the IL-21 receptor and has potent regulatory effects on cells of the immune system, including natural killer (NK) cells and cytotoxic cells, and can destroy virally infected or cancer cells. Thus, without being bound by any particular theory of operation, it is believed that IL-21 can increase the viability and / or anti-tumor efficacy of the TIL compositions provided herein.

[0624] In some embodiments, the IL-21 is human IL-21. In some embodiments, the IL-21 associated with the modified TIL is full-length IL-21, a fragment or variant of IL-21. In some embodiments, the IL-21 is human IL-21 or a variant human IL-21. In exemplary embodiments, the IL-21 is a biologically active human IL-21 variant. In some embodiments, the IL-21 comprises 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 mutations compared to wild-type IL-21.

[0625] [Table 8]

[0626] In some embodiments, the TIL composition comprises an immunomodulatory fusion protein or nanoparticle composition comprising IL-21 or a bioactive variant thereof. Exemplary fusion proteins comprising IL-21 are shown in Figures 36 and 37 and Tables 58 and 59.

[0627] In exemplary embodiments, the TIL compositions provided herein comprise a nucleic acid encoding an immunomodulatory fusion protein comprising IL-21, wherein the nucleic acid is operably linked to an NFAT promoter, an EF-1a promoter, an MND promoter, or an SSFV promoter, as described herein.

[0628] 5.IL-2 In some embodiments, the modified TILs are associated with IL-2 or a variant thereof. In an exemplary embodiment, IL-2 is included as part of an immunomodulatory fusion protein (e.g., a membrane-anchored immunomodulatory fusion protein) described herein.

[0629] In certain embodiments, the cytokine-ABD comprises an IL-2 molecule or a fragment thereof. As used herein, "interleukin 2," "IL-2," "IL2," and "TCGF" (e.g., Genbank Accession Nos. NM_000586 and NP_000577 (human)) all refer to members of the cytokine family that bind to the IL-2 receptor. IL-2 enhances activation-induced cell death ...

Claims

1. 1. A method for expanding tumor infiltrating lymphocytes (TILs) into a therapeutic TIL population, said method comprising: (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 a plurality of tumor fragments; (b) adding the first TIL population to a closed system; (c) performing a first expansion by culturing the first TIL population in a cell culture medium comprising IL-2 to produce a second TIL population, wherein the first expansion is performed in a sealed container that provides a first gas permeable surface area, and the first expansion is performed for about 3-14 days to obtain the second TIL population, and the transition from step (b) to step (c) occurs without opening the system; (d) performing a second expansion by supplementing the cell culture medium of the second TIL population with additional IL-2, OKT-3, and antigen presenting cells (APCs) to produce a third TIL population, wherein the second expansion is performed for about 7-14 days to obtain the third TIL population, the third TIL population being a therapeutic TIL population, and wherein the second expansion is performed in a sealed container that provides a second gas permeable surface area, and the transition from step (c) to step (d) occurs without opening the system. (e) harvesting the therapeutic TIL population obtained from step (d), wherein the transition from step (d) to step (e) occurs without opening the system; (f) transferring the harvested TIL population from step (e) to an infusion bag, wherein the transition from step (e) to (f) occurs without opening the system; (g) at any time prior to transfer to the infusion bag in step (f), modifying a portion of the first, second, or third population of TILs to produce modified TILs, each of which comprises an immunomodulatory composition associated with its surface membrane; The method, wherein the immunomodulatory composition comprises one or more membrane-anchored immunomodulatory fusion proteins, each comprising one or more immunomodulatory agents and a cell membrane anchor portion, and the one or more immunomodulatory agents comprise IL-12, IL-15, or IL-18.

2. 10. The method of claim 1, wherein the first expansion is performed for about 3 to 11 days and / or the second expansion is performed for about 7 to 11 days.

3. 2. The method of claim 1, 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 papillomavirus, head and neck cancer (including head and neck squamous cell carcinoma (HNSCC)), renal cancer, and renal cell carcinoma.

4. 1. A method for expanding tumor infiltrating lymphocytes (TILs) into a therapeutic TIL population, comprising: (a) performing a first priming expansion by culturing a first TIL population in a cell culture medium comprising IL-2, optionally OKT-3, and optionally antigen-presenting cells (APCs) to produce a second TIL population, wherein said first priming expansion is performed for a first period of about 1 to 7 / 8 days to obtain said second TIL population, and producing said second TIL population, wherein said second TIL population is greater in number than said first TIL population; (b) performing a second rapid expansion of the second TIL population by contacting the second TIL population with a cell culture medium comprising IL-2, OKT-3, and APC to produce a third TIL population, wherein the second rapid expansion is performed for a second period of about 1 to 11 days to obtain the third TIL population, wherein the third TIL population is a therapeutic TIL population; (c) harvesting the therapeutic TIL population obtained from step (b); (d) any time before or after harvesting in step (c), modifying a portion of the first, second, or third population of TILs to produce modified TILs, each of which comprises an immunomodulatory composition associated with its surface membrane; The method, wherein the immunomodulatory composition comprises one or more membrane-anchored immunomodulatory fusion proteins, each comprising one or more immunomodulatory agents and a cell membrane anchor portion, and the one or more immunomodulatory agents comprise IL-12, IL-15, or IL-18.

5. The method of any one of claims 1 to 4, wherein the immunomodulatory composition comprises IL-12 and IL-15.

6. i) the IL-12 comprises a human IL-12 p35 subunit attached to a human IL-12 p40 subunit; ii) the IL-15 comprises human IL-15, and / or iii) the IL-18 comprises human IL-18; The method according to any one of claims 1 to 4.

7. The method according to any one of claims 1 to 4, wherein the cell membrane anchor portion comprises a CD8a transmembrane intracellular domain, a B7-1 transmembrane domain, a B7-2 transmembrane domain, or a CD8a transmembrane domain.

8. 5. The method of claim 1, wherein the modification comprises introducing a heterologous nucleic acid encoding the one or more membrane-anchored immunomodulatory fusion proteins into a portion of the TIL and expressing the one or more membrane-anchored immunomodulatory fusion proteins on the surface of the modified TIL.

9. 9. The method of claim 8, wherein the heterologous nucleic acid comprises an adenoviral vector, a retroviral vector, a lentiviral vector, an adeno-associated vector (AAV), or a transposon / transposase selected from piggyBac, sleeping beauty, Helraiser, and Tol2.

10. 9. The method of claim 8, wherein the heterologous nucleic acid comprises an NFAT promoter, an EF-1a promoter, an MND promoter, or an SSFV promoter.

11. 5. The method of any one of claims 1-4, wherein the modified TILs further comprise a genetic modification that silences or reduces expression of one or more immune checkpoint genes in at least a portion of the therapeutic TIL population.

12. The one or more immune checkpoint genes are PD-1, CTLA-4, LAG-3, HAVCR2 (TIM-3), Cish, TGFβ, PKA, CBL-B, PPP2CA, PPP2CB, PTPN6, PTPN22, PDCD1, BTLA, CD160, TIGIT, TET2, CD96, CRTAM, LAIR1, SIGLEC7, SIGLEC9, CD244, TNFRSF10B, TNFRSF10A, CASP8, CASP10, CASP3, CA 12. The method of claim 11, wherein the protein is selected from the group including SP6, 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.

13. 12. The method of claim 11, wherein the genetic modification is produced using one or more methods selected from RNA interference (e.g., shRNA), CRISPR, TALE, zinc finger, Cas-CLOVER, and combinations thereof.

14. a nucleic acid encoding one or more membrane-anchored immunomodulatory fusion proteins, each comprising one or more immunomodulatory agents and a cell membrane anchor portion; the one or more immunomodulatory agents comprise IL-12, IL-15, IL-18, or a variant thereof; A nucleic acid, wherein the cell membrane anchor portion comprises a CD8a transmembrane intracellular domain, a B7-1 transmembrane domain, a B7-2 transmembrane domain, or a CD8a transmembrane domain.

15. 15. The nucleic acid of claim 14, wherein the one or more membrane-anchored immunomodulatory fusion proteins are expressed under the control of an NFAT promoter, an EF-1a promoter, an MND promoter, or an SSFV promoter.

16. A retroviral or lentiviral expression vector comprising the nucleic acid of claim 14 or 15.

17. A gene-edited tumor infiltrating lymphocyte (TIL) population modified with the nucleic acid of claim 14 or 15.

18. further comprising a genetic modification that silences or reduces expression of one or more immune checkpoint genes in at least a portion of the gene-edited TIL population; the one or more immune checkpoint genes are PD-1, CTLA-4, LAG-3, HAVCR2 (TIM-3), Cish, TGFβ, PKA, CBL-B, PPP2CA, PPP2CB, PTPN6, PTPN22, PDCD1, BTLA, CD160, TIGIT, TET2, CD96, CRTAM, LAIR1, SIGLEC7, SIGLEC9, CD244, TNFRSF10B, TNFRSF10A, CASP8, CASP10, CASP3, CASP6, 18. The gene-edited TIL population of claim 17, wherein the TILs are selected from the group comprising: 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.

19. 20. The gene-edited TIL population of claim 18, wherein the genetic modifications are produced using one or more methods selected from RNA interference (e.g., shRNA), CRISPR, TALE, zinc finger, Cas-CLOVER, and combinations thereof.

20. 20. A pharmaceutical composition comprising the gene-edited TIL population of any one of claims 17 to 19 for use in a method for treating cancer in a patient or subject in need thereof.

21. The patient or subject is i) a non-myeloablative lymphodepletion regimen prior to the use of said TILs; and / or ii) an IL-2 regimen starting the day after or on the same day as the use of said TILs; 21. The pharmaceutical composition for use according to claim 20, wherein the patient is further treated with

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