Expansion process and agents for tumor-infiltrating lymphocytes

Abstract

The present invention provides a method of treating cancer in a patient or subject in need thereof, comprising administering a population of modified tumor infiltrating lymphocytes (TILs), which have been modified by adding an epigenetic reprogramming agent to the cell culture medium used to expand the TILs.

Description

[Technical Field]

[0001] CROSS-REFERENCE TO RELATED APPLICATIONS This application claims priority to U.S. Provisional Patent Application No. 63 / 248,350, filed September 24, 2021, U.S. Provisional Patent Application No. 63 / 249,459, filed September 28, 2021, and U.S. Provisional Patent Application No. 63 / 304,361, filed January 28, 2022, each of which is incorporated by reference in its entirety.

[0002] Sequence Listing This application contains a Sequence Listing that has been submitted electronically in XML file format and is incorporated herein by reference in its entirety. The XML file, created on September 23, 2022, is named 116983-5095-WO_Sequence_Listing.XML and is 312 kilobytes in size. [Background technology]

[0003] Treatment of melanoma remains challenging, especially for patients who do not respond to commonly used initial therapies, including nivolumab monotherapy, pembrolizumab monotherapy, nivolumab plus ipilimumab monotherapy, ipilimumab monotherapy, dabrafenib plus trametinib monotherapy, vemurafenib monotherapy, or pegylated interferon (pre-interferon) alpha-2b. Adoptive cell therapy using autologous tumor-infiltrating lymphocytes (TILs) has shown durable responses in subsets of patients with metastatic melanoma (Sarnaik AA et al., J Clin Oncol 2021), cervical cancer (Jazaeri AA et al., ASCO 2019#182), and other epithelial malignancies.

[0004] Although TILs can be reactivated and expanded ex vivo, their epigenetic programming can keep them in a more differentiated and less functional state. Thus, strategies aimed at expanding TILs with less differentiated, more stem-like attributes are needed to improve persistence, functionality, and better effective tumor responses. Summary of the Invention

[0005] Provided herein are methods for generating modified TILs that can be used to treat cancer patients or subjects by adding epigenetic reprogramming agents to the cell culture medium used to expand the TILs.

[0006] The present invention provides a method of treating cancer in a patient or subject in need thereof, comprising administering a population of modified tumor-infiltrating lymphocytes (TILs), wherein the population of TILs has been modified by adding an epigenetic reprogramming agent to the cell culture medium used to expand the TILs.

[0007] The present invention provides a method of treating cancer in a 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 or by processing a tumor sample obtained from the subject into a tumor digest; (b) 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; (c) 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, and the second expansion is optionally performed in a sealed container that provides a second gas-permeable surface area, and the transition from step (b) to step (c) optionally occurs without opening the system to produce a third TIL population; (d) harvesting the third population of TILs obtained from step (c), wherein the transition from step (c) to step (d) optionally occurs without opening the system; (e) administering to the subject a therapeutically effective dose of the third population of TILs obtained in step (d); (f) in step (b) and / or step (c), adding an epigenetic reprogramming agent to the cell culture medium, and optionally adding a cell permeabilization agent.

[0008] In aspects, the present invention provides 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 or by processing a tumor sample obtained from the subject into a tumor digest; (b) 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; (c) 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 the second expansion is optionally performed in a sealed container that provides a second gas-permeable surface area, and the transition from step (b) to step (c) optionally occurs without opening the system to produce a third TIL population; (d) harvesting the third population of TILs obtained from step (c), wherein the transition from step (c) to step (d) optionally occurs without opening the system; (e) transferring the harvested third population of TILs from step (d) to an infusion bag, wherein the transition from step (d) to (e) optionally occurs without opening the system; (f) cryopreserving the infusion bag containing the harvested TIL population from step (e) using a cryopreservation process; (g) administering a therapeutically effective dose of the third population of TILs from the infusion bag in step (f) to the subject; (h) in step (b) and / or step (c), adding an epigenetic reprogramming agent to the cell culture medium, and optionally adding a cell permeabilization agent.

[0009] In another aspect, the present invention provides 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) 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; (c) 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 the second expansion is optionally performed in a sealed container that provides a second gas-permeable surface area, and the transition from step (b) to step (c) optionally occurs without opening the system to produce a third TIL population; (d) harvesting the third population of TILs obtained from step (c), wherein the transition from step (c) to step (d) optionally occurs without opening the system; (e) transferring the harvested third population of TILs from step (d) to an infusion bag, wherein the transition from step (d) to (e) optionally occurs without opening the system; (f) cryopreserving the infusion bag containing the harvested TIL population from step (e) using a cryopreservation process; (g) administering a therapeutically effective dose of the third population of TILs from the infusion bag in step (f) to the subject; (h) in step (b) and / or step (c), adding an epigenetic reprogramming agent to the cell culture medium, and optionally adding a cell permeabilization agent.

[0010] In another aspect, the present invention provides 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; (c) enzymatically digesting the plurality of tumor fragments to obtain a first population of TILs; (d) 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; (e) 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 a third TIL population, and the second expansion is optionally performed in a sealed container that provides a second gas-permeable surface area, and the transition from step (d) to step (e) optionally occurs without opening the system to produce a third TIL population; (f) harvesting the third population of TILs obtained from step (e), wherein the transition from step (e) to step (f) optionally occurs without opening the system; (g) transferring the harvested third population of TILs from step (f) to an infusion bag, wherein the transition from step (f) to (g) optionally occurs without opening the system; (h) cryopreserving the infusion bag containing the harvested TIL population from step (g) using a cryopreservation process; (i) administering a therapeutically effective dose of the third population of TILs from the infusion bag of step (h) to a subject or patient with cancer; (j) in step (d) and / or step (e), adding an epigenetic reprogramming agent to the cell culture medium, and optionally adding a cell permeabilization agent.

[0011] In another aspect, the present invention provides 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; (b) performing an initial expansion (or first expansion by priming) of a first TIL population in a first cell culture medium to obtain a second TIL population, optionally OKT-3 (anti-CD3 antibody), and optionally antigen-presenting cells (APCs), wherein the first expansion by priming occurs over a period of 1 to 8 days; (c) performing a second rapid expansion of the second TIL population in a second cell culture medium to obtain a third TIL population, wherein the second cell culture medium comprises IL-2, OKT-3 (an anti-CD3 antibody), and 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; (d) harvesting a third population of TILs; (e) administering a therapeutically effective portion of the third population of TILs to a subject or patient with cancer; (h) in step (b) and / or step (c), adding an epigenetic reprogramming agent to the cell culture medium, and optionally adding a cell permeabilization agent.

[0012] In another aspect, the present invention provides 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 or processing the tumor into a tumor digest; (c) contacting the tumor fragment or tumor digest 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, optionally OKT-3 (anti-CD3 antibody), and optionally antigen-presenting cells (APCs), wherein the first expansion by priming occurs over a period of 1 to 8 days; (e) performing a second rapid expansion of the second TIL population in a second cell culture medium to obtain a third TIL population, wherein the second cell culture medium comprises IL-2, OKT-3 (an anti-CD3 antibody), and APCs, and the rapid expansion is performed over a period of no more than 14 days, 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) in step (d) and / or step (e), adding an epigenetic reprogramming agent to the cell culture medium, and optionally adding a cell permeabilization agent.

[0013] In another aspect, the present invention provides 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) culturing the first TIL population in a culture medium containing IL-2 for 1 to 3 days; (c) performing a first priming expansion by culturing the first TIL population in a first 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 time of about 1 to 11 days to obtain a second TIL population, wherein the second TIL population is more numerous than the first TIL population; (d) performing a second rapid expansion of the second TIL population by culturing the second TIL population in a second 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 time of not more than about 14 days to obtain a therapeutic TIL population, wherein the third TIL population is a therapeutic TIL population; (e) harvesting a third population of TILs; (f) administering a therapeutically effective portion of the third population of TILs to a subject or patient with cancer; (g) in step (b) and / or step (c), adding an epigenetic reprogramming agent to the cell culture medium, and optionally adding a cell permeabilization agent.

[0014] In another aspect, the present invention provides 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 or by processing a tumor sample obtained from the subject into a tumor digest; (b) performing a first priming expansion by culturing the first TIL population in a first 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 more numerous than the first TIL population; and producing a second TIL population. (c) 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 third 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 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; (d) harvesting the therapeutic TIL population obtained from step (c); (e) transferring the harvested TIL population from step (d) into an infusion bag; (f) in step (b) and / or step (c), adding an epigenetic reprogramming agent to the cell culture medium, and optionally adding a cell permeation agent.

[0015] In another aspect, the present invention provides 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 or by processing a tumor sample obtained from the subject into a tumor digest; (b) 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; (c) 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 (b) to step (c) optionally occurs without opening the system; (d) harvesting the third population of TILs obtained from step (c), wherein the transition from step (c) to step (d) optionally occurs without opening the system; (e) transferring the harvested third population of TILs from step (d) to an infusion bag, wherein the transition from step (d) to (e) optionally occurs without opening the system; (h) in step (b) and / or step (c), adding an epigenetic reprogramming agent to the cell culture medium, and optionally adding a cell permeabilization agent.

[0016] In another aspect, the present invention provides 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 or by processing a tumor sample obtained from the subject into a tumor digest; (b) 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; (c) 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 the second expansion is optionally performed in a sealed container that provides a second gas-permeable surface area, and the transition from step (b) to step (c) optionally occurs without opening the system to produce a third TIL population; (d) harvesting the third population of TILs obtained from step (c), wherein the transition from step (c) to step (d) optionally occurs without opening the system; (e) transferring the harvested third population of TILs from step (d) to an infusion bag, wherein the transition from step (d) to (e) optionally occurs without opening the system; (h) in step (b) and / or step (c), adding an epigenetic reprogramming agent to the cell culture medium, and optionally adding a cell permeabilization agent.

[0017] In another aspect, the present invention provides 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 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) 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; (c) 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 the second expansion is optionally performed in a sealed container that provides a second gas-permeable surface area, and the transition from step (b) to step (c) optionally occurs without opening the system to produce a third TIL population; (d) harvesting the third population of TILs obtained from step (c), wherein the transition from step (c) to step (d) optionally occurs without opening the system; (e) transferring the harvested third population of TILs from step (d) to an infusion bag, wherein the transition from step (d) to (e) optionally occurs without opening the system; (f) in step (b) and / or step (c), adding an epigenetic reprogramming agent to the cell culture medium, and optionally adding a cell permeabilization agent.

[0018] In another aspect, the present invention provides a method for 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) processing the tumor into a plurality of tumor fragments; (c) enzymatically digesting the plurality of tumor fragments to obtain a first population of TILs; (d) 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; (e) 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 a third TIL population, the third TIL population being a therapeutic TIL population, and wherein the second expansion is optionally performed in a sealed container that provides a second gas-permeable surface area, and wherein the transition from step (d) to step (e) optionally occurs without opening the system; (f) harvesting the third population of TILs obtained from step (e), wherein the transition from step (e) to step (f) optionally occurs without opening the system; (g) transferring the harvested third population of TILs from step (f) to an infusion bag, wherein the transition from step (f) to (g) optionally occurs without opening the system; (j) in step (d) and / or step (e), adding an epigenetic reprogramming agent to the cell culture medium, and optionally adding a cell permeabilization agent.

[0019] In another aspect, the present invention provides 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 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) performing an initial expansion (or first expansion by priming) of a first TIL population in a first cell culture medium to obtain a second TIL population, optionally OKT-3 (anti-CD3 antibody), and optionally antigen-presenting cells (APCs), wherein the first expansion by priming occurs over a period of 1 to 8 days; (c) performing a second rapid expansion of the second TIL population in a second cell culture medium to obtain a third TIL population, wherein the third TIL population is a therapeutic TIL population, and 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; (d) harvesting a third population of TILs; (e) in step (b) and / or step (c), adding an epigenetic reprogramming agent to the cell culture medium, and optionally adding a cell permeabilization agent.

[0020] In another aspect, the present invention provides a method for 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) fragmenting the tumor into tumor fragments or tumor digests; (c) contacting the tumor fragment or tumor digest 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 second rapid expansion of the second TIL population in a second cell culture medium to obtain a third TIL population, wherein the third TIL population is a therapeutic TIL population, and 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 to obtain a third TIL population; (f) harvesting a third population of TILs; (g) in step (d) and / or step (e), adding an epigenetic reprogramming agent to the cell culture medium, and optionally adding a cell permeabilization agent.

[0021] In another aspect, the present invention provides 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 or by processing a tumor sample obtained from the subject into a tumor digest; (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 11 days to obtain a second TIL population, wherein the second TIL population is more numerous 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 the third TIL population, wherein the third TIL population is a therapeutic TIL population; (d) harvesting the therapeutic TIL population obtained from step (c); (g) adding an epigenetic reprogramming agent to the cell culture medium in step (b) and / or step (c), and optionally adding a cell permeabilization agent. In some embodiments, in step (b), the cell culture medium further comprises 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 (d).

[0022] In another aspect, the present invention provides 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 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) culturing the first TIL population in a culture medium containing IL-2 for 1 to 3 days; (c) performing a first priming expansion by culturing the first TIL population in a first 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 time of about 1 to 11 days to obtain a second TIL population, wherein the second TIL population is more numerous than the first TIL population; (d) performing a second rapid expansion of the second TIL population by culturing the second TIL population in a second culture medium comprising IL-2, OKT-3, and APC to produce a third TIL population, wherein the third TIL population is a therapeutic TIL population, and the second rapid expansion is performed for a second period of time of not more than about 14 days to obtain the therapeutic TIL population; (e) harvesting a therapeutic TIL population; (f) in step (b), step (c) and / or step (d), adding an epigenetic reprogramming agent to the cell culture medium, and optionally adding a cell permeabilization agent.

[0023] In some embodiments, the subject being treated has a cancer 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.

[0024] In another aspect, the present invention provides 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 11 days to obtain a second TIL population, wherein the second TIL population is more numerous 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 third population of TILs obtained from step (b); and (d) adding an epigenetic reprogramming agent to the cell culture medium in step (a) and / or step (b), and optionally adding a cell permeabilization agent. In some embodiments, in step (a), the cell culture medium further comprises 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).

[0025] In another aspect, the present invention provides a method for 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 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) in step (a) and / or step (b), adding an epigenetic reprogramming agent to the cell culture medium, and optionally adding a cell permeabilization agent.

[0026] In another aspect, the present invention provides a method for 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) in step (a) and / or step (b), adding an epigenetic reprogramming agent to the cell culture medium, and optionally adding a cell permeabilization agent.

[0027] In some embodiments, the second TIL population and / or the third TIL population have an increased frequency of CD8 TILs and / or an increased ratio of CD4 TILs to CD8 TILs compared to a corresponding TIL population expanded in cell culture medium without the epigenetic reprogramming agent.

[0028] In some embodiments, the second TIL population is at least 5-fold more numerous than the first TIL population, and the first cell culture medium comprises IL-2.

[0029] In some embodiments, the third TIL population is at least 50-fold more numerous than the second TIL population 7-8 days after the start of rapid expansion.

[0030] In some embodiments, the cell permeation agent comprises an octyl ester or a disodium salt. In some embodiments, the octyl ester is selected from (2S)-octyl-α-hydroxyglutarate and (2R)-octyl-α-hydroxyglutarate. In some embodiments, the disodium salt is selected from R-2-hydroxyglutaric acid disodium salt and S-2-hydroxyglutaric acid disodium salt.

[0031] In some embodiments, the epigenetic reprogramming agent comprises one or more of a DNA hypomethylating agent, a MEK inhibitor, an HDAC inhibitor, an EZH2 inhibitor, a bromodomain inhibitor, an AKT inhibitor, and / or a TET inhibitor.

[0032] In some embodiments, the DNA hypomethylating agent is selected from the group consisting of decitabine, azacitidine, GSK-3484862, RG-108, GSK-3685032, DHAC, SGI-1027, CM-272, zebularine, hinokitiol, guadecitabine, gamma-oryzanol, CM-579, DC-517, 5-fluoro-2′-deoxycytidine, 5-methyldeoxycytidine, DC-05, 6-methyl-5-azacytidine, procainamide, procaine, hydrazine, EGCG, FdCyd, CP-4200, nanomycin A, and pharmaceutically acceptable salts thereof.

[0033] In some embodiments, the HDAC inhibitor is selected from the group consisting of rosirinostat, vorinostat, trichostatin A, belinostat, panabinostat, panobinostat, xinostat, divinostat, resminostat, abexinostat, xinostat, practinostat, CHR-3996, valproic acid, butyric acid, phenylbutyric acid, encethiostat, tacedinaline, mocetinostat, romidespin, nicotinamide, sirtinol, cambinol, EX-527, apicidin, depsipeptide, MS275, BML-210, splitomicin, RGFP966, and pharmaceutically acceptable salts thereof.

[0034] In some embodiments, the TET inhibitor is selected from the group consisting of C35, Bobcat339, D(R)-2-hydroxyglutarate (D2HG), D(R)-2-hydroxyglutarate (D2HG) and L-2-hydroxyglutarate (L2HG).

[0035] In some embodiments, the TET inhibitor comprises C35.

[0036] In some embodiments, the bromodomain inhibitor is one or more selected from JQ1, ZEN-3694, I-BET762, OTX015, I-BET151, RVX-208, MS417, ABBV-075, ABBV-744, SJ432, AZD5153, INCB054329, INCB054329, FT-1101, CPI-0610, RO6870810, BAY1238097, RVX000222, and pharmaceutically acceptable salts thereof.

[0037] In some embodiments, the EZH2 inhibitor is selected from the group consisting of 3-deazaneplanocin A, tazemetostat, GSK343, GSK926, GSK126, EPZ005687, and pharmaceutically acceptable salts thereof.

[0038] In some embodiments, the AKT inhibitor is selected from the group consisting of ipatasertib, GSK690693, GSK2141795, GSK2110183, AZD5363, GDC-0068, AT7867, CCT128930, MK-2206, BAY1125976, perifosine, oridonin, herbacetin, teheranolide, isoliquirigenin, scutellarin, honokiol, and pharmaceutically acceptable salts thereof.

[0039] In some embodiments, the MEK inhibitor inhibits MEK1 and / or MEK2. In some embodiments, the MEK inhibitor is selected from the group consisting of trametinib, cobimetinib, binimetinib, selumetinib, PD-325901, CI-1040, TAK-733, GDC-0623, pimasertinib, refametinib, BI-847325, and pharmaceutically acceptable salts thereof.

[0040] In some embodiments, the second TIL population and / or the third TIL population have increased expression of IL-7 receptor compared to a corresponding TIL population expanded in cell culture medium without the epigenetic reprogramming agent.

[0041] In some embodiments, the second TIL population and / or the third TIL population have increased expression of at least one of CD25, CD28, ICOS, Ki-67, and GZMB compared to a corresponding TIL population expanded in cell culture medium without the epigenetic reprogramming agent.

[0042] In some embodiments, the second TIL population and / or the third TIL population have reduced expression of at least one of PDI and TIGIT compared to a corresponding TIL population expanded in cell culture medium without the epigenetic reprogramming agent.

[0043] In some embodiments, the second TIL population and / or the third TIL population have increased expression of at least one of TCFI, EOMES, and KLF2 compared to a corresponding TIL population expanded in cell culture medium without the epigenetic reprogramming agent.

[0044] In some embodiments, the epigenetic reprogramming agent is a DNA hypomethylating agent. In some embodiments, the DNA hypomethylating agent is selected from the group consisting of decitabine, azacitidine, GSK-3484862, RG-108, GSK-3685032, DHAC, SGI-1027, CM-272, zebularine, hinokitiol, guadecitabine, gamma-oryzanol, CM-579, DC-517, 5-fluoro-2′-deoxycytidine, 5-methyldeoxycytidine, DC-05, 6-methyl-5-azacytidine, procainamide, procaine, hydrazine, EGCG, FdCyd, CP-4200, nanomycin A, and pharmaceutically acceptable salts thereof. In some embodiments, the epigenetic reprogramming agent is decitabine.

[0045] In some embodiments, the epigenetic reprogramming agent is a MEK inhibitor. In some embodiments, the MEK inhibitor is selected from the group consisting of trametinib, cobimetinib, binimetinib, selumetinib, PD-325901, CI-1040, TAK-733, GDC-0623, pimasertinib, refametinib, BI-847325, and pharmaceutically acceptable salts thereof. In some embodiments, the epigenetic reprogramming agent is trametinib.

[0046] In some embodiments, the epigenetic reprogramming agent is an HDAC inhibitor. In some embodiments, the HDAC inhibitor is selected from the group consisting of rosirinostat, vorinostat, trichostatin A, belinostat, panabinostat, panobinostat, xinostat, divinostat, resminostat, abexinostat, xinostat, practinostat, CHR-3996, valproic acid, butyric acid, phenylbutyric acid, enethiostat, tacedinaline, mocetinostat, romidespin, nicotinamide, sirtinol, cambinol, EX-527, apicidin, depsipeptide, MS275, BML-210, splitomicin, RGFP966, and pharmaceutically acceptable salts thereof. In some embodiments, the epigenetic reprogramming agent is licorinistat.

[0047] In some embodiments, the epigenetic reprogramming agent is a bromodomain inhibitor. In some embodiments, the bromodomain inhibitor is selected from JQ1, ZEN-3694, I-BET762, OTX015, I-BET151, RVX-208, MS417, ABBV-075, ABBV-744, SJ432, AZD5153, INCB054329, INCB054329, FT-1101, CPI-0610, RO6870810, BAY1238097, RVX000222, and pharmaceutically acceptable salts thereof. In some embodiments, the epigenetic reprogramming agent is JQ1.

[0048] In some embodiments, the epigenetic reprogramming agent is an EZH2 inhibitor, hi some embodiments, the EZH2 inhibitor is selected from the group consisting of 3-deazaneplanocin A, tazemetostat, GSK343, GSK926, GSK126, EPZ005687, and pharmaceutically acceptable salts thereof.

[0049] In some embodiments, the AKT inhibitor is selected from the group consisting of ipatasertib, GSK690693, GSK2141795, GSK2110183, AZD5363, GDC-0068, AT7867, CCT128930, MK-2206, BAY1125976, perifosine, oridonin, herbacetin, teheranolide, isoliquirigenin, scutellarin, honokiol, and pharmaceutically acceptable salts thereof. In some embodiments, the epigenetic reprogramming agent is ipatasertib.

[0050] In some embodiments, the epigenetic reprogramming agent is a TET inhibitor. In some embodiments, the TET inhibitor is selected from the group consisting of C35, Bobcat339, D(R)-2-hydroxyglutarate (D2HG), D(R)-2-hydroxyglutarate (D2HG), and L-2-hydroxyglutarate (L2HG). In some embodiments, the epigenetic reprogramming agent is C35.

[0051] In some embodiments, the epigenetic reprogramming agent is a combination of a DNA hypomethylating agent and a MEK inhibitor. In some embodiments, the MEK inhibitor is selected from the group consisting of trametinib, cobimetinib, binimetinib, selumetinib, PD-325901, CI-1040, TAK-733, GDC-0623, pimasertinib, refametinib, BI-847325, and pharmaceutically acceptable salts thereof. In some embodiments, the MEK inhibitor is trametinib. In some embodiments, the DNA hypomethylating agent is selected from the group consisting of decitabine, azacitidine, GSK-3484862, RG-108, GSK-3685032, DHAC, SGI-1027, CM-272, zebularine, hinokitiol, guadecitabine, gamma-oryzanol, CM-579, DC-517, 5-fluoro-2′-deoxycytidine, 5-methyldeoxycytidine, DC-05, 6-methyl-5-azacytidine, procainamide, procaine, hydrazine, EGCG, FdCyd, CP-4200, nanomycin A, and pharmaceutically acceptable salts thereof. In some embodiments, the DNA hypomethylating agent is decitabine.

[0052] In some embodiments, the epigenetic reprogramming agent is a combination of a DNA hypomethylating agent and an HDAC inhibitor. In some embodiments, the HDAC inhibitor is selected from the group consisting of rosirinostat, vorinostat, trichostatin A, belinstat, panabinostat, panobinostat, xinostat, divinostat, resminostat, abexinostat, xinostat, practinostat, CHR-3996, valproic acid, butyric acid, phenylbutyric acid, entionstat, tacedinaline, mocetinostat, romidespin, nicotinamide, sirtinol, cambinol, EX-527, apicidin, depsipeptide, MS275, BML-210, splitomicin, RGFP966, and pharmaceutically acceptable salts thereof. In some embodiments, the HDAC inhibitor is rosirinostat. In some embodiments, the DNA hypomethylating agent is selected from the group consisting of decitabine, azacitidine, GSK-3484862, RG-108, GSK-3685032, DHAC, SGI-1027, CM-272, zebularine, hinokitiol, guadecitabine, gamma-oryzanol, CM-579, DC-517, 5-fluoro-2′-deoxycytidine, 5-methyldeoxycytidine, DC-05, 6-methyl-5-azacytidine, procainamide, procaine, hydrazine, EGCG, FdCyd, CP-4200, nanomycin A, and pharmaceutically acceptable salts thereof. In some embodiments, the DNA hypomethylating agent is decitabine.

[0053] In some embodiments, the epigenetic reprogramming agent is a combination of a DNA hypomethylating agent and an EZH2 inhibitor, hi some embodiments, the EZH2 inhibitor is selected from the group consisting of 3-deazaneplanocin A, tazemetostat, GSK343, GSK926, GSK126, EPZ005687, and pharmaceutically acceptable salts thereof. In some embodiments, the DNA hypomethylating agent is selected from the group consisting of decitabine, azacitidine, GSK-3484862, RG-108, GSK-3685032, DHAC, SGI-1027, CM-272, zebularine, hinokitiol, guadecitabine, gamma-oryzanol, CM-579, DC-517, 5-fluoro-2′-deoxycytidine, 5-methyldeoxycytidine, DC-05, 6-methyl-5-azacytidine, procainamide, procaine, hydrazine, EGCG, FdCyd, CP-4200, nanomycin A, and pharmaceutically acceptable salts thereof. In some embodiments, the DNA hypomethylating agent is decitabine.

[0054] In some embodiments, the epigenetic reprogramming agent is a combination of a DNA hypomethylating agent and a bromodomain inhibitor. In some embodiments, the bromodomain inhibitor is selected from JQ1, ZEN-3694, I-BET762, OTX015, I-BET151, RVX-208, MS417, ABBV-075, ABBV-744, SJ432, AZD5153, INCB054329, INCB054329, FT-1101, CPI-0610, RO6870810, BAY1238097, RVX000222, and pharmaceutically acceptable salts thereof. In some embodiments, the bromodomain inhibitor is JQ1. In some embodiments, the DNA hypomethylating agent is selected from the group consisting of decitabine, azacitidine, GSK-3484862, RG-108, GSK-3685032, DHAC, SGI-1027, CM-272, zebularine, hinokitiol, guadecitabine, gamma-oryzanol, CM-579, DC-517, 5-fluoro-2′-deoxycytidine, 5-methyldeoxycytidine, DC-05, 6-methyl-5-azacytidine, procainamide, procaine, hydrazine, EGCG, FdCyd, CP-4200, nanomycin A, and pharmaceutically acceptable salts thereof. In some embodiments, the DNA hypomethylating agent is decitabine.

[0055] In some embodiments, the epigenetic reprogramming agent is a combination of a DNA hypomethylating agent and an AKT inhibitor. In some embodiments, the AKT inhibitor is selected from the group consisting of ipatasertib, GSK690693, GSK2141795, GSK2110183, AZD5363, GDC-0068, AT7867, CCT128930, MK-2206, BAY1125976, perifosine, oridonin, herbacetin, teheranolide, isoliquirigenin, scutellarin, honokiol, and pharmaceutically acceptable salts thereof. In some embodiments, the AKT inhibitor is ipatasertib. In some embodiments, the DNA hypomethylating agent is selected from the group consisting of decitabine, azacitidine, GSK-3484862, RG-108, GSK-3685032, DHAC, SGI-1027, CM-272, zebularine, hinokitiol, guadecitabine, gamma-oryzanol, CM-579, DC-517, 5-fluoro-2′-deoxycytidine, 5-methyldeoxycytidine, DC-05, 6-methyl-5-azacytidine, procainamide, procaine, hydrazine, EGCG, FdCyd, CP-4200, nanomycin A, and pharmaceutically acceptable salts thereof. In some embodiments, the DNA hypomethylating agent is decitabine.

[0056] In some embodiments, the epigenetic reprogramming agent is a combination of a DNA hypomethylating agent and a TET inhibitor. In some embodiments, the TET inhibitor is selected from the group consisting of C35, Bobcat339, D(R)-2-hydroxyglutarate (D2HG), D(R)-2-hydroxyglutarate (D2HG), and L-2-hydroxyglutarate (L2HG). In some embodiments, the epigenetic reprogramming agent is C35. In some embodiments, the DNA hypomethylating agent is selected from the group consisting of decitabine, azacitidine, GSK-3484862, RG-108, GSK-3685032, DHAC, SGI-1027, CM-272, zebularine, hinokitiol, guadecitabine, gamma-oryzanol, CM-579, DC-517, 5-fluoro-2′-deoxycytidine, 5-methyldeoxycytidine, DC-05, 6-methyl-5-azacytidine, procainamide, procaine, hydrazine, EGCG, FdCyd, CP-4200, nanomycin A, and pharmaceutically acceptable salts thereof. In some embodiments, the DNA hypomethylating agent is decitabine.

[0057] In some embodiments, the epigenetic reprogramming agent is a combination of a MEK inhibitor and an HDAC inhibitor. In some embodiments, the HDAC inhibitor is selected from the group consisting of rosirinostat, vorinostat, trichostatin A, belinstat, panabinostat, panobinostat, xinostat, divinostat, resminostat, abexinostat, xinostat, practinostat, CHR-3996, valproic acid, butyric acid, phenylbutyric acid, entionstat, tacedinaline, mocetinostat, romidespin, nicotinamide, sirtinol, cambinol, EX-527, apicidin, depsipeptide, MS275, BML-210, splitomicin, RGFP966, and pharmaceutically acceptable salts thereof. In some embodiments, the HDAC inhibitor is rosirinostat. In some embodiments, the MEK inhibitor is selected from the group consisting of trametinib, cobimetinib, binimetinib, selumetinib, PD-325901, CI-1040, TAK-733, GDC-0623, pimasertinib, refametinib, BI-847325, and pharmaceutically acceptable salts thereof. In some embodiments, the MEK inhibitor is trametinib.

[0058] In some embodiments, the epigenetic reprogramming agent is a combination of a MEK inhibitor and an EZH2 inhibitor. In some embodiments, the EZH2 inhibitor is selected from the group consisting of 3-deazaneplanocin A, tazemetostat, GSK343, GSK926, GSK126, EPZ005687, and pharmaceutically acceptable salts thereof. In some embodiments, the MEK inhibitor is selected from the group consisting of trametinib, cobimetinib, binimetinib, selumetinib, PD-325901, CI-1040, TAK-733, GDC-0623, pimasertinib, refametinib, BI-847325, and pharmaceutically acceptable salts thereof. In some embodiments, the MEK inhibitor is trametinib.

[0059] In some embodiments, the epigenetic reprogramming agent is a combination of a MEK inhibitor and a bromodomain inhibitor. In some embodiments, the bromodomain inhibitor is selected from JQ1, ZEN-3694, I-BET762, OTX015, I-BET151, RVX-208, MS417, ABBV-075, ABBV-744, SJ432, AZD5153, INCB054329, INCB054329, FT-1101, CPI-0610, RO6870810, BAY1238097, RVX000222, and pharmaceutically acceptable salts thereof. In some embodiments, the bromodomain inhibitor is JQ1. In some embodiments, the MEK inhibitor is selected from the group consisting of trametinib, cobimetinib, binimetinib, selumetinib, PD-325901, CI-1040, TAK-733, GDC-0623, pimasertinib, refametinib, BI-847325, and pharmaceutically acceptable salts thereof. In some embodiments, the MEK inhibitor is trametinib.

[0060] In some embodiments, the epigenetic reprogramming agent is a combination of a MEK inhibitor and an AKT inhibitor. In some embodiments, the AKT inhibitor is selected from the group consisting of ipatasertib, GSK690693, GSK2141795, GSK2110183, AZD5363, GDC-0068, AT7867, CCT128930, MK-2206, BAY1125976, perifosine, oridonin, herbacetin, teheranolide, isoliquirigenin, scutellarin, honokiol, and pharmaceutically acceptable salts thereof. In some embodiments, the AKT inhibitor is ipatasertib. In some embodiments, the MEK inhibitor is selected from the group consisting of trametinib, cobimetinib, binimetinib, selumetinib, PD-325901, CI-1040, TAK-733, GDC-0623, pimasertinib, refametinib, BI-847325, and pharmaceutically acceptable salts thereof. In some embodiments, the MEK inhibitor is trametinib.

[0061] In some embodiments, the epigenetic reprogramming agent is a combination of a MEK inhibitor and a TET inhibitor. In some embodiments, the TET inhibitor is selected from the group consisting of C35, Bobcat339, D(R)-2-hydroxyglutarate (D2HG), D(R)-2-hydroxyglutarate (D2HG), and L-2-hydroxyglutarate (L2HG). In some embodiments, the epigenetic reprogramming agent is C35. In some embodiments, the MEK inhibitor is selected from the group consisting of trametinib, cobimetinib, binimetinib, selumetinib, PD-325901, CI-1040, TAK-733, GDC-0623, pimasertinib, refametinib, BI-847325, and pharmaceutically acceptable salts thereof. In some embodiments, the MEK inhibitor is trametinib.

[0062] In some embodiments, the epigenetic reprogramming agent is a combination of an HDAC inhibitor and an EZH2 inhibitor, hi some embodiments, the EZH2 inhibitor is selected from the group consisting of 3-deazaneplanocin A, tazemetostat, GSK343, GSK926, GSK126, EPZ005687, and pharmaceutically acceptable salts thereof. In some embodiments, the HDAC inhibitor is selected from the group consisting of rosirinostat, vorinostat, trichostatin A, belinstat, panabinostat, panobinostat, xinostat, divinostat, resminostat, abexinostat, xinostat, practinostat, CHR-3996, valproic acid, butyric acid, phenylbutyric acid, encethiostat, tacedinaline, mocetinostat, romidespin, nicotinamide, sirtinol, cambinol, EX-527, apicidin, depsipeptide, MS275, BML-210, splitomicin, RGFP966, and pharmaceutically acceptable salts thereof. In some embodiments, the HDAC inhibitor is rosirinostat.

[0063] In some embodiments, the epigenetic reprogramming agent is a combination of an HDAC inhibitor and a bromodomain inhibitor. In some embodiments, the bromodomain inhibitor is selected from JQ1, ZEN-3694, I-BET762, OTX015, I-BET151, RVX-208, MS417, ABBV-075, ABBV-744, SJ432, AZD5153, INCB054329, INCB054329, FT-1101, CPI-0610, RO6870810, BAY1238097, RVX000222, and pharmaceutically acceptable salts thereof. In some embodiments, the bromodomain inhibitor is JQ1. In some embodiments, the HDAC inhibitor is selected from the group consisting of rosirinostat, vorinostat, trichostatin A, belinstat, panabinostat, panobinostat, xinostat, divinostat, resminostat, abexinostat, xinostat, practinostat, CHR-3996, valproic acid, butyric acid, phenylbutyric acid, encethiostat, tacedinaline, mocetinostat, romidespin, nicotinamide, sirtinol, cambinol, EX-527, apicidin, depsipeptide, MS275, BML-210, splitomicin, RGFP966, and pharmaceutically acceptable salts thereof. In some embodiments, the HDAC inhibitor is rosirinostat.

[0064] In some embodiments, the epigenetic reprogramming agent is a combination of an HDAC inhibitor and an AKT inhibitor. In some embodiments, the AKT inhibitor is selected from the group consisting of ipatasertib, GSK690693, GSK2141795, GSK2110183, AZD5363, GDC-0068, AT7867, CCT128930, MK-2206, BAY1125976, perifosine, oridonin, herbacetin, teheranolide, isoliquirigenin, scutellarin, honokiol, and pharmaceutically acceptable salts thereof. In some embodiments, the AKT inhibitor is ipatasertib. In some embodiments, the HDAC inhibitor is selected from the group consisting of rosirinostat, vorinostat, trichostatin A, belinstat, panabinostat, panobinostat, xinostat, divinostat, resminostat, abexinostat, xinostat, practinostat, CHR-3996, valproic acid, butyric acid, phenylbutyric acid, encethiostat, tacedinaline, mocetinostat, romidespin, nicotinamide, sirtinol, cambinol, EX-527, apicidin, depsipeptide, MS275, BML-210, splitomicin, RGFP966, and pharmaceutically acceptable salts thereof. In some embodiments, the HDAC inhibitor is rosirinostat.

[0065] In some embodiments, the epigenetic reprogramming agent is a combination of an HDAC inhibitor and a TET inhibitor. In some embodiments, the TET inhibitor is selected from the group consisting of C35, Bobcat339, D(R)-2-hydroxyglutarate (D2HG), D(R)-2-hydroxyglutarate (D2HG), and L-2-hydroxyglutarate (L2HG). In some embodiments, the epigenetic reprogramming agent is C35. In some embodiments, the HDAC inhibitor is selected from the group consisting of rosirinostat, vorinostat, trichostatin A, belinstat, panabinostat, panobinostat, xinostat, divinostat, resminostat, abexinostat, xinostat, practinostat, CHR-3996, valproic acid, butyric acid, phenylbutyric acid, encethiostat, tacedinaline, mocetinostat, romidespin, nicotinamide, sirtinol, cambinol, EX-527, apicidin, depsipeptide, MS275, BML-210, splitomicin, RGFP966, and pharmaceutically acceptable salts thereof. In some embodiments, the HDAC inhibitor is rosirinostat.

[0066] In some embodiments, the epigenetic reprogramming agent is a combination of an EZH2 inhibitor and a bromodomain inhibitor. In some embodiments, the bromodomain inhibitor is selected from JQ1, ZEN-3694, I-BET762, OTX015, I-BET151, RVX-208, MS417, ABBV-075, ABBV-744, SJ432, AZD5153, INCB054329, INCB054329, FT-1101, CPI-0610, RO6870810, BAY1238097, RVX000222, and pharmaceutically acceptable salts thereof. In some embodiments, the bromodomain inhibitor is JQ1. In some embodiments, the EZH2 inhibitor is selected from the group consisting of 3-deazaneplanocin A, tazemetostat, GSK343, GSK926, GSK126, EPZ005687, and pharmaceutically acceptable salts thereof.

[0067] In some embodiments, the epigenetic reprogramming agent is a combination of an EZH2 inhibitor and an AKT inhibitor. In some embodiments, the AKT inhibitor is selected from the group consisting of ipatasertib, GSK690693, GSK2141795, GSK2110183, AZD5363, GDC-0068, AT7867, CCT128930, MK-2206, BAY1125976, perifosine, oridonin, herbacetin, teheranolide, isoliquirigenin, scutellarin, honokiol, and pharmaceutically acceptable salts thereof. In some embodiments, the AKT inhibitor is ipatasertib. In some embodiments, the EZH2 inhibitor is selected from the group consisting of 3-deazaneplanocin A, tazemetostat, GSK343, GSK926, GSK126, EPZ005687, and pharmaceutically acceptable salts thereof.

[0068] In some embodiments, the epigenetic reprogramming agent is a combination of an EZH2 inhibitor and a TET inhibitor. In some embodiments, the TET inhibitor is selected from the group consisting of C35, Bobcat339, D(R)-2-hydroxyglutarate (D2HG), D(R)-2-hydroxyglutarate (D2HG), and L-2-hydroxyglutarate (L2HG). In some embodiments, the epigenetic reprogramming agent is C35. In some embodiments, the EZH2 inhibitor is selected from the group consisting of 3-deazaneplanocin A, tazemetostat, GSK343, GSK926, GSK126, EPZ005687, and pharmaceutically acceptable salts thereof.

[0069] In some embodiments, the epigenetic reprogramming agent is a combination of a bromodomain inhibitor and an AKT inhibitor. In some embodiments, the AKT inhibitor is selected from the group consisting of ipatasertib, GSK690693, GSK2141795, GSK2110183, AZD5363, GDC-0068, AT7867, CCT128930, MK-2206, BAY1125976, perifosine, oridonin, herbacetin, teheranolide, isoliquirigenin, scutellarin, honokiol, and pharmaceutically acceptable salts thereof. In some embodiments, the AKT inhibitor is ipatasertib. In some embodiments, the bromodomain inhibitor is selected from JQ1, ZEN-3694, I-BET762, OTX015, I-BET151, RVX-208, MS417, ABBV-075, ABBV-744, SJ432, AZD5153, INCB054329, INCB054329, FT-1101, CPI-0610, RO6870810, BAY1238097, RVX000222, and pharmaceutically acceptable salts thereof. In some embodiments, the bromodomain inhibitor is JQ1.

[0070] In some embodiments, the epigenetic reprogramming agent is a combination of a bromodomain inhibitor and a TET inhibitor. In some embodiments, the TET inhibitor is selected from the group consisting of C35, Bobcat339, D(R)-2-hydroxyglutarate (D2HG), and L-2-hydroxyglutarate (L2HG). In some embodiments, the epigenetic reprogramming agent is C35. In some embodiments, the bromodomain inhibitor is selected from JQ1, ZEN-3694, I-BET762, OTX015, I-BET151, RVX-208, MS417, ABBV-075, ABBV-744, SJ432, AZD5153, INCB054329, INCB054329, FT-1101, CPI-0610, RO6870810, BAY1238097, RVX000222, and pharmaceutically acceptable salts thereof. In some embodiments, the bromodomain inhibitor is JQ1.

[0071] In some embodiments, the epigenetic reprogramming agent is a combination of an AKT inhibitor and a TET inhibitor. In some embodiments, the TET inhibitor is selected from the group consisting of C35, Bobcat339, D(R)-2-hydroxyglutarate (D2HG), D(R)-2-hydroxyglutarate (D2HG), and L-2-hydroxyglutarate (L2HG). In some embodiments, the epigenetic reprogramming agent is C35. In some embodiments, the AKT inhibitor is selected from the group consisting of ipatasertib, GSK690693, GSK2141795, GSK2110183, AZD5363, GDC-0068, AT7867, CCT128930, MK-2206, BAY1125976, perifosine, oridonin, herbacetin, teheranolide, isoliquirigenin, scutellarin, honokiol, and pharmaceutically acceptable salts thereof. In some embodiments, the AKT inhibitor is ipatasertib.

[0072] The present invention provides the use according to any of the methods set out in any one of the preceding claims. [Brief explanation of the drawings]

[0073] [Figure 1] An exemplary Gen2 (Process 2A) chart providing an overview of steps A-F. [Figure 2A] 1 is a process flow diagram of several embodiments of Gen2 (Process 2A) for TIL fabrication. [Figure 2B] 1 is a process flow diagram of several embodiments of Gen2 (Process 2A) for TIL fabrication. [Figure 2C] 1 is a process flow diagram of several embodiments of Gen2 (Process 2A) for TIL fabrication. [Figure 3] 1 shows a diagram of several embodiments of an exemplary manufacturing process (approximately 22 days) for cryopreserved TILs. [Figure 4]1 shows diagrams of several embodiments 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 some embodiments of Process 1C with some embodiments of Gen2 (Process 2A) for TIL fabrication. [Figure 7] An exemplary GEN3 TIL manufacturing process. [Figure 8A] 1 shows a comparison of several embodiments of the 2A process (an approximately 22 day process) and the Gen3 process (an approximately 14-16 day process) for TIL fabrication. [Figure 8B] An exemplary Process Gen3 chart is shown, providing an overview of steps A-F (approximately a 14-16 day process). [Figure 8C] A chart is shown providing three exemplary Gen3 processes along with an overview of steps A-F (approximately a 14- to 16-day process) for each of the three process variations. [Figure 8D] 1 shows an exemplary modified Gen2-like process providing an overview of steps A-F (approximately a 22 day process). [Figure 8E] An exemplary Gen3 process (approximately a 14-18 day process from steps A-E) is shown, providing an overview of steps A-E. [Figure 8F] Three exemplary Gen3 processes are shown, along with an overview of steps A through F (approximately 14- to 18-day processes) for each of the three process variations. [Figure 8G] 1 shows an exemplary modified Gen2-like process providing an overview of steps A-F (approximately a 22 day process). [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 several embodiments of the Gen3 process, referred to as Gen3.1. [Figure 13] 1 is a table illustrating various features of embodiments of the Gen2, Gen2.1, and Gen3.1 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, which 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 intracellular signaling domains of six receptors and signaling proteins to form a signaling complex. The TNFRSF-binding domains shown as cylinders can be, for example, scFv domains comprising VH and VL chains connected by a linker that may contain hydrophilic residues and Gly and Ser sequences for flexibility, and Glu and Lys for solubility. [Figure 19]Schematic of an exemplary embodiment of the Gen3 process (16-day process). [Figure 20] 1 provides a process overview of an exemplary embodiment of the Gen3.1 process (16-day process). [Figure 21] Schematic of an exemplary embodiment of the Gen3.1 Test (Optimized Gen3.1) 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, with exemplary differences highlighted. [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 several embodiments of the Gen2, Gen2.1, and Gen3 processes (16-day processes). [Figure 29] Comparison of several embodiments of the Gen2, Gen2.1, and Gen3 processes (16-day processes). [Figure 30] Components of a Gen3 embodiment. [Figure 31] Flowchart comparison of Gen3 embodiments (Gen3.0, Gen3.1 control, Gen3.1 test). [Figure 32] The components of an exemplary embodiment of the Gen3 process (Optimized Gen3, 16-17 day process) are shown. [Figure 33] Approval criteria table. [Figure 34]Schematic showing different time points at which epigenetic reprogramming agents can be added to the cell culture medium during the expansion step described herein. Non-solid lines indicate optional processes. [Figure 35] Schematic representation of decitabine (DAC) added to cultures during ex vivo expansion, either before and during the REP stage, or only during the REP stage. [Figure 36A] At the end of the 22 day expansion process, fold expansion and survival are shown when DAC was added before and during the REP phase, or only during the REP phase. [Figure 36B] The frequencies of CD8+, CD4+, and CD4+ (Foxp3+) cells after the expansion process on cryopreserved cells are shown. *P<0.05, **P<0.01. TILs were left untreated or treated with increasing concentrations of DAC. Treatment was added either before and during REP, or only during the REP stage. [Figure 37A] Figure 1 shows the subsets of CD8+ TILs after expansion in control and DAC-treated TILs. Frequencies of Tcm (CD45RA-CCR7+), Tem (CD45RA-CCR7-), and Temra (CD45+CCR7-) cells. *P<0.05, **P<0.01. [Figure 37B] Figure 1 shows the subsets of CD4+ TILs after expansion in control and DAC-treated TILs. Frequencies of Tcm (CD45RA-CCR7+), Tem (CD45RA-CCR7-), and Temra (CD45+CCR7-) cells. *P<0.05, **P<0.01. [Figure 38A] Expression of CD25, ICOS, CD28, and IL-7R on CD8+ TILs is shown. Control or DAC-treated cryopreserved TILs were thawed and stained for flow cytometry analysis. Similar results were observed for CD4+ TILs. *P<0.05, **P<0.01, ***P<0.001, ****P<0.0001. [Figure 38B]Expression of inhibitory receptors PD-1 and TIGIT on CD8+ TILs is shown. Control or DAC-treated cryopreserved TILs were thawed and stained for flow cytometry analysis. Similar results were observed for CD4+ TILs. *P<0.05, **P<0.01, ***P<0.001, ****P<0.0001. [Figure 39] Expression of transcription factors in TILs treated with DAC. Control or DAC-treated cryopreserved TILs were thawed and stained for flow cytometry analysis. Expression of Eomes, KLF2, BATF, and T-bet in CD8+ TILs is shown. *P<0.05, **P<0.01. [Figure 40] Cytokine expression in control or DAC-treated TILs after in vitro stimulation. Cryopreserved control and DAC-treated TILs were stimulated overnight with anti-CD3 / CD28 beads at a bead-to-cell ratio of 1:5. Expression of IFNγ, TNFα, and GZMB in CD8+ TILs is shown. *P<0.05, **P<0.01. [Figure 41A] Cryopreserved control and REP-treated TILs with 100 nM DAC were co-cultured with KILR® THP-1 cells (Eurofins DiscoverX, Fremont, CA, USA) at an E:T cell ratio of 10:1 for 24 hours to measure cytotoxicity in an allogeneic setting. [Figure 41B] Control and DAC-treated TILs were stimulated every 5 days with TransAct™ (Miltentyi Biotech, Germany). The day after the third stimulation, cells were washed and cocultured with KILR THP-1 cells at an effector-to-target cell ratio of 10:1 for 24 hours to measure cytotoxicity. *P<0.05. [Figure 42A] Expression of IL-7R, PD-1, and TIM3 in TILs after repeated stimulation. Control and DAC-treated TILs were stimulated every 5 days with TransAct™ (Miltentyi Biotech, Germany). The day after the third stimulation, cells were washed and stained for flow cytometry analysis. *P<0.05, **P<0.01. [Figure 42B]Expression levels of transcription factors in TILs after repeated stimulation. Control and DAC-treated TILs were stimulated every 5 days with TransAct™ (Miltentyi Biotech, Germany). The day after the third stimulation, cells were washed and stained for flow cytometry analysis. *P<0.05, **P<0.01.

[0074] Brief description of the sequence listing SEQ ID NO: 1 is the amino acid sequence of the heavy chain of muromonab.

[0075] SEQ ID NO: 2 is the amino acid sequence of the light chain of muromonab.

[0076] SEQ ID NO: 3 is the amino acid sequence of recombinant human IL-2 protein.

[0077] SEQ ID NO: 4 is the amino acid sequence of aldesleukin.

[0078] SEQ ID NO: 5 is an IL-2 form.

[0079] SEQ ID NO: 6 is the amino acid sequence of nemvaleukin alpha.

[0080] SEQ ID NO: 7 is an IL-2 form.

[0081] SEQ ID NO: 8 is a mucin domain polypeptide.

[0082] SEQ ID NO: 9 is the amino acid sequence of recombinant human IL-4 protein.

[0083] SEQ ID NO: 10 is the amino acid sequence of recombinant human IL-7 protein.

[0084] SEQ ID NO: 11 is the amino acid sequence of recombinant human IL-15 protein.

[0085] SEQ ID NO: 12 is the amino acid sequence of recombinant human IL-21 protein.

[0086] SEQ ID NO: 13 is the IL-2 sequence.

[0087] SEQ ID NO: 14 is the IL-2 mutein sequence.

[0088] SEQ ID NO: 15 is the sequence of an IL-2 mutein.

[0089] SEQ ID NO: 16 is HCDR1_IL-2 of IgG.IL2R67A.H1.

[0090] SEQ ID NO: 17 is HCDR2 of IgG.IL2R67A.H1.

[0091] SEQ ID NO: 18 is the HCDR3 of IgG.IL2R67A.H1.

[0092] SEQ ID NO: 19 is HCDR1_IL-2 Kabat of IgG.IL2R67A.H1.

[0093] SEQ ID NO: 20 is the HCDR2 Kabat of IgG.IL2R67A.H1.

[0094] SEQ ID NO: 21 is the HCDR3 Kabat of IgG.IL2R67A.H1.

[0095] SEQ ID NO: 22 is the HCDR1_IL-2 clotia of IgG.IL2R67A.H1.

[0096] SEQ ID NO: 23 is the HCDR2 clone of IgG.IL2R67A.H1.

[0097] SEQ ID NO: 24 is the HCDR3 clone of IgG.IL2R67A.H1.

[0098] SEQ ID NO: 25 is HCDR1_IL-2 IMGT of IgG.IL2R67A.H1.

[0099] SEQ ID NO: 26 is HCDR2 IMGT of IgG.IL2R67A.H1.

[0100] SEQ ID NO: 27 is the HCDR3 IMGT of IgG.IL2R67A.H1.

[0101] SEQ ID NO: 28 is the V H It's a chain.

[0102] SEQ ID NO: 29 is the heavy chain of IgG.IL2R67A.H1.

[0103] SEQ ID NO: 30 is the LCDR1 Kabat of IgG.IL2R67A.H1.

[0104] SEQ ID NO: 31 is the LCDR2 Kabat of IgG.IL2R67A.H1.

[0105] SEQ ID NO: 32 is the LCDR3 Kabat of IgG.IL2R67A.H1.

[0106] SEQ ID NO: 33 is the LCDR1 chothia of IgG.IL2R67A.H1.

[0107] SEQ ID NO: 34 is the LCDR2 chothia of IgG.IL2R67A.H1.

[0108] SEQ ID NO: 35 is the LCDR3 chothia of IgG.IL2R67A.H1.

[0109] SEQ ID NO: 36 is V L It's a chain.

[0110] SEQ ID NO: 37 is the light chain.

[0111] SEQ ID NO: 38 is the light chain.

[0112] SEQ ID NO: 39 is the light chain.

[0113] SEQ ID NO: 40 is the amino acid sequence of human 4-1BB.

[0114] SEQ ID NO: 41 is the amino acid sequence of mouse 4-1BB.

[0115] SEQ ID NO: 42 is the heavy chain of the 4-1BB agonist monoclonal antibody utomilumab (PF-05082566).

[0116] SEQ ID NO: 43 is the light chain of the 4-1BB agonist monoclonal antibody utomilumab (PF-05082566).

[0117] SEQ ID NO: 44 represents the heavy chain variable region (V) of the 4-1BB agonist monoclonal antibody utomilumab (PF-05082566). H )

[0118] SEQ ID NO: 45 is the light chain variable region (V) of the 4-1BB agonist monoclonal antibody utomilumab (PF-05082566). L )

[0119] SEQ ID NO: 46 is the heavy chain CDR1 of the 4-1BB agonist monoclonal antibody utomilumab (PF-05082566).

[0120] SEQ ID NO: 47 is the heavy chain CDR2 of the 4-1BB agonist monoclonal antibody utomilumab (PF-05082566).

[0121] SEQ ID NO: 48 is the heavy chain CDR3 of the 4-1BB agonist monoclonal antibody utomilumab (PF-05082566).

[0122] SEQ ID NO: 49 is the light chain CDR1 of the 4-1BB agonist monoclonal antibody utomilumab (PF-05082566).

[0123] SEQ ID NO: 50 is the light chain CDR2 of the 4-1BB agonist monoclonal antibody utomilumab (PF-05082566).

[0124] SEQ ID NO: 51 is the light chain CDR3 of the 4-1BB agonist monoclonal antibody utomilumab (PF-05082566).

[0125] SEQ ID NO: 52 is the heavy chain of the 4-1BB agonist monoclonal antibody urelumab (BMS-663513).

[0126] SEQ ID NO: 53 is the light chain of the 4-1BB agonist monoclonal antibody urelumab (BMS-663513).

[0127] SEQ ID NO: 54 is the heavy chain variable region (VH) of the 4-1BB agonist monoclonal antibody urelumab (BMS-663513).

[0128] SEQ ID NO: 55 is the light chain variable region (VL) of the 4-1BB agonist monoclonal antibody urelumab (BMS-663513).

[0129] SEQ ID NO: 56 is the heavy chain CDR1 of the 4-1BB agonist monoclonal antibody urelumab (BMS-663513).

[0130] SEQ ID NO: 57 is the heavy chain CDR2 of the 4-1BB agonist monoclonal antibody urelumab (BMS-663513).

[0131] SEQ ID NO: 58 is the heavy chain CDR3 of the 4-1BB agonist monoclonal antibody urelumab (BMS-663513).

[0132] SEQ ID NO: 59 is the light chain CDR1 of the 4-1BB agonist monoclonal antibody urelumab (BMS-663513).

[0133] SEQ ID NO: 60 is the light chain CDR2 of the 4-1BB agonist monoclonal antibody urelumab (BMS-663513).

[0134] SEQ ID NO: 61 is the light chain CDR3 of the 4-1BB agonist monoclonal antibody urelumab (BMS-663513).

[0135] SEQ ID NO: 62 is the Fc domain of the TNFRSF agonist fusion protein.

[0136] SEQ ID NO: 63 is the linker of the TNFRSF agonist fusion protein.

[0137] SEQ ID NO: 64 is the linker of the TNFRSF agonist fusion protein.

[0138] SEQ ID NO: 65 is the linker of the TNFRSF agonist fusion protein.

[0139] SEQ ID NO: 66 is the linker of the TNFRSF agonist fusion protein.

[0140] SEQ ID NO: 67 is the linker for the TNFRSF agonist fusion protein.

[0141] SEQ ID NO: 68 is the linker for the TNFRSF agonist fusion protein.

[0142] SEQ ID NO: 69 is the linker for the TNFRSF agonist fusion protein.

[0143] SEQ ID NO: 70 is the linker of the TNFRSF agonist fusion protein.

[0144] SEQ ID NO: 71 is the linker for the TNFRSF agonist fusion protein.

[0145] SEQ ID NO: 72 is the linker for the TNFRSF agonist fusion protein.

[0146] SEQ ID NO: 73 is the Fc domain of the TNFRSF agonist fusion protein.

[0147] SEQ ID NO: 74 is the linker for the TNFRSF agonist fusion protein.

[0148] SEQ ID NO: 75 is the linker of the TNFRSF agonist fusion protein.

[0149] SEQ ID NO: 76 is the linker for the TNFRSF agonist fusion protein.

[0150] SEQ ID NO: 77 is the 4-1BB ligand (4-1BBL) amino acid sequence.

[0151] SEQ ID NO: 78 is the soluble portion of the 4-1BBL polypeptide.

[0152] SEQ ID NO: 79 is the heavy chain variable region (V) of 4-1BB agonist antibody 4B4-1-1 version 1 H )

[0153] SEQ ID NO: 80 represents the light chain variable region (V) of 4-1BB agonist antibody 4B4-1-1 version 1 L )

[0154] SEQ ID NO: 81 is the heavy chain variable region (V H )

[0155] SEQ ID NO: 82 is the light chain variable region (V) of 4-1BB agonist antibody 4B4-1-1 version 2 L )

[0156] SEQ ID NO: 83 identifies the heavy chain variable region (V H )

[0157] SEQ ID NO: 84 identifies the light chain variable region (V L )

[0158] SEQ ID NO: 85 is the amino acid sequence of human OX40.

[0159] SEQ ID NO: 86 is the amino acid sequence of mouse OX40.

[0160] SEQ ID NO: 87 is the heavy chain of the OX40 agonist monoclonal antibody tabolixizumab (MEDI-0562).

[0161] SEQ ID NO: 88 is the light chain of the OX40 agonist monoclonal antibody tabolixizumab (MEDI-0562).

[0162] SEQ ID NO: 89 is the heavy chain variable region (V) of the OX40 agonist monoclonal antibody tabolixizumab (MEDI-0562). H )

[0163] SEQ ID NO: 90 is the light chain variable region (V) of the OX40 agonist monoclonal antibody tabolixizumab (MEDI-0562). L )

[0164] SEQ ID NO: 91 is the heavy chain CDR1 of the OX40 agonist monoclonal antibody tabolixizumab (MEDI-0562).

[0165] SEQ ID NO: 92 is the heavy chain CDR2 of the OX40 agonist monoclonal antibody tabolixizumab (MEDI-0562).

[0166] SEQ ID NO: 93 is the heavy chain CDR3 of the OX40 agonist monoclonal antibody tabolixizumab (MEDI-0562).

[0167] SEQ ID NO: 94 is the light chain CDR1 of the OX40 agonist monoclonal antibody tabolixizumab (MEDI-0562).

[0168] SEQ ID NO: 95 is the light chain CDR2 of the OX40 agonist monoclonal antibody tabolixizumab (MEDI-0562).

[0169] SEQ ID NO: 96 is the light chain CDR3 of the OX40 agonist monoclonal antibody tabolixizumab (MEDI-0562).

[0170] SEQ ID NO: 97 is the heavy chain of the OX40 agonist monoclonal antibody 11D4.

[0171] SEQ ID NO: 98 is the light chain of the OX40 agonist monoclonal antibody 11D4.

[0172] SEQ ID NO: 99 is the heavy chain variable region (V) of the OX40 agonist monoclonal antibody 11D4. H )

[0173] SEQ ID NO: 100 is the light chain variable region (V) of the OX40 agonist monoclonal antibody 11D4. L )

[0174] SEQ ID NO: 101 is the heavy chain CDR1 of the OX40 agonist monoclonal antibody 11D4.

[0175] SEQ ID NO: 102 is the heavy chain CDR2 of the OX40 agonist monoclonal antibody 11D4.

[0176] SEQ ID NO: 103 is the heavy chain CDR3 of the OX40 agonist monoclonal antibody 11D4.

[0177] SEQ ID NO: 104 is the light chain CDR1 of the OX40 agonist monoclonal antibody 11D4.

[0178] SEQ ID NO: 105 is the light chain CDR2 of the OX40 agonist monoclonal antibody 11D4.

[0179] SEQ ID NO: 106 is the light chain CDR3 of the OX40 agonist monoclonal antibody 11D4.

[0180] SEQ ID NO: 107 is the heavy chain of the OX40 agonist monoclonal antibody 18D8.

[0181] SEQ ID NO: 108 is the light chain of the OX40 agonist monoclonal antibody 18D8.

[0182] SEQ ID NO: 109 is the heavy chain variable region (V) of the OX40 agonist monoclonal antibody 18D8. H )

[0183] SEQ ID NO: 110 is the light chain variable region (V) of the OX40 agonist monoclonal antibody 18D8. L )

[0184] SEQ ID NO: 111 is the heavy chain CDR1 of the OX40 agonist monoclonal antibody 18D8.

[0185] SEQ ID NO: 112 is the heavy chain CDR2 of the OX40 agonist monoclonal antibody 18D8.

[0186] SEQ ID NO: 113 is the heavy chain CDR3 of the OX40 agonist monoclonal antibody 18D8.

[0187] SEQ ID NO: 114 is the light chain CDR1 of the OX40 agonist monoclonal antibody 18D8.

[0188] SEQ ID NO: 115 is the light chain CDR2 of the OX40 agonist monoclonal antibody 18D8.

[0189] SEQ ID NO: 116 is the light chain CDR3 of the OX40 agonist monoclonal antibody 18D8.

[0190] SEQ ID NO: 117 is the heavy chain variable region (V) of the OX40 agonist monoclonal antibody Hu119-122. H )

[0191] SEQ ID NO: 118 is the light chain variable region (V) of the OX40 agonist monoclonal antibody Hu119-122. L )

[0192] SEQ ID NO: 119 is the heavy chain CDR1 of the OX40 agonist monoclonal antibody Hu119-122.

[0193] SEQ ID NO: 120 is the heavy chain CDR2 of the OX40 agonist monoclonal antibody Hu119-122.

[0194] SEQ ID NO: 121 is the heavy chain CDR3 of the OX40 agonist monoclonal antibody Hu119-122.

[0195] SEQ ID NO: 122 is the light chain CDR1 of the OX40 agonist monoclonal antibody Hu119-122.

[0196] SEQ ID NO: 123 is the light chain CDR2 of the OX40 agonist monoclonal antibody Hu119-122.

[0197] SEQ ID NO: 124 is the light chain CDR3 of the OX40 agonist monoclonal antibody Hu119-122.

[0198] SEQ ID NO: 125 is the heavy chain variable region (V) of the OX40 agonist monoclonal antibody Hu106-222. H )

[0199] SEQ ID NO: 126 is the light chain variable region (V) of the OX40 agonist monoclonal antibody Hu106-222. L )

[0200] SEQ ID NO: 127 is the heavy chain CDR1 of the OX40 agonist monoclonal antibody Hu106-222.

[0201] SEQ ID NO: 128 is the heavy chain CDR2 of the OX40 agonist monoclonal antibody Hu106-222.

[0202] SEQ ID NO: 129 is the heavy chain CDR3 of the OX40 agonist monoclonal antibody Hu106-222.

[0203] SEQ ID NO: 130 is the light chain CDR1 of the OX40 agonist monoclonal antibody Hu106-222.

[0204] SEQ ID NO: 131 is the light chain CDR2 of the OX40 agonist monoclonal antibody Hu106-222.

[0205] SEQ ID NO: 132 is the light chain CDR3 of the OX40 agonist monoclonal antibody Hu106-222.

[0206] SEQ ID NO: 133 is the OX40 ligand (OX40L) amino acid sequence.

[0207] SEQ ID NO: 134 is the soluble portion of the OX40L polypeptide.

[0208] SEQ ID NO: 135 is an alternative soluble portion of the OX40L polypeptide.

[0209] SEQ ID NO: 136 is the heavy chain variable region (V H )

[0210] SEQ ID NO: 137 is the light chain variable region (V) of OX40 agonist monoclonal antibody 008. L )

[0211] SEQ ID NO: 138 is the heavy chain variable region (V) of OX40 agonist monoclonal antibody 011. H )

[0212] SEQ ID NO: 139 is the light chain variable region (V) of the OX40 agonist monoclonal antibody 011. L )

[0213] SEQ ID NO: 140 is the heavy chain variable region (V) of the OX40 agonist monoclonal antibody 021. H )

[0214] SEQ ID NO: 141 is the light chain variable region (V) of the OX40 agonist monoclonal antibody 021. L )

[0215] SEQ ID NO: 142 is the heavy chain variable region (V) of the OX40 agonist monoclonal antibody 023.H )

[0216] SEQ ID NO: 143 is the light chain variable region (V) of the OX40 agonist monoclonal antibody 023. L )

[0217] SEQ ID NO: 144 is the heavy chain variable region (V H )

[0218] SEQ ID NO: 145 is the light chain variable region (V) of the OX40 agonist monoclonal antibody L )

[0219] SEQ ID NO: 146 is the heavy chain variable region (V H )

[0220] SEQ ID NO: 147 is the light chain variable region (V) of the OX40 agonist monoclonal antibody L )

[0221] SEQ ID NO: 148 is the heavy chain variable region (V H )

[0222] SEQ ID NO: 149 is the heavy chain variable region (V) of a humanized OX40 agonist monoclonal antibody. H )

[0223] SEQ ID NO: 150 is the light chain variable region (V) of a humanized OX40 agonist monoclonal antibody. L )

[0224] SEQ ID NO: 151 is the light chain variable region (V) of a humanized OX40 agonist monoclonal antibody. L )

[0225] SEQ ID NO: 152 is the heavy chain variable region (V H )

[0226] SEQ ID NO: 153 is the heavy chain variable region (V) of a humanized OX40 agonist monoclonal antibody. H )

[0227] SEQ ID NO: 154 is the light chain variable region (V) of a humanized OX40 agonist monoclonal antibody. L )

[0228] SEQ ID NO: 155 is the light chain variable region (V) of a humanized OX40 agonist monoclonal antibody. L )

[0229] SEQ ID NO: 156 is the heavy chain variable region (V H )

[0230] SEQ ID NO: 157 is the light chain variable region (V) of the OX40 agonist monoclonal antibody L )

[0231] SEQ ID NO: 158 is the heavy chain amino acid sequence of the PD-1 inhibitor nivolumab.

[0232] SEQ ID NO: 159 is the light chain amino acid sequence of the PD-1 inhibitor nivolumab.

[0233] SEQ ID NO: 160 represents the heavy chain variable region (V) of the PD-1 inhibitor nivolumab H ) amino acid sequence.

[0234] SEQ ID NO: 161 represents the light chain variable region (V) of the PD-1 inhibitor nivolumab L ) amino acid sequence.

[0235] SEQ ID NO: 162 is the heavy chain CDR1 amino acid sequence of the PD-1 inhibitor nivolumab.

[0236] SEQ ID NO: 163 is the heavy chain CDR2 amino acid sequence of the PD-1 inhibitor nivolumab.

[0237] SEQ ID NO: 164 is the heavy chain CDR3 amino acid sequence of the PD-1 inhibitor nivolumab.

[0238] SEQ ID NO: 165 is the light chain CDR1 amino acid sequence of the PD-1 inhibitor nivolumab.

[0239] SEQ ID NO: 166 is the light chain CDR2 amino acid sequence of the PD-1 inhibitor nivolumab.

[0240] SEQ ID NO: 167 is the light chain CDR3 amino acid sequence of the PD-1 inhibitor nivolumab.

[0241] SEQ ID NO: 168 is the heavy chain amino acid sequence of the PD-1 inhibitor pembrolizumab.

[0242] SEQ ID NO: 169 is the light chain amino acid sequence of the PD-1 inhibitor pembrolizumab.

[0243] SEQ ID NO: 170 represents the heavy chain variable region (V) of the PD-1 inhibitor pembrolizumab H ) amino acid sequence.

[0244] SEQ ID NO: 171 is the light chain variable region (V) of the PD-1 inhibitor pembrolizumab L ) amino acid sequence.

[0245] SEQ ID NO: 172 is the heavy chain CDR1 amino acid sequence of the PD-1 inhibitor pembrolizumab.

[0246] SEQ ID NO: 173 is the heavy chain CDR2 amino acid sequence of the PD-1 inhibitor pembrolizumab.

[0247] SEQ ID NO: 174 is the heavy chain CDR3 amino acid sequence of the PD-1 inhibitor pembrolizumab.

[0248] SEQ ID NO: 175 is the light chain CDR1 amino acid sequence of the PD-1 inhibitor pembrolizumab.

[0249] SEQ ID NO: 176 is the light chain CDR2 amino acid sequence of the PD-1 inhibitor pembrolizumab.

[0250] SEQ ID NO: 177 is the light chain CDR3 amino acid sequence of the PD-1 inhibitor pembrolizumab.

[0251] SEQ ID NO: 178 is the heavy chain amino acid sequence of the PD-L1 inhibitor durvalumab.

[0252] SEQ ID NO: 179 is the light chain amino acid sequence of the PD-L1 inhibitor durvalumab.

[0253] SEQ ID NO: 180 represents the heavy chain variable region (V) of the PD-L1 inhibitor durvalumab H ) amino acid sequence.

[0254] SEQ ID NO: 181 represents the light chain variable region (V) of the PD-L1 inhibitor durvalumab L ) amino acid sequence.

[0255] SEQ ID NO: 182 is the heavy chain CDR1 amino acid sequence of the PD-L1 inhibitor durvalumab.

[0256] SEQ ID NO: 183 is the heavy chain CDR2 amino acid sequence of the PD-L1 inhibitor durvalumab.

[0257] SEQ ID NO: 184 is the heavy chain CDR3 amino acid sequence of the PD-L1 inhibitor durvalumab.

[0258] SEQ ID NO: 185 is the light chain CDR1 amino acid sequence of the PD-L1 inhibitor durvalumab.

[0259] SEQ ID NO: 186 is the light chain CDR2 amino acid sequence of the PD-L1 inhibitor durvalumab.

[0260] SEQ ID NO: 187 is the light chain CDR3 amino acid sequence of the PD-L1 inhibitor durvalumab.

[0261] SEQ ID NO: 188 is the heavy chain amino acid sequence of the PD-L1 inhibitor avelumab.

[0262] SEQ ID NO: 189 is the light chain amino acid sequence of the PD-L1 inhibitor avelumab.

[0263] SEQ ID NO: 190 represents the heavy chain variable region (V) of the PD-L1 inhibitor avelumab H ) amino acid sequence.

[0264] SEQ ID NO: 191 is the light chain variable region (V) of the PD-L1 inhibitor avelumab L ) amino acid sequence.

[0265] SEQ ID NO: 192 is the heavy chain CDR1 amino acid sequence of the PD-L1 inhibitor avelumab.

[0266] SEQ ID NO: 193 is the heavy chain CDR2 amino acid sequence of the PD-L1 inhibitor avelumab.

[0267] SEQ ID NO: 194 is the heavy chain CDR3 amino acid sequence of the PD-L1 inhibitor avelumab.

[0268] SEQ ID NO: 195 is the light chain CDR1 amino acid sequence of the PD-L1 inhibitor avelumab.

[0269] SEQ ID NO: 196 is the light chain CDR2 amino acid sequence of the PD-L1 inhibitor avelumab.

[0270] SEQ ID NO: 197 is the light chain CDR3 amino acid sequence of the PD-L1 inhibitor avelumab.

[0271] SEQ ID NO: 198 is the heavy chain amino acid sequence of the PD-L1 inhibitor atezolizumab.

[0272] SEQ ID NO: 199 is the light chain amino acid sequence of the PD-L1 inhibitor atezolizumab.

[0273] SEQ ID NO: 200 represents the heavy chain variable region (V) of the PD-L1 inhibitor atezolizumab H ) amino acid sequence.

[0274] SEQ ID NO: 201 is the light chain variable region (V) of the PD-L1 inhibitor atezolizumab L ) amino acid sequence.

[0275] SEQ ID NO: 202 is the heavy chain CDR1 amino acid sequence of the PD-L1 inhibitor atezolizumab.

[0276] SEQ ID NO: 203 is the heavy chain CDR2 amino acid sequence of the PD-L1 inhibitor atezolizumab.

[0277] SEQ ID NO: 204 is the heavy chain CDR3 amino acid sequence of the PD-L1 inhibitor atezolizumab.

[0278] SEQ ID NO: 205 is the light chain CDR1 amino acid sequence of the PD-L1 inhibitor atezolizumab.

[0279] SEQ ID NO: 206 is the light chain CDR2 amino acid sequence of the PD-L1 inhibitor atezolizumab.

[0280] SEQ ID NO: 207 is the light chain CDR3 amino acid sequence of the PD-L1 inhibitor atezolizumab.

[0281] SEQ ID NO: 208 is the heavy chain amino acid sequence of the CTLA-4 inhibitor ipilimumab.

[0282] SEQ ID NO: 209 is the light chain amino acid sequence of the CTLA-4 inhibitor ipilimumab.

[0283] SEQ ID NO: 210 represents the heavy chain variable region (V) of the CTLA-4 inhibitor ipilimumab H ) amino acid sequence.

[0284] SEQ ID NO: 211 represents the light chain variable region (V) of the CTLA-4 inhibitor ipilimumab L ) amino acid sequence.

[0285] SEQ ID NO: 212 is the heavy chain CDR1 amino acid sequence of the CTLA-4 inhibitor ipilimumab.

[0286] SEQ ID NO: 213 is the heavy chain CDR2 amino acid sequence of the CTLA-4 inhibitor ipilimumab.

[0287] SEQ ID NO: 214 is the heavy chain CDR3 amino acid sequence of the CTLA-4 inhibitor ipilimumab.

[0288] SEQ ID NO: 215 is the light chain CDR1 amino acid sequence of the CTLA-4 inhibitor ipilimumab.

[0289] SEQ ID NO: 216 is the light chain CDR2 amino acid sequence of the CTLA-4 inhibitor ipilimumab.

[0290] SEQ ID NO: 217 is the light chain CDR3 amino acid sequence of the CTLA-4 inhibitor ipilimumab.

[0291] SEQ ID NO: 218 is the heavy chain amino acid sequence of the CTLA-4 inhibitor tremelimumab.

[0292] SEQ ID NO: 219 is the light chain amino acid sequence of the CTLA-4 inhibitor tremelimumab.

[0293] SEQ ID NO: 220 represents the heavy chain variable region (V) of the CTLA-4 inhibitor tremelimumab H ) amino acid sequence.

[0294] SEQ ID NO: 221 represents the light chain variable region (V) of the CTLA-4 inhibitor tremelimumab L ) amino acid sequence.

[0295] SEQ ID NO: 222 is the heavy chain CDR1 amino acid sequence of the CTLA-4 inhibitor tremelimumab.

[0296] SEQ ID NO: 223 is the heavy chain CDR2 amino acid sequence of the CTLA-4 inhibitor tremelimumab.

[0297] SEQ ID NO: 224 is the heavy chain CDR3 amino acid sequence of the CTLA-4 inhibitor tremelimumab.

[0298] SEQ ID NO: 225 is the light chain CDR1 amino acid sequence of the CTLA-4 inhibitor tremelimumab.

[0299] SEQ ID NO: 226 is the light chain CDR2 amino acid sequence of the CTLA-4 inhibitor tremelimumab.

[0300] SEQ ID NO: 227 is the light chain CDR3 amino acid sequence of the CTLA-4 inhibitor tremelimumab.

[0301] SEQ ID NO: 228 is the heavy chain amino acid sequence of the CTLA-4 inhibitor zalifrelimab.

[0302] SEQ ID NO: 229 is the light chain amino acid sequence of the CTLA-4 inhibitor zalifrelimab.

[0303] SEQ ID NO: 230 is the heavy chain variable region (V) of the CTLA-4 inhibitor zalifrelimab H ) amino acid sequence.

[0304] SEQ ID NO: 231 is the light chain variable region (V) of the CTLA-4 inhibitor zalifrelimab L ) amino acid sequence.

[0305] SEQ ID NO: 232 is the heavy chain CDR1 amino acid sequence of the CTLA-4 inhibitor zalifrelimab.

[0306] SEQ ID NO: 233 is the heavy chain CDR2 amino acid sequence of the CTLA-4 inhibitor zalifrelimab.

[0307] SEQ ID NO: 234 is the heavy chain CDR3 amino acid sequence of the CTLA-4 inhibitor zalifrelimab.

[0308] SEQ ID NO: 235 is the light chain CDR1 amino acid sequence of the CTLA-4 inhibitor zalifrelimab.

[0309] SEQ ID NO: 236 is the light chain CDR2 amino acid sequence of the CTLA-4 inhibitor zalifrelimab.

[0310] SEQ ID NO: 237 is the light chain CDR3 amino acid sequence of the CTLA-4 inhibitor zalifrelimab. DETAILED DESCRIPTION OF THE INVENTION

[0311] I. Introduction Adoptive cell therapy using TILs cultured ex vivo by rapid expansion protocols (REPs) has been successful in patients with cancers such as melanoma after host immunosuppression. Current infusion acceptance parameters rely on TIL composition (e.g., CD28, CD8, or CD4 positivity) as a readout, as well as the numerical fold expansion and viability of the REP product. While TILs can be reactivated and expanded ex vivo, their epigenetic programming in the suppressive tumor microenvironment can maintain TILs in a more differentiated, less functional state when administered.

[0312] The present invention relates to the use of epigenetic reprogramming agents in the cell culture medium during ex vivo expansion of TILs to counteract the effects of the suppressive tumor microenvironment and improve the quality of the expanded TILs with respect to persistence, functionality, and anti-tumor potential.

[0313] 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.

[0314] As used herein, the terms "co-administration," "co-administering," "administered in combination with," "administering in combination with," "simultaneous," and "concurrent" encompass administration of two or more active pharmaceutical ingredients (e.g., multiple TILs) to a subject such that both active pharmaceutical ingredients and / or their metabolites are present in the subject at the same time. Concurrent administration includes simultaneous administration in separate compositions, administration at different times in separate compositions, or administration in a composition in which two or more active pharmaceutical ingredients are present. Concurrent administration in separate compositions and administration in a composition in which both agents are present are preferred.

[0315] The term "in vivo" refers to events that take place inside a subject's body.

[0316] 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.

[0317] 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.

[0318] 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.

[0319] As used herein, "tumor infiltrating lymphocytes" or "TILs" refers to a population of cells that are initially acquired as leukocytes that have left the bloodstream of a subject and migrated to a tumor. TILs include CD8 + Cytotoxic T cells (lymphocytes), Th1 and Th17 CD4 + These include, but are not limited to, T cells, natural killer cells, dendritic cells, and M1 macrophages. TILs include both primary and secondary TILs. "Primary TILs" are those obtained from a patient tissue sample as outlined herein (sometimes referred to as "freshly harvested"), and "secondary TILs" are any expanded or propagated TIL cell populations discussed herein, including, but not limited to, bulk TILs and expanded TILs ("REP TILs" or "post-REP TILs"). TIL cell populations may include genetically modified TILs.

[0320] As used herein, a "population of cells" (including TILs) refers to several cells that share a common trait. Generally, a population is roughly 1 x 10 6 ~1×10 10 The number of TILs ranges from approximately 1 x 10 to 1 x 10, with different TIL populations containing different numbers. For example, the initial growth of primary TILs in the presence of IL-2 is approximately 1 x 10 8 REP expansion typically yields a bulk TIL population of 1.5 x 10 cells for injection. 9 ~1.5×10 10 This is done to provide a population of cells.

[0321] 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.

[0322] By "thawed cryopreserved TILs" herein is meant a population of TILs that have been previously cryopreserved and then processed to return to room temperature or above, including but not limited to, cell culture temperature or a temperature at which the TILs can be administered to a patient.

[0323] 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.

[0324] 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.

[0325] 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.

[0326] 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.

[0327] 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.

[0328] 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.

[0329] 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.

[0330] 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.

[0331] 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.

[0332] 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.

[0333] [Table 1]

[0334] 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. US2019 / 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. US2014 / 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.

[0335] In some embodiments, a suitable IL-2 form for use in the present invention is THOR-707, available from Synthorx, Inc. The preparation and properties of THOR-707 and additional alternative forms of IL-2 suitable for use in the present invention are described in U.S. Patent Application Publication Nos. US2020 / 0181220 A1 and US2020 / 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) conjugate comprising an isolated and purified IL-2 polypeptide and a conjugate moiety that binds to the isolated and purified IL-2 polypeptide at an amino acid position selected from K35, T37, R38, T41, F42, K43, F44, Y45, E61, E62, E68, K64, P65, V69, L72, and Y107, wherein the numbering of the amino acid residues corresponds to SEQ ID NO:5. In some embodiments, the amino acid position is selected from T37, R38, T41, F42, F44, Y45, E61, E62, E68, K64, P65, V69, L72, and Y107. In some embodiments, the amino acid position is selected from T37, R38, T41, F42, F44, Y45, E61, E62, E68, P65, V69, L72, and Y107. In some embodiments, the amino acid position is selected from T37, T41, F42, F44, Y45, P65, V69, L72, and Y107. In some embodiments, the amino acid position is selected from R38 and K64. In some embodiments, the amino acid position is selected from E61, E62, and E68. In some embodiments, the amino acid position is E62. In some embodiments, an amino acid residue selected from K35, T37, R38, T41, F42, K43, F44, Y45, E61, E62, E68, K64, P65, V69, L72, and Y107 is further mutated to lysine, cysteine, or histidine. In some embodiments, the amino acid residue is mutated to cysteine. In some embodiments, the amino acid residue is mutated to lysine. In some embodiments, K35, T37, R38, T41, F42, K43, F44, Y45, E61, E62, E68, K64, P65, V69, L72, and Y107 are ...and Y107 are further mutated to an unnatural amino acid. In some embodiments, the unnatural amino acid is selected from N6-azidoethoxy-L-lysine (AzK), N6-propargylethoxy-L-lysine (PraK), BCN-L-lysine, norbornene lysine, TCO-lysine, methyltetrazine lysine, allyloxycarbonyl lysine, 2-amino-8-oxononanoic acid, 2-amino-8-oxooctanoic acid, p-acetyl-L-phenylalanine, p-azidomethyl-L-phenylalanine (pAMF), p-iodo-L-phenylalanine, m-acetylphenylalanine, 2-amino-8-oxononanoic acid, p-propargyloxyphenylalanine, p-propargyl-phenylalanine, 3-methyl-phenylalanine, L-dopa, fluorinated phenylalanine, isopropyl and selenocysteine, or selenocysteine. In some embodiments, the IL-2 conjugate has reduced affinity for the IL-2 receptor alpha (IL-2Rα) subunit compared to a wild-type IL-2 polypeptide. In some embodiments, the reduced affinity is about a 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 95%, 99%, or greater than a 99% decrease in binding affinity for IL-2Rα compared to a wild-type IL-2 polypeptide. In some embodiments, the reduced affinity is about a 1-fold, 2-fold, 3-fold, 4-fold, 5-fold, 6-fold, 7-fold, 8-fold, 9-fold, 10-fold, 30-fold, 50-fold, 100-fold, 200-fold, 300-fold, 500-fold, or greater decrease in binding affinity for IL-2Rα compared to a wild-type IL-2 polypeptide.1000-fold or more. In some embodiments, the conjugate moiety impairs or blocks the binding of IL-2 to IL-2Rα. In some embodiments, the conjugate moiety comprises a water-soluble polymer. In some embodiments, the additional conjugate 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(olefin 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 conjugate moiety comprises a protein. In some embodiments, the additional conjugate moiety comprises a protein. In some embodiments, each of the proteins independently comprises albumin, transferrin, or transthyretin. In some embodiments, each of the proteins independently comprises an Fc portion. In some embodiments, each of the proteins independently comprises an Fc portion of an IgG. In some embodiments, the conjugate moiety comprises a polypeptide. In some embodiments, the additional conjugate moiety comprises a polypeptide. In some embodiments, each of the proteins independently comprisesThe conjugate moiety may comprise an XTEN peptide, a glycine-rich homoamino acid polymer (HAP), a PAS polypeptide, an elastin-like polypeptide (ELP), a CTP peptide, or a gelatin-like protein (GLK) polymer. In some embodiments, the isolated and purified IL-2 polypeptide is modified by glutamylation. In some embodiments, the conjugate moiety is directly attached to the isolated and purified IL-2 polypeptide. In some embodiments, the conjugate 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 (sulfo-DST), 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)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-xylenesulfonic acid, N,N'-ethylene-bis(iodoacetamide),or N,N'-hexamethylenebis(iodoacetamide). In some embodiments, the linker comprises a heterobifunctional linker. In some embodiments, the heterobifunctional linker is N-succinimidyl 3-(2-pyridyldithio)propionate (sPDP), long-chain N-succinimidyl 3-(2-pyridyldithio)propionate (LC-sPDP), water-soluble long-chain N-succinimidyl 3-(2-pyridyldithio)propionate (sulfo-LC-sPDP), succinimidyloxycarbonyl-α-methyl-α-(2-pyridyldithio)toluene (sMPT), sulfosuccinimidyl-6-[α-methyl-α-(2-pyridyldithio)toluamido]hexanoate (sulfo-LC-sMPT), succinimidyl-4-(N-maleimidomethyl)cyclohexane-1-carboxylate (sMCC), sulfosuccinimidyl-4-(N-maleimidomethyl)cyclohexane- 1-carboxylate (sulfo-sMCC), m-maleimidobenzoyl-N-hydroxysuccinimide ester (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 (MCH), 3-(2-pyridyldithio)propionyl hydrazide (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 (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 glyoxal (APG). In some embodiments, the linker comprises a cleavable linker, optionally comprising a dipeptide linker.In some embodiments, the dipeptide linker comprises Val-Cit, Phe-Lys, Val-Ala, or Val-Lys. In some embodiments, the linker comprises a non-cleavable linker. In some embodiments, the linker optionally comprises a maleimido group comprising maleimidocaproyl (mc), succinimidyl-4-(N-maleimidomethyl)cyclohexane-1-carboxylate (sMCC), or sulfosuccinimidyl-4-(N-maleimidomethyl)cyclohexane-1-carboxylate (sulfo-sMCC). In some embodiments, the linker further comprises a spacer. In some embodiments, the spacer comprises p-aminobenzyl alcohol (PAB), p-aminobenzyloxycarbonyl (PABC), a derivative, or an analog thereof. In some embodiments, the conjugate moiety is capable of extending the serum half-life of the IL-2 conjugate. In some embodiments, the additional conjugate 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, the IL-2 form suitable for use in the present invention is pegylated as disclosed in U.S. Patent Application Publication Nos. US2020 / 0181220 A1 and US2020 / 0330601 A1. In some embodiments, an IL-2 form suitable 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 conjugate 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, IL-2 forms 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, 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 conjugate 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, an IL-2 form suitable 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 conjugate 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, an IL-2 form suitable 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 conjugate 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.

[0336] In some embodiments, a form of IL-2 suitable for use in the present invention is nembareukin alfa, also known as ALKS-4230 (SEQ ID NO: 6), available from Alkermes, Inc. Nembareukin alfa is a nucleotide analogue of IL-2 that is linked via a peptidyl linker ( 60 GG 61 ) and fused to human interleukin-2 fragment (62-132) via a peptidyl linker ( 133 GSGGGS 138 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. US2021 / 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.

[0337] [Table 2]

[0338] 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. US2020 / 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 V 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.

[0339] 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.

[0340] 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.

[0341] 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.

[0342] In some embodiments, the IL-2 molecule described herein is an IL-2 mutein. In some cases, the IL-2 mutein comprises an R67A substitution. In some embodiments, the IL-2 mutein comprises the amino acid sequence of SEQ ID NO: 14 or SEQ ID NO: 15. In some embodiments, the IL-2 mutein comprises the amino acid sequence of Table 1 of U.S. Patent Application Publication No. US2020 / 0270334 A1, the disclosure of which is incorporated herein by reference.

[0343] 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.

[0344] [Table 3-1] [Table 3-2]

[0345] 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).

[0346] 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).

[0347] 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).

[0348] 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: 21).

[0349] 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.

[0350] 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.

[0351] 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.

[0352] 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.

[0353] 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.

[0354] 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.

[0355] 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.

[0356] 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.

[0357] 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).

[0358] 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.

[0359] 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.

[0360] As used herein, "tumor infiltrating lymphocytes" or "TILs" refers to a population of cells that are initially acquired as leukocytes that have left the bloodstream of a subject and migrated to a tumor. TILs include CD8 + Cytotoxic T cells (lymphocytes), Th1 and Th17 CD4 + These include, but are not limited to, T cells, natural killer cells, dendritic cells, and M1 macrophages. TILs include both primary and secondary TILs. "Primary TILs" are those obtained from a patient tissue sample as outlined herein (sometimes referred to as "freshly harvested"). "Secondary TILs" are any TIL cell populations that have been expanded or propagated as discussed herein, including, but not limited to, bulk TILs, expanded TILs ("REP TILs"), and "reREP TILs" as discussed herein. reREP TILs can include, for example, second expanded TILs or second additional expanded TILs (e.g., those described in step D of FIG. 8, including TILs designated as reREP TILs).

[0361] TILs can generally be defined either biochemically using cell surface markers or functionally by their ability to infiltrate tumors and achieve therapy. TILs can generally be classified by expression of one or more of the following biomarkers: CD4, CD8, TCRαβ, CD27, CD28, CD56, CCR7, CD45Ra, CD95, PD-1, and CD25. Additionally and alternatively, TILs can be functionally defined by their ability to infiltrate solid tumors upon reintroduction into patients. TILs can be further characterized by potency; for example, TILs can be considered potent if their interferon (IFN) release is greater than about 50 pg / mL, greater than about 100 pg / mL, greater than about 150 pg / mL, or greater than about 200 pg / mL. For example, TILs can be considered potent if interferon (IFNγ) release is greater than about 50 pg / mL, greater than about 100 pg / mL, greater than about 150 pg / mL, or greater than about 200 pg / mL, greater than about 300 pg / mL, greater than about 400 pg / mL, greater than about 500 pg / mL, greater than about 600 pg / mL, greater than about 700 pg / mL, greater than about 800 pg / mL, greater than about 900 pg / mL, or greater than about 1000 pg / mL.

[0362] 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.

[0363] 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.

[0364] 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.

[0365] 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.

[0366] 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," "consisting essentially of," and "consisting of."

[0367] The terms "antibody" and its plural "antibodies" refer to whole immunoglobulins and any antigen-binding fragment ("antigen-binding portion") or single chains thereof. "Antibody" also refers to a glycoprotein comprising at least two heavy (H) chains and two light (L) chains interconnected by disulfide bonds, or antigen-binding portions thereof. Each heavy chain contains a heavy chain variable region (referred to herein as V H Each light chain is composed of a light chain variable region (abbreviated herein as V) and a heavy chain constant region. The heavy chain constant region is composed of three domains: CH1, CH2, and CH3. L The light chain constant region consists of one domain, C L The V of the antibody H and V L The regions can be further subdivided into regions of hypervariability, called complementarity-determining regions (CDRs) or hypervariable regions (HVRs), which may be interspersed with more conserved regions called framework regions (FRs). H and 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.

[0368] 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.

[0369] 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.

[0370] 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 can be joined by a synthetic linker, allowing 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, an scFv protein domain comprises a VH portion and a VL portion. scFv molecules are designated either as VL-L-VH, where the VL domain is the N-terminal portion of the scFv molecule, or as VH-L-VL, where the VH domain is the N-terminal portion of the scFv molecule. Methods for producing scFv molecules and designing suitable peptide linkers are described in U.S. Pat. No. 4,704,692, U.S. Pat. No. 4,946,778, R. Raag and M. Whitlow, "Single Chain Fvs." FASEB Vol. 9:73-80 (1995), and 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.

[0371] 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.

[0372] 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.

[0373] 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.

[0374] As used herein, "isotype" refers to the antibody class (e.g., IgM or IgG1) that is encoded by heavy chain constant region genes.

[0375] 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."

[0376] 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.

[0377] The terms "humanized antibody," "humanized antibodies," and "humanization" are intended to refer to antibodies in which CDR sequences derived from the germline of another mammalian species, such as mouse, have been grafted onto human framework sequences. Additional framework region modifications can be made within the human framework sequences. Humanized forms of non-human (e.g., murine) antibodies are chimeric antibodies that contain minimal sequence derived from non-human immunoglobulin. In most cases, humanized antibodies are human immunoglobulins (recipient antibodies) in which residues from a hypervariable region of the recipient are replaced by residues from a hypervariable region of a non-human species (donor antibody) such as mouse, rat, rabbit, or non-human primate having the desired specificity, affinity, and capacity. In some instances, Fv framework (FR) residues of the human immunoglobulin are replaced by corresponding non-human residues. Furthermore, humanized antibodies may contain residues that are not found in the recipient or donor antibody. These modifications are made to further refine antibody performance. Generally, a humanized antibody will comprise substantially all of at least one, and typically two, variable domains, in which all or substantially all of the hypervariable loops correspond to those of a non-human immunoglobulin and all or substantially all of the FR regions are those of a human immunoglobulin sequence. The humanized antibody will also optionally comprise at least a portion of an immunoglobulin constant region (Fc), typically that of a human immunoglobulin. For further details, see Jones, et al., Nature 1986, 321, 522-525; Riechmann, et al., Nature 1988, 332, 323-329; and Presta, Curr. Op. Struct. Biol. 1992, 2, 593-596. The antibodies described herein can also be modified to use any Fc variant known to confer improved (e.g., reduced) effector function and / or FcR binding.Fc variants are described in, for example, International Patent Application Publication Nos. WO1988 / 07089A1, WO1996 / 14339A1, WO1998 / 05787A1, WO1998 / 23289A1, WO1999 / 51642A1, WO99 / 58572A1, WO2000 / 09560A2, WO2000 / 32767A1, WO2000 / 42072A2, and WO2002 / 4 4215A2, WO2002 / 060919A2, WO2003 / 074569A2, WO2004 / 016750A2, WO2004 / 029207A2, WO2004 / 03 5752A2, WO2004 / 063351A2, WO2004 / 074455A2, WO2004 / 099249A2, WO2005 / 040217A2, WO2005 / 07 0963A1, WO2005 / 077981A2, WO2005 / 092925A2, WO2005 / 123780A2, WO2006 / 019447A1, WO2006 / 047350A2, and WO2006 / 085967A2, as well as U.S. Pat. Nos. 5,648,260, 5,739,277, 5,834,250, 5,869,046, 6,096 ,871, 6,121,022, 6,194,551, 6,242,195, 6,277,375, 6,528,624, 6,538,124, 6,737,056, 6,821,505, 6,998,253, and 7,083,784 (the disclosures of which are incorporated herein by reference).

[0378] 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.

[0379] A "diabody" is a small antibody fragment that has two antigen-binding sites. The fragments bind to the same polypeptide chain (VH -V L or V L -V H ) in the light chain variable domain (V L ) connected to the heavy chain variable domain (V H ). When a linker that is too short to pair the two domains on the same chain is used, the domains are forced to pair with complementary domains on another chain and create two antigen-binding sites. Bispecific antibodies are more fully described, for example, in European Patent No. EP 404,097, International Patent Publication No. WO 93 / 11161, and Bolliger, et al., Proc. Natl. Acad. Sci. USA 1993, 90, 6444-6448.

[0380] 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 may increase the affinity of an antibody for an antigen. Additionally or alternatively, antibodies can be generated with altered types of glycosylation, such as hypofucosylated antibodies with reduced amounts of fucosyl residues or antibodies with increased bisecting GlcNac structures. Such altered glycosylation patterns have been demonstrated to increase antibody potency. Such carbohydrate modifications can be achieved, for example, by expressing the antibody in a host cell with altered glycosylation machinery. Cells with altered glycosylation machinery have been described in the art and can be used as host cells to express the recombinant antibodies of the present invention, thereby producing antibodies with altered glycosylation. For example, the cell lines Ms704, Ms705, and Ms709 lack the fucosyltransferase gene FUT8 (alpha(1,6) fucosyltransferase), such that antibodies expressed in these cell lines lack fucose on their carbohydrates. The Ms704, Ms705, and Ms709 FUT8- / - cell lines were generated by targeted disruption of the FUT8 gene in CHO / DG44 cells using two replacement vectors (see, e.g., U.S. Patent Publication No. 2004 / 0110704 or Yamane-Ohnuki, et al., Biotechnol. Bioeng., 2004, 87, 614-622).As another example, European Patent No. EP 1,176,195 describes cell lines with a functionally disrupted FUT8 gene encoding a fucosyltransferase, thereby resulting in antibodies expressed in such cell lines exhibiting hypofucosylation by reducing or eliminating alpha-1,6 bond-related enzymes. It also describes cell lines with reduced or no enzymatic activity for adding fucose to N-acetylglucosamine linked to the Fc region of antibodies, such as the rat myeloma cell line YB2 / 0 (ATCC CRL 1662). International Patent Publication No. WO 03 / 035835 describes a variant CHO cell line, Lec13 cells, that has a reduced ability to attach fucose to Asn(297)-linked carbohydrates, which also results in hypofucosylation of antibodies expressed in the host cells (see also Shields, et al., J. Biol. Chem. 2002, 277, 26733-26740). International Patent Publication No. WO 99 / 54342 describes cell lines engineered to express glycoprotein-modifying glycosyltransferases (e.g., beta(1,4)-N-acetylglucosaminyltransferase III (GnTIII)), such that antibodies expressed in the engineered cell lines exhibit increased bisecting GlcNac structures, resulting in increased ADCC activity of the antibodies (see also Umana, et al., Nat. Biotech. 1999, 17, 176-180). Alternatively, fucosidase enzymes can be used to cleave fucose residues from antibodies. For example, the fucosidase alpha-L-fucosidase removes fucosyl residues from antibodies, as described in Tarentino, et al., Biochem. 1975, 14, 5516-5523.

[0381] "PEGylation" refers to a modified antibody or fragment thereof that has been reacted with polyethylene glycol (PEG), such as a reactive ester or aldehyde derivative of PEG, under conditions that result in one or more PEG groups being attached to the antibody or antibody fragment. PEGylation can, for example, increase the biological (e.g., serum) half-life of the antibody. Preferably, PEGylation is carried out via an acylation reaction or an alkylation reaction with a reactive PEG molecule (or an analogous reactive water-soluble polymer). As used herein, the term "polyethylene glycol" refers to a mono (C1-C 10 PEG is intended to encompass any of the forms of PEG that have been used to derivatize other proteins, such as alkoxy- or aryloxy-polyethylene glycol, or polyethylene glycol-maleimide. The antibody to be pegylated may be an aglycosylated antibody. Methods for pegylation are known in the art and can be applied to the antibodies of the invention, for example, as described in European Patent Nos. EP 0 154 316 and EP 0 401 384, and U.S. Pat. No. 5,824,778 (the disclosures of each of which are incorporated herein by reference).

[0382] 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 authorisation or licensing 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 non-clinical studies. As used herein, the term "biosimilar" also refers to a biopharmaceutical that has been or can be compared to a non-EEA-approved comparator. Particular biosimilars are proteins, such as antibodies, antibody fragments (e.g., antigen-binding portions), and fusion proteins. Protein biosimilars may have amino acid sequences with minor modifications to the amino acid structure (e.g., including amino acid deletions, additions, and / or substitutions) that do not significantly affect the function of the polypeptide. A biosimilar may have an amino acid sequence that has 97% or more sequence identity, e.g., 97%, 98%, 99%, or 100%, to the amino acid sequence of its reference pharmaceutical. A biosimilar may include one or more post-translational modifications, such as, but not limited to, glycosylation, oxidation, deamidation, and / or cleavage, that differ from the post-translational modifications of the reference pharmaceutical, provided that the differences do not result in a change in the safety and / or efficacy of the pharmaceutical. A biosimilar may have the same or a different glycosylation pattern as the reference pharmaceutical, provided that the differences do not result in a change in the safety and / or efficacy of the pharmaceutical.In particular, but not exclusively, biosimilars may have different glycosylation patterns if the differences address or are intended to address safety concerns associated with the reference drug. Additionally, biosimilars may deviate from the reference drug, for example, in its strength, dosage form, formulation, excipients, and / or presentation, provided that the drug's safety and efficacy are not compromised. Biosimilars may contain differences, for example, in their pharmacokinetic (PK) and / or pharmacodynamic (PD) profiles, compared to the reference drug, but are still considered sufficiently similar to the reference drug to be approved or deemed suitable for approval. In certain circumstances, biosimilars exhibit different binding characteristics compared to the reference drug, and these different binding characteristics are not considered by regulatory authorities, such as the EMA, to be a barrier to approval as a similar biological product. The term "biosimilar" is also used interchangeably by regulatory agencies in other countries and regions.

[0383] As used herein, the term "epigenetic reprogramming" refers to the remodeling of epigenetic marks during cellular development. Epigenetic reprogramming affects cellular function throughout successive generations of cells without altering the underlying DNA sequence. Epigenetic reprogramming involves the modulation of DNA and / or histone methylation to result in transcriptional reorganization in cells. Reprogramming can be artificially induced by the introduction of exogenous factors, usually transcription factors, in cell culture media, for example, during ex vivo expansion of TILs. Various drug compounds can be used for epigenetic reprogramming, which may involve modulating the activity of one or more pathways and / or proteins involved in transcription in cells. Compounds that can reconfigure transcription in cells include, but are not limited to, DNA hypomethylating agents, mitogen-activated protein kinase (MEK) inhibitors, histone deacetylase (HDAC) inhibitors, enhancer of zeste homolog 2 (EZH2) inhibitors, bromodomain inhibitors, protein kinase B (AKT) inhibitors, and / or ten-eleven translocation protein (TET) inhibitors.

[0384] The term "DNA hypomethylating agent" refers to a drug that inhibits or otherwise reduces DNA methylation. Most DNA hypomethylating agents block the activity of DNA methyltransferase and therefore function as DNA methyltransferase inhibitors. DNA methyltransferase activity may be reduced by a statistically significant amount, including, for example, at least about a 5%, 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 95%, or 100% reduction in activity compared to a suitable control. Examples of DNA hypomethylating agents include decitabine, azacitidine, GSK-3484862, RG-108, GSK-3685032, DHAC, SGI-1027, CM-272, zebularine, hinokitiol, guadecitabine, gamma-oryzanol, CM-579, DC-517, 5-fluoro-2′-deoxycytidine, 5-methyldeoxycytidine, DC-05, 6-methyl-5-azacytidine, procainamide, procaine, hydrazine, EGCG, FdCyd, CP-4200, nanomycin A, and pharmaceutically acceptable salts, cocrystals, and solvents thereof. In some embodiments, the DNA hypomethylating agent may be decitabine, including, for example, a co-crystal solvate, or a pharmaceutically acceptable salt thereof (see, e.g., Aribi A, et al. Cancer (2007) 109(4):713-7).

[0385] i. Decitabine In one embodiment, the DNA hypomethylating agent is decitabine. Teheranolide has the chemical structure and name shown below: 4-amino-1-[(2R,4S,5R)-4-hydroxy-5-(hydroxymethyl)oxolan-2-yl]-1,3,5-triazin-2-one

[0386] [ka]

[0387] ii. Azacitidine In one embodiment, the DNA hypomethylating agent is azacitidine, which has the chemical structure and name shown below: 4-amino-1-[(2R,3R,4S,5R)-3,4-dihydroxy-5-(hydroxymethyl)oxolan-2-yl]-1,3,5-triazin-2-one

[0388] [ka]

[0389] iii.GSK-3484862 In one embodiment, the DNA hypomethylating agent is GSK-3484862, which has the chemical structure and name shown below: (2R)-2-[3,5-dicyano-6-(dimethylamino)-4-ethylpyridin-2-yl]sulfanyl-2-phenylacetamide

[0390] [ka]

[0391] iv.RG-108 In one embodiment, the DNA hypomethylating agent is RG-108, which has the chemical structure and name shown below: (2S)-2-(1,3-dioxoisoindol-2-yl)-3-(1H-indol-3-yl)propanoic acid

[0392] [ka]

[0393] v.GSK-3685032 In one embodiment, the DNA hypomethylating agent is GSK-3685032, which has the chemical structure and name shown below: 2-[6-(4-aminopiperidin-1-yl)-3,5-dicyano-4-ethylpyridin-2-yl]sulfanyl-2-phenylacetamide

[0394] [ka]

[0395] vi.DHAC In one embodiment, the DNA hypomethylating agent is DHAC, which has the chemical structure and name shown below: 6-amino-3-[3,4-dihydroxy-5-(hydroxymethyl)oxolan-2-yl]-1,4-dihydro-1,3,5-triazin-2-one

[0396] [ka]

[0397] vii.SGI-1027 In one embodiment, the DNA hypomethylating agent is SGI-1027, which has the chemical structure and name shown below: N-[4-[(2-amino-6-methylpyrimidin-4-yl)amino]phenyl]-4-(quinolin-4-ylamino)benzamide

[0398] [ka]

[0399] viii.CM-272 In one embodiment, the DNA hypomethylating agent is CM-272, which has the chemical structure and name shown below: 6-methoxy-2-(5-methylfuran-2-yl)-N-(1-methylpiperidin-4-yl)-7-(3-pyrrolidin-1-ylpropoxy)quinolin-4-amine

[0400] [ka]

[0401] ix. Zebularine In one embodiment, the DNA hypomethylating agent is Zebularine, which has the chemical structure and name shown below: 1-[(2R,3R,4S,5R)-3,4-dihydroxy-5-(hydroxymethyl)oxolan-2-yl]pyrimidin-2-one

[0402] [ka]

[0403] x. Hinokitiol In one embodiment, the DNA hypomethylating agent is hinokitiol, which has the chemical structure and name shown below: 2-hydroxy-6-propan-2-ylcyclohepta-2,4,6-trien-1-one

[0404] [ka]

[0405] xi. Guadecitabine In one embodiment, the DNA hypomethylating agent is guadecitabine, which has the chemical structure and name shown below: [(2R,3S,5R)-5-(2-amino-6-oxo-1H-purin-9-yl)-3-hydroxyoxolan-2-yl]methyl [(2R,3S,5R)-5-(4-amino-2-oxo-1,3,5-triazin-1-yl)-2-(hydroxymethyl)oxolan-3-yl]phosphate

[0406] [ka]

[0407] xii. Gamma-oryzanol In one embodiment, the DNA hypomethylating agent is gamma-oryzanol, which has the chemical structure and name shown below: [(1S,3R,6S,8R,11S,12S,15R,16R)-7,7,12,16-tetramethyl-15-[(2R)-6-methylhept-5-en-2-yl]-6-pentacyclo[9.7.0.0] 1,3 .0 3,8 .0 12,16 ]octadecanyl](E)-3-(4-hydroxy-3-methoxyphenyl)prop-2-enoate

[0408] [ka]

[0409] xiii.CM-579 In one embodiment, the DNA hypomethylating agent is CM-579, which has the chemical structure and name shown below: 6-methoxy-2-(5-methylfuran-2-yl)-N-(1-methylpiperidin-4-yl)methyl]-7-(3-pyrrolidin-1-ylpropoxy)quinolin-4-amine

[0410] [ka]

[0411] xiv.DC-517 In one embodiment, the DNA hypomethylating agent is DC-517, which has the chemical structure and name shown below: 1-[1,3-di(carbazol-9-yl)propan-2-yloxy]-3-(propan-2-ylamino)propan-2-ol

[0412] [ka]

[0413] xv. 5-Fluoro-2′-deoxycytidine In one embodiment, the DNA hypomethylating agent is 5-fluoro-2'-deoxycytidine, which has the chemical structure and name shown below: 4-amino-5-fluoro-1-[(2R,4S,5R)-4-hydroxy-5-(hydroxymethyl)oxolan-2-yl]pyrimidin-2-one

[0414] [ka]

[0415] xvi. 5-Methyldeoxycytidine In one embodiment, the DNA hypomethylating agent is 5-methyldeoxycytidine, which has the chemical structure and name shown below: 4-amino-1-[(2R,4S,5R)-4-hydroxy-5-(hydroxymethyl)oxolan-2-yl]-5-methylpyrimidin-2-one

[0416] [ka]

[0417] xvii.DC-05 In one embodiment, the DNA hypomethylating agent is DC-05, which has the chemical structure and name shown below: 1-carbazol-9-yl-3-[2-(1H-indol-3-yl)ethylamino]propan-2-ol

[0418] [ka]

[0419] xviii. 6-Methyl-5-azacytidine In one embodiment, the DNA hypomethylating agent is 6-methyl-5-azacytidine, which has the chemical structure and name shown below: 4-amino-1-[(2R,3R,4S,5R)-3,4-dihydroxy-5-(hydroxymethyl)oxolan-2-yl]-6-methyl-1,3,5-triazin-2-one

[0420] [ka]

[0421] 19. Procainamide In one embodiment, the DNA hypomethylating agent is procainamide, which has the chemical structure and name shown below: 4-amino-N-[2-(diethylamino)ethyl]benzamide

[0422] [ka]

[0423] xx. Procaine In one embodiment, the DNA hypomethylating agent is procaine, which has the chemical structure and name shown below: 2-(diethylamino)ethyl 4-aminobenzoate

[0424] [ka]

[0425] xxi. Hydralazine In one embodiment, the DNA hypomethylating agent is hydralazine, which has the chemical structure and name shown as phthalazin-1-ylhydrazine.

[0426] [ka]

[0427] xxii.EGCG In one embodiment, the DNA hypomethylating agent is EGCG, which has the chemical structure and name shown below: [(2R,3R)-5,7-dihydroxy-2-(3,4,5-trihydroxyphenyl)-3,4-dihydro-2H-chromen-3-yl]3,4,5-trihydroxybenzoate

[0428] [ka]

[0429] xxiii.FdCyd In one embodiment, the DNA hypomethylating agent is FdCyD, which has the chemical structure and name shown below: 4-amino-5-fluoro-1-[(2R,4S,5R)-4-hydroxy-5-(hydroxymethyl)oxolan-2-yl]pyrimidin-2-one

[0430] [ka]

[0431] xxiv.CP-4200 In one embodiment, the DNA hypomethylating agent is CP-4200, which has the chemical structure and name shown below: 5-azacytidine-5'-elaidate

[0432] [ka]

[0433] xxv. Nanomycin A In one embodiment, the DNA hypomethylating agent is nanomycin A. Nanomycin A has the chemical structure and name shown below: 2-[(1S,3R)-9-hydroxy-1-methyl-5,10-dioxo-3,4-dihydro-1H-benzo[g]isochromen-3-yl]acetic acid

[0434] [ka]

[0435] As used herein, the term "MEK inhibitor" refers to a compound that reduces, inhibits, or otherwise decreases one or more of the biological activities of mitogen-activated protein kinase (MEK) (MEK1 and / or MEK2). Activity may be decreased by a statistically significant amount, including, for example, at least about a 5%, 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 95%, or 100% decrease in MEK activity compared to an appropriate control. MEK is a dual-specificity kinase that phosphorylates tyrosine and threonine residues on ERK1 and ERK2, which are required for activation. Two related genes encode MEK1 and MEK2, which differ in their binding to ERK and possibly their activation profiles. MEK is a substrate of several protein kinases, including B-Raf, in the MAPK / ERK pathway. MEK inhibitors include, but are not limited to, trametinib (Mekinist®, GSK1120212), cobimetinib (Cotellic®), binimetinib (Mektovi®, MEK162, ARRY-162, ARRY-438162), selumetinib, PD-325901, CI-1040, TAK-733, GDC-0623, pimasertinib, refametinib, BI-847325, and pharmaceutically acceptable salts, cocrystals, and solvates thereof. In some embodiments, the MEK inhibitor is trametinib (e.g., Flaherty et al., N. Engl. J. Med. 2012, 367:1694-1703), including, for example, a cocrystal solvate thereof, or a pharmaceutically acceptable salt thereof. In some embodiments, the MEK inhibitor is trametinib as a DMSO solvate or co-crystal.

[0436] As used herein, the term "HDAC" refers to a compound that reduces, inhibits, or otherwise decreases one or more of the biological activities of histone deacetylase (HDAC). The activity can be decreased by a statistically significant amount, including, for example, at least about a 5%, 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 95%, or 100% decrease in HDAC activity compared to an appropriate control. Examples of HDAC inhibitors include, but are not limited to, rosillinostat, vorinostat, trichostatin A, belinostat, panabinostat, panobinostat, xinostat, divinostat, resminostat, abexinostat, xinostat, practinostat, CHR-3996, valproic acid, butyric acid, phenylbutyric acid, encethiostat, tacedinaline, mocetinostat, romidespin, nicotinamide, sirtinol, cambinol, EX-527, apicidin, depsipeptide, MS275, BML-210, splitomicin, RGFP966, and co-crystal solvates or pharmaceutically acceptable salts thereof. In some embodiments, the HDAC inhibitor is licorinistat (e.g., Dan T. Vogl, et al. Clin Cancer Res. 2017, 23(13)3307-15), including, for example, a co-crystal solvate thereof, or a pharmaceutically acceptable salt thereof.

[0437] As used herein, the term "EZH2 inhibitor" refers to a compound that reduces, inhibits, or otherwise decreases one or more of the biological activities of enhancer of zeste homolog 2 (EZH2). Activity can be reduced by a statistically significant amount, including, for example, at least about a 5%, 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 95%, or 100% reduction in EZH2 activity compared to an appropriate control. Examples of EZH2 inhibitors include, but are not limited to, 3-deazaneplanocin A, tazemetostat, GSK343, GSK926, GSK126, EPZ005687, and co-crystal solvates or pharmaceutically acceptable salts thereof.

[0438] As used herein, the term "AKT inhibitor" refers to a compound that reduces, inhibits, or otherwise decreases one or more of the biological activities of protein kinase B (AKT). Activity can be decreased by a statistically significant amount, including, for example, at least about a 5%, 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 95%, or 100% decrease in the activity of AKT compared to a suitable control. Examples of AKT inhibitors include, but are not limited to, ipatasertib, GSK690693, GSK2141795, GSK2110183, AZD5363, GDC-0068, AT7867, CCT128930, MK-2206, BAY1125976, perifosine, oridonin, herbacetin, teheranolide, isoliquirigenin, scutellarin, honokiol, and pharmaceutically acceptable salts thereof. In some embodiments, the AKT inhibitor is ipatasertib (e.g., Lin et al., Clin. Cancer Res. (2013) 19(7):1760-72), and co-crystal solvates or pharmaceutically acceptable salts thereof.

[0439] As used herein, the term "bromodomain inhibitor" refers to a compound that reduces, inhibits, or otherwise decreases the interaction between bromodomain-containing proteins and acetyl groups during DNA transcription. Activity can be reduced by a statistically significant amount, including, for example, at least about a 5%, 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 95%, or 100% reduction in bromodomain activity compared to an appropriate control. Bromodomains bind to acetylated lysines on histone tails, and recognition of the acetyl group is crucial for recruiting other chromatin factors and transcriptional machinery, and thus for regulating gene transcription. See, e.g., Perez-Salvia and Esteller, Epigenetics: 2017, 12(5), 323-329. Examples of bromodomain inhibitors include, but are not limited to, JQ1, ZEN-3694, I-BET762, OTX015, I-BET151, RVX-208, MS417, ABBV-075, ABBV-744, SJ432, AZD5153, INCB054329, INCB054329, FT-1101, CPI-0610, RO6870810, BAY1238097, RVX000222, and pharmaceutically acceptable salts thereof.

[0440] As used herein, the term "TET inhibitor" refers to a compound that reduces, inhibits, or otherwise decreases one or more biological activities of the ten-eleven translocation protein (TET). Activity can be reduced by a statistically significant amount, including, for example, at least about a 5%, 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 95%, or 100% reduction in TET activity compared to an appropriate control. Examples of TET inhibitors include, but are not limited to, Bobcat339, D(R)-2-hydroxyglutarate (D2HG), L-2-hydroxyglutarate (L2HG), and C35 (e.g., Singh, et al., PNAS February 18, 2020 117(7)3621-3626), and pharmaceutically acceptable salts thereof.

[0441] III. Gen2 TIL Manufacturing Process An exemplary family of TIL processes known as Gen2 (also known as Process 2A) that includes some of these features is shown in Figures 1 and 2. An embodiment of Gen2 is shown in Figure 2.

[0442] As discussed herein, the present invention may include steps related to restimulating cryopreserved TILs prior to transplantation into a patient to improve their metabolic activity, and thus their relative health, and methods for testing said metabolic health. As generally outlined herein, TILs are generally harvested from a patient sample and manipulated to expand their numbers prior to transplantation into a patient. In some embodiments, TILs may optionally be genetically engineered, as discussed below.

[0443] In some embodiments, TILs can be cryopreserved, and upon thawing, they can be restimulated to enhance their metabolism before infusion into patients.

[0444] In some embodiments, as discussed in detail below and in the Examples and Figures, the first expansion (including the process referred to as pre-REP and the process shown as step A in FIG. 1 ) is shortened to 3-14 days, and the second expansion (including the process referred to as REP and the process shown as step B in FIG. 1 ) is shortened to 7-14 days. In some embodiments, the first expansion (e.g., the expansion described as step B in FIG. 1 ) is shortened to 11 days, and the second expansion (e.g., the expansion described as step D in FIG. 1 ) is shortened to 11 days. In some embodiments, as discussed in detail below and in the Examples and Figures, the combined first and second expansion (e.g., the expansion described as steps B and D in FIG. 1 ) is shortened to 22 days.

[0445] The "step" designations A, B, C, etc. below refer to Figure 1 and to certain specific embodiments described herein. The order of steps below and in Figure 1 is exemplary, and any combination or order of steps, as well as additional steps, repeated steps, and / or omission of steps, are contemplated by the present application and methods disclosed herein.

[0446] A. Step A: Obtaining a patient tumor sample Generally, TILs are initially obtained from a patient's tumor sample ("primary TILs") and then expanded into larger populations for further manipulation as described herein, optionally cryopreserved, restimulated as outlined herein, and optionally assessed for phenotypic and metabolic parameters as indicators of TIL health.

[0447] Patient tumor samples can be obtained using methods known in the art, generally via surgical resection, needle biopsy, core biopsy, mini-biopsy, or other means for obtaining a sample containing a mixture of tumor cells and TIL cells. In some embodiments, multi-lesion sampling is used. In some embodiments, surgical resection, needle biopsy, core biopsy, mini-biopsy, or other means for obtaining a sample containing a mixture of tumor cells and TIL cells includes multi-lesion sampling (i.e., obtaining samples from one or more tumor sites and / or locations in a patient, as well as one or more tumors at the same or adjacent locations). Generally, tumor samples can be derived from any solid tumor, including primary tumors, invasive tumors, or metastatic tumors. Tumor samples can be liquid tumors, such as tumors obtained from hematological malignancies. Solid tumors can be of lung tissue. In some embodiments, useful TILs are obtained from non-small cell lung cancer (NSCLC). Solid tumors can be of skin tissue. In some embodiments, useful TILs are obtained from melanoma.

[0448] Once obtained, tumor samples are generally cut into sections of 1 to approximately 8 mm using sharp dissection. 3 fragmented into small pieces of about 2-3 mm 3are particularly useful. In some embodiments, TILs are cultured from these fragments using an enzymatic tumor digest. Such a tumor digest can be produced by incubation in an enzymatic medium (e.g., Roswell Park Memorial Institute (RPMI) 1640 buffer, 2 mM glutamic acid, 10 mcg / mL gentamicin, 30 units / mL DNase, and 1.0 mg / mL collagenase) followed by mechanical dissociation (e.g., using a tissue dissociator). Tumor digests can be produced by placing the tumor in the enzymatic medium, mechanically dissociating the tumor for approximately 1 minute, followed by incubation at 37°C in 5% CO2 for 30 minutes, and then repeating cycles of mechanical dissociation and incubation under the aforementioned conditions until only small tissue fragments are present. At the end of this process, if the cell suspension contains a large number of red blood cells or dead cells, density gradient separation using FICOLL branched hydrophilic polysaccharides can be performed to remove these cells. Alternative methods known in the art can be used, such as those described in U.S. Patent Application Publication No. 2012 / 0244133A1, the disclosure of which is incorporated herein by reference. Any of the foregoing methods can be used in any of the embodiments described herein for methods of expanding TILs or methods of treating cancer.

[0449] The tumor dissociation enzyme mixture can include one or more dissociation (digestion) enzymes, such as, but not limited to, collagenase (including any blend or type of collagenase), Accutase™, Accumax™, hyaluronidase, neutral protease (dispase), chymotrypsin, chymopapain, trypsin, caseinase, elastase, papain, type XIV protease (pronase), deoxyribonuclease I (DNase), trypsin inhibitor, any other dissociation or proteolytic enzyme, and any combination thereof. In some embodiments, the dissociation enzyme is reconstituted from lyophilized enzyme. In some embodiments, the lyophilized enzyme is reconstituted with a volume of sterile buffer, such as HBSS. In some cases, collagenase (such as animal-free type 1 collagenase) is reconstituted with 10 mL of sterile HBSS or another buffer. The lyophilized stock enzyme can be at a concentration of 2892 PZ U / vial. In some embodiments, collagenase is reconstituted in 5 mL to 15 mL of buffer. In some embodiments, the collagenase stock after reconstitution is in the range of about 100 U / mL to about 400 U / mL, e.g., about 100 U / mL to about 400 U / mL, about 100 U / mL to about 350 U / mL, about 100 U / mL to about 300 U / mL, about 150 U / mL to about 400 U / mL, about 100 U / mL, about 150 U / mL, about 200 U / mL, about 210 U / mL, about 220 U / mL, about 230 U / mL, about 240 U / mL, about 250 U / mL, about 260 U / mL, about 270 U / mL, about 280 U / mL, about 289.2 U / mL, about 300 U / mL, U / mL, about 350 PZ U / mL, or about 400 PZ U / mL. In some embodiments, the neutral protease is reconstituted in 1 mL of sterile HBSS or another buffer. The lyophilized stock enzyme may be at a concentration of 175 DMC U / vial.In some embodiments, the neutral protease stock after reconstitution is in the range of about 100 DMC / mL to about 400 DMC / mL, e.g., about 100 DMC / mL to about 400 DMC / mL, about 100 DMC / mL to about 350 DMC / mL, about 100 DMC / mL to about 300 DMC / mL, about 150 DMC / mL to about 400 DMC / mL, about 100 DMC / mL, about 110 DMC / mL, C / mL, about 120 DMC / mL, about 130 DMC / mL, about 140 DMC / mL, about 150 DMC / mL, about 160 DMC / mL, about 170 DMC / mL, about 175 DMC / mL, about 180 DMC / mL, about 190 DMC / mL, about 200 DMC / mL, about 250 DMC / mL, about 300 DMC / mL, about 350 DMC / mL, or about 400 DMC / mL. In some embodiments, DNAse I is reconstituted in 1 mL of sterile HBSS or another buffer. The lyophilized stock enzyme was at a concentration of 4 KU / vial. In some embodiments, the DNase I stock after reconstitution ranges from about 1 KU / mL to 10 KU / mL, e.g., about 1 KU / mL, about 2 KU / mL, about 3 KU / mL, about 4 KU / mL, about 5 KU / mL, about 6 KU / mL, about 7 KU / mL, about 8 KU / mL, about 9 KU / mL, or about 10 KU / mL. In some embodiments, the enzyme stock is variable and the concentration may need to be determined. In some embodiments, the concentration of the lyophilized stock can be verified. In some embodiments, the final amount of enzyme added to the digest cocktail is adjusted based on the determined stock concentration. In some embodiments, the enzyme mixture comprises about 10.2 ul of neutral protease (0.36 DMC U / mL), 21.3 ul of collagenase (1.2 PZ / mL), and 250 ul of DNAse I (200 U / mL) in about 4.7 ml of sterile HBSS.

[0450] As noted above, in some embodiments, the TILs are derived from solid tumors. In some embodiments, the solid tumor is not fragmented. In some embodiments, the solid tumor is not fragmented and is subjected to enzymatic digestion as a whole tumor. In some embodiments, the tumor is digested in an enzyme mixture comprising collagenase, DNase, and hyaluronidase. In some embodiments, the tumor is digested in an enzyme mixture comprising collagenase, DNase, and hyaluronidase for 1-2 hours. In some embodiments, the tumor is digested in an enzyme mixture comprising collagenase, DNase, and hyaluronidase for 1-2 hours at 37°C and 5% CO2. In some embodiments, the tumor is digested in an enzyme mixture comprising collagenase, DNase, and hyaluronidase for 1-2 hours at 37°C and 5% CO2 with rotation. In some embodiments, the tumor is digested overnight with constant rotation. In some embodiments, the tumor is digested overnight at 37°C and 5% CO2 with constant rotation. In some embodiments, the whole tumor is combined with the enzymes to form a tumor digestion reaction mixture.

[0451] In some embodiments, the tumor is reconstituted with lyophilized enzyme in a sterile buffer. In some embodiments, the buffer is sterile HBSS.

[0452] In some embodiments, the enzyme mixture includes collagenase. In some embodiments, the collagenase is collagenase IV. In some embodiments, the working stock of collagenase is a 10x working stock of 100 mg / mL.

[0453] In some embodiments, the enzyme mixture includes DNAse. In some embodiments, the working stock of DNAse is a 10x working stock of 10,000 IU / mL.

[0454] In some embodiments, the enzyme mixture comprises hyaluronidase. In some embodiments, the working stock of hyaluronidase is a 10x working stock of 10 mg / mL.

[0455] In some embodiments, the enzyme mixture comprises 10 mg / mL collagenase, 1000 IU / mL DNAse, and 1 mg / mL hyaluronidase.

[0456] In some embodiments, the enzyme mixture comprises 10 mg / mL collagenase, 500 IU / mL DNAse, and 1 mg / mL hyaluronidase.

[0457] Generally, the harvested cell suspension is referred to as a "primary cell population" or "freshly harvested" cell population.

[0458] In some embodiments, fragmentation comprises physical fragmentation, including, for example, dissection and digestion. In some embodiments, fragmentation is physical fragmentation. In some embodiments, fragmentation is dissection. In some embodiments, fragmentation is by digestion. In some embodiments, TILs may be initially cultured from enzymatic tumor digests and tumor fragments obtained from digesting or fragmenting a tumor sample obtained from a patient.

[0459] In some embodiments where the tumor is a solid tumor, after a tumor sample is obtained, for example, in step A (provided in FIG. 1), the tumor undergoes physical fragmentation. In some embodiments, fragmentation occurs before cryopreservation. In some embodiments, fragmentation occurs after cryopreservation. In some embodiments, fragmentation occurs after obtaining the tumor in the absence of any cryopreservation. In some embodiments, the tumor is fragmented and 10, 20, 30, 40 or more fragments or pieces are placed in each container for the first expansion. In some embodiments, the tumor is fragmented and 30 or 40 fragments or pieces are placed in each container for the first expansion. In some embodiments, the tumor is fragmented and 40 fragments or pieces are placed in each container for the first expansion. In some embodiments, the plurality of fragments comprises about 4 to about 50 fragments, each fragment being about 27 mm. 3 In some embodiments, the plurality of pieces has a volume of about 1300 mm 3~approx. 1500mm 3 In some embodiments, the plurality of pieces comprises about 30 to about 60 pieces with a total volume of about 1350 mm 3 In some embodiments, the plurality of fragments comprises about 50 fragments with a total mass of about 1 gram to about 1.5 grams. In some embodiments, the plurality of fragments comprises about 4 fragments.

[0460] In some embodiments, the TILs are obtained from tumor fragments. In some embodiments, the tumor fragments are obtained by sharp dissection. In some embodiments, the tumor fragments are approximately 1 mm 3 ~10mm 3 In some embodiments, the tumor fragment is about 1 mm 3 ~8mm 3 In some embodiments, the tumor fragment is about 1 mm 3 In some embodiments, the tumor fragment is about 2 mm 3 In some embodiments, the tumor fragment is about 3 mm 3 In some embodiments, the tumor fragment is about 4 mm 3 In some embodiments, the tumor fragment is about 5 mm 3 In some embodiments, the tumor fragment is about 6 mm 3 In some embodiments, the tumor fragment is about 7 mm 3 In some embodiments, the tumor fragment is about 8 mm 3 In some embodiments, the tumor fragment is about 9 mm 3 In some embodiments, the tumor fragment is about 10 mm 3 In some embodiments, the tumor is 1-4 mm x 1-4 mm x 1-4 mm. In some embodiments, the tumor is 1 mm x 1 mm x 1 mm. In some embodiments, the tumor is 2 mm x 2 mm x 2 mm. In some embodiments, the tumor is 3 mm x 3 mm x 3 mm. In some embodiments, the tumor is 4 mm x 4 mm x 4 mm.

[0461] In some embodiments, the tumor is resected to minimize the amount of hemorrhagic, necrotic, and / or fatty tissue on each piece. In some embodiments, the tumor is resected to minimize the amount of hemorrhagic tissue on each piece. In some embodiments, the tumor is resected to minimize the amount of necrotic tissue on each piece. In some embodiments, the tumor is resected to minimize the amount of fatty tissue on each piece.

[0462] In some embodiments, tumor fragmentation is performed to maintain the internal structure of the tumor. In some embodiments, tumor fragmentation is performed without sawing with a scalpel. In some embodiments, TILs are obtained from tumor digests. In some embodiments, tumor digests are generated by incubation in an enzyme medium, such as, but not limited to, RPMI 1640 (2 mM GlutaMAX, 10 mg / mL gentamicin, 30 U / mL DNase, and 1.0 mg / mL collagenase), followed by mechanical dissociation (GentleMACS, Miltenyi Biotec, Auburn, CA). After placing the tumor in the enzyme medium, the tumor may be mechanically dissociated for approximately 1 minute. The solution may then be incubated at 37°C in 5% CO2 for 30 minutes, after which it may be mechanically disrupted again for approximately 1 minute. After again incubating at 37°C in 5% CO2 for 30 minutes, the tumor may be mechanically disrupted a third time for approximately 1 minute. In some embodiments, if large tissue debris was present, one or two additional rounds of mechanical dissociation were applied to the sample after the third mechanical disruption, with incubation for an additional 30 minutes at 37° C. in 5% CO In some embodiments, if the cell suspension contains a large number of red blood cells or dead cells, density gradient separation using Ficoll can be performed at the end of the final incubation to remove these cells.

[0463] In some embodiments, the cell suspension harvested prior to the first expansion step is referred to as a "primary cell population" or "freshly harvested" cell population.

[0464] In some embodiments, the cells may optionally be frozen after sampling and cryopreserved before undergoing expansion as described in step B, which is described in more detail below and also illustrated in Figures 1 and 8.

[0465] 1. Pleural fluid T cells and TILs In some embodiments, the sample is a pleural fluid sample. In some embodiments, the source of T cells or TILs for expansion by the processes described herein is a pleural fluid sample. In some embodiments, the sample is a pleural fluid-derived sample. In some embodiments, the source of TILs for expansion by the processes described herein is a pleural fluid-derived sample. See, e.g., the methods described in U.S. Patent Publication No. 2014 / 0295426, which is incorporated by reference in its entirety for all purposes.

[0466] In some embodiments, any pleural fluid or pleural effusion that appears to be and / or contains TILs can be utilized. Such samples can be derived from primary or metastatic lung cancer, such as NSCLC or SCLC. In some embodiments, samples can be derived from secondary metastatic cancer cells from another organ, such as the breast, ovary, colon, or prostate. In some embodiments, the sample used in the expansion methods described herein is a pleural effusion. In some embodiments, the sample used in the expansion methods described herein is a pleural transudate. Other biological samples can include other serous fluids containing TILs, including, for example, ascites from the abdomen or pancreatic cyst fluid. Ascites and pleural fluids have very similar chemical systems, and both the abdomen and lungs have mesothelial lines and fluid forms in the pleural and abdominal spaces of the same material in malignant tumors; in some embodiments, such fluids contain TILs. In some embodiments in which the disclosed methods utilize pleural effusion, the same methods can be performed with similar results using other cyst fluids containing TILs.

[0467] In some embodiments, the pleural fluid is in an unprocessed form removed directly from the patient. In some embodiments, the unprocessed pleural fluid is placed in a standard collection tube, such as an EDTA or heparin tube, before further processing steps. In some embodiments, the unprocessed pleural fluid is placed in a standard CellSave® tube (Veridex) before further processing steps. In some embodiments, the sample is placed in a CellSave tube immediately after collection from the patient to avoid a decrease in the number of viable TILs. The number of viable TILs can decrease significantly within 24 hours if left in unprocessed pleural fluid, even at 4°C. In some embodiments, the sample is placed in an appropriate collection tube within 1 hour, 5 hours, 10 hours, 15 hours, or up to 24 hours after removal from the patient. In some embodiments, the sample is placed in an appropriate collection tube at 4°C within 1 hour, 5 hours, 10 hours, 15 hours, or up to 24 hours after removal from the patient.

[0468] In some embodiments, the pleural fluid sample from a selected subject may be diluted. In some embodiments, the dilution is 1:10 pleural fluid to diluent. In some embodiments, the dilution is 1:9 pleural fluid to diluent. In some embodiments, the dilution is 1:8 pleural fluid to diluent. In some embodiments, the dilution is 1:5 pleural fluid to diluent. In some embodiments, the dilution is 1:2 pleural fluid to diluent. In some embodiments, the dilution is 1:1 pleural fluid to diluent. In some embodiments, the diluent includes saline, phosphate-buffered saline, another buffer, or a physiologically acceptable diluent. In some embodiments, the sample is placed into a CellSave tube immediately after collection from the patient and diluted to avoid a significant loss of viable TILs, which can occur within 24 to 48 hours if left in unprocessed pleural fluid, even at 4°C. In some embodiments, the pleural fluid sample is placed into a suitable collection tube within 1 hour, 5 hours, 10 hours, 15 hours, 24 hours, 36 hours, or up to 48 hours after removal from the patient and dilution. In some embodiments, the pleural fluid sample is placed into a suitable collection tube within 1 hour, 5 hours, 10 hours, 15 hours, 24 hours, 36 hours, or up to 48 hours after removal from the patient and dilution at 4° C.

[0469] In yet other embodiments, the pleural fluid sample is concentrated by conventional means prior to further processing steps. In some embodiments, this pretreatment of the pleural fluid is preferred in situations where the pleural fluid must be frozen for transport to the laboratory where the method will be performed or for subsequent analysis (e.g., more than 24-48 hours after collection). In some embodiments, the pleural fluid sample is prepared by centrifuging the pleural fluid sample after it is collected from the subject and resuspending the centrate or pellet in a buffer. In some embodiments, the pleural fluid sample is subjected to multiple centrifugations and resuspensions before being frozen for transport or subsequent analysis and / or processing.

[0470] In some embodiments, the pleural fluid sample is concentrated prior to further processing steps by using a filtration method. In some embodiments, the pleural fluid sample used in further processing steps is prepared by filtering the fluid through a filter containing a known, essentially uniform pore size that allows pleural fluid to pass through the membrane but retains tumor cells. In some embodiments, the membrane pore diameter can be at least 4 μM. In other embodiments, the pore diameter can be 5 μM or greater, and in other embodiments, 6, 7, 8, 9, or 10 μM. After filtration, the TIL-containing cells retained by the membrane can be rinsed from the membrane into an appropriate physiologically acceptable buffer. The thus-enriched TIL-containing cells can then be used in further processing steps of the method.

[0471] In some embodiments, a pleural fluid sample (e.g., including unprocessed pleural fluid), diluted pleural fluid, or a resuspended cell pellet is contacted with a lysis reagent that specifically lyses nonnucleated red blood cells present in the sample. In some embodiments, this step is performed before further processing steps in situations where the pleural fluid contains a significant number of RBCs. Suitable lysis reagents include a single lysis reagent or a lysis reagent and a quenching reagent, or a lysis agent, a quenching reagent, and a fixation reagent. Suitable lysis systems are commercially available and include the BD Pharm Lyse™ system (Becton Dickenson). Other lysis systems include the Versalyse™ system, the FACSlyse™ system (Becton Dickenson), the Immunoprep™ system, or the Erythrolyse II system (Beckman Coulter, Inc.), or an ammonium chloride system. In some embodiments, the lysis reagent can vary depending on key requirements, such as efficient lysis of red blood cells and preservation of TILs and their phenotypic characteristics in the pleural fluid. In addition to utilizing a single reagent for lysis, lysis systems useful in the methods described herein can include a second reagent, e.g., one that quenches or delays the effect of the lysis reagent during the remaining steps of the method, such as Stabilyse™ reagent (Beckman Coulter, Inc.) Depending on the choice of lysis reagent or the preferred implementation of the method, conventional fixative reagents can also be used.

[0472] In some embodiments, pleural fluid samples that have been unprocessed, diluted, or multiple centrifuged or processed as described herein are stored frozen at a temperature of about -140°C before being further processed and / or expanded as provided herein.

[0473] B. Step B: First Expansion In some embodiments, the methods provide for obtaining young TILs that are capable of increasing their replication cycle upon administration to a subject / patient and thus may provide additional therapeutic benefit over older TILs (i.e., TILs that have undergone more rounds of replication prior to administration to a subject / patient). The characteristics of young TILs are described in the literature, for example, Donia, et al., Scand. J. Immunol. 2012, 75, 157-167, Dudley, et al., Clin. al.,Clin.Cancer Res.2013,19,OF1-OF9, Besser,et al.,J.Immunother.2009,32:415-423,Robbins,et al.,J.Immunol.2004,173,7125-7130,Shen,et al. al.,J.Immunother.,2007,30,123-129, Zhou,et al., J. Immunother. 2005, 28, 53-62, and Tran, et al., J. Immunother., 2008, 31, 742-751, the disclosures of each of which are incorporated herein by reference.

[0474] The diverse antigen receptors of T and B lymphocytes are produced by somatic recombination of a limited number of gene segments. These gene segments: V (variable), D (diversity), J (joining), and C (constant) determine the binding specificity and downstream applications of immunoglobulins and T cell receptors (TCRs). The present invention provides methods for generating TILs that exhibit and increase T cell repertoire diversity. In some embodiments, TILs obtained by the present methods exhibit increased T cell repertoire diversity. In some embodiments, TILs obtained by the present methods exhibit increased T cell repertoire diversity compared to freshly harvested TILs and / or TILs prepared using methods other than those provided herein, including, for example, methods other than those embodied in FIG. 1. In some embodiments, TILs obtained by the present methods exhibit increased T cell repertoire diversity compared to freshly harvested TILs and / or TILs prepared using a method designated Process 1C as illustrated in FIG. 5 and / or FIG. 6. In some embodiments, the TILs obtained in the first expansion exhibit increased T cell repertoire diversity. In some embodiments, the increased diversity is increased immunoglobulin diversity and / or T cell receptor diversity. In some embodiments, the diversity is in immunoglobulins and in immunoglobulin heavy chains. In some embodiments, the diversity is in immunoglobulins and in immunoglobulin light chains. In some embodiments, the diversity is in T cell receptors. In some embodiments, the diversity is in one of the T cell receptors selected from the group consisting of alpha, beta, gamma, and delta receptors. In some embodiments, expression of T cell receptor (TCR) alpha and / or beta is increased. In some embodiments, expression of T cell receptor (TCR) alpha is increased. In some embodiments, expression of T cell receptor (TCR) beta is increased. In some embodiments, expression of TCRab (i.e., TCRα / β) is increased.

[0475] For example, after dissection or digestion of tumor fragments, such as those described in step A of Figure 1, the resulting cells are cultured in serum containing IL-2 under conditions that favor the growth of TILs over tumor and other cells. In some embodiments, tumor digests are incubated in 2 mL wells in medium containing inactivated human AB serum with 6000 IU / mL of IL-2. This primary cell population is cultured for several days, generally 3-14 days, resulting in a bulk TIL population, generally about 1 x 10 8 In some embodiments, this primary cell population is cultured for 7-14 days, resulting in a bulk TIL population, generally about 1 x 10 8 In some embodiments, this primary cell population is cultured for 10-14 days, resulting in a bulk TIL population, generally about 1 x 10 8 In some embodiments, this primary cell population is cultured for about 11 days, resulting in a bulk TIL population, generally about 1 x 10 8 Generate bulk TIL cells.

[0476] In a preferred embodiment, TIL expansion can be performed using an initial bulk TIL expansion step (e.g., such as that described in step B of FIG. 1, which may include a process referred to as pre-REP) as described below and herein, followed by step D below and a second expansion as described herein (step D, which includes a process referred to as the rapid expansion protocol (REP) step), followed by optional cryopreservation, followed by a second step D below and described herein (which includes a process referred to as the restimulation REP step). TILs obtained from this process can optionally be characterized for phenotypic characteristics and metabolic parameters as described herein.

[0477] In embodiments in which TIL cultures are initiated in 24-well plates, e.g., using Costar 24-well cell culture clusters, flat bottom (Corning Incorporated, Corning, NY), 1 x 10 cells are cultured in 2 mL of complete medium (CM) containing IL-2 (6000 IU / mL, Chiron Corp., Emeryville, CA).6 Tumor digested cells or one tumor fragment can be seeded into each well. In some embodiments, the tumor fragment is approximately 1 mm 3 ~10mm 3 is.

[0478] In some embodiments, the first expansion culture medium is referred to as "CM," an abbreviation for culture medium. In some embodiments, the CM in step B consists of RPMI 1640 with GlutaMAX, supplemented with 10% human AB serum, 25 mM Hepes, and 10 mg / mL gentamicin. When the culture is in a 40 mL volume and 10 cm 2 In embodiments initiated in gas-permeable flasks with gas-permeable silicon bottoms (e.g., G-REX10, Wilson Wolf Manufacturing, New Brighton, MN), each flask contains 10-40 x 10 cells in 10-40 mL of CM with IL-2. 6 Live tumor cells or 5–30 tumor fragments were loaded. Both G-REX10 and 24-well plates were incubated in a humidified incubator at 37°C with 5% CO2. Five days after the start of culture, half of the medium was removed and replaced with fresh CM and IL-2. After five days, half of the medium was replaced every 2–3 days.

[0479] After preparation of tumor fragments, the resulting cells (i.e., fragments) are cultured in serum containing IL-2 under conditions that favor the growth of TILs over tumor and other cells. In some embodiments, tumor digests are incubated in 2 mL wells (or in some cases, in the presence of an APC cell population, as outlined herein) in medium containing inactivated human AB serum with 6000 IU / mL of IL-2. This primary cell population is cultured for several days, generally 10-14 days, resulting in a bulk TIL population, generally about 1 x 10 8In some embodiments, the growth medium during the first expansion contains IL-2 or a variant thereof. In some embodiments, the IL is recombinant human IL-2 (rhIL-2). In some embodiments, the IL-2 stock solution contains 20-30 x 10 cells per 1 mg vial. 6 IU / mg specific activity. In some embodiments, the IL-2 stock solution contains 20×10 6 IU / mg specific activity. In some embodiments, the IL-2 stock solution contains 25×10 6 IU / mg specific activity. In some embodiments, the IL-2 stock solution contains 30×10 6 IU / mg specific activity. In some embodiments, the IL-2 stock solution contains 4-8 x 10 6 In some embodiments, the IL-2 stock solution has a final concentration of 5-7 x 10 IU / mg IL-2. 6 In some embodiments, the IL-2 stock solution has a final concentration of 6×10 IU / mg of IL-2. 6IU / mg IL-2. In some embodiments, the IL-2 stock solution is prepared as described in Example 5. In some embodiments, the first expansion culture medium contains about 10,000 IU / mL IL-2, about 9,000 IU / mL IL-2, about 8,000 IU / mL IL-2, about 7,000 IU / mL IL-2, about 6,000 IU / mL IL-2, or about 5,000 IU / mL IL-2. In some embodiments, the first expansion culture medium contains about 9,000 IU / mL to about 5,000 IU / mL IL-2. In some embodiments, the first expansion culture medium contains about 8,000 IU / mL to about 6,000 IU / mL IL-2. In some embodiments, the first expansion culture medium comprises about 7,000 IU / mL to about 6,000 IU / mL of IL-2. In some embodiments, the first expansion culture medium comprises about 6,000 IU / mL of IL-2. In some embodiments, the cell culture medium further comprises IL-2. In some embodiments, the cell culture medium comprises about 3000 IU / mL of IL-2. In some embodiments, the cell culture medium further comprises IL-2. In some embodiments, the cell culture medium comprises about 3000 IU / mL of IL-2. In some embodiments, the cell culture medium comprises about 1000 IU / mL, about 1500 IU / mL, about 2000 IU / mL, about 2500 IU / mL, about 3000 IU / mL, about 3500 IU / mL, about 4000 IU / mL, about 4500 IU / mL, about 5000 IU / mL, about 5500 IU / mL, about 6000 IU / mL, about 6500 IU / mL, about 7000 IU / mL, about 7500 IU / mL, or about 8000 IU / mL of IL-2. In some embodiments, the cell culture medium contains 1000-2000 IU / mL, 2000-3000 IU / mL, 3000-4000 IU / mL, 4000-5000 IU / mL, 5000-6000 IU / mL, 6000-7000 IU / mL, 7000-8000 IU / mL, or about 8000 IU / mL of IL-2.

[0480] In some embodiments, the first expansion culture medium contains about 500 IU / mL IL-15, about 400 IU / mL IL-15, about 300 IU / mL IL-15, about 200 IU / mL IL-15, about 180 IU / mL IL-15, about 160 IU / mL IL-15, about 140 IU / mL IL-15, about 120 IU / mL IL-15, or about 100 IU / mL IL-15. In some embodiments, the first expansion culture medium contains about 500 IU / mL to about 100 IU / mL IL-15. In some embodiments, the first expansion culture medium contains about 400 IU / mL to about 100 IU / mL IL-15. In some embodiments, the first expansion culture medium comprises about 300 IU / mL to about 100 IU / mL of IL-15. In some embodiments, the first expansion culture medium comprises about 200 IU / mL of IL-15. In some embodiments, the cell culture medium comprises about 180 IU / mL of IL-15. In some embodiments, the cell culture medium further comprises IL-15. In some embodiments, the cell culture medium comprises about 180 IU / mL of IL-15.

[0481] In some embodiments, the first expansion culture medium comprises about 20 IU / mL IL-21, about 15 IU / mL IL-21, about 12 IU / mL IL-21, about 10 IU / mL IL-21, about 5 IU / mL IL-21, about 4 IU / mL IL-21, about 3 IU / mL IL-21, about 2 IU / mL IL-21, about 1 IU / mL IL-21, or about 0.5 IU / mL IL-21. In some embodiments, the first expansion culture medium comprises about 20 IU / mL to about 0.5 IU / mL IL-21. In some embodiments, the first expansion culture medium comprises about 15 IU / mL to about 0.5 IU / mL IL-21. In some embodiments, the first expansion culture medium comprises about 12 IU / mL to about 0.5 IU / mL of IL-21. In some embodiments, the first expansion culture medium comprises about 10 IU / mL to about 0.5 IU / mL of IL-21. In some embodiments, the first expansion culture medium comprises about 5 IU / mL to about 1 IU / mL of IL-21. In some embodiments, the first expansion culture medium comprises about 2 IU / mL of IL-21. In some embodiments, the cell culture medium comprises about 1 IU / mL of IL-21. In some embodiments, the cell culture medium comprises about 0.5 IU / mL of IL-21. In some embodiments, the cell culture medium further comprises IL-21. In some embodiments, the cell culture medium comprises about 1 IU / mL of IL-21.

[0482] In some embodiments, the cell culture medium comprises an anti-CD3 agonist antibody, such as an OKT-3 antibody. In some embodiments, the cell culture medium comprises about 30 ng / mL of the OKT-3 antibody. In some embodiments, the cell culture medium comprises about 0.1 ng / mL, about 0.5 ng / mL, about 1 ng / mL, about 2.5 ng / mL, about 5 ng / mL, about 7.5 ng / mL, about 10 ng / mL, about 15 ng / mL, about 20 ng / mL, about 25 ng / mL, about 30 ng / mL, about 35 ng / mL, about 40 ng / mL, about 50 ng / mL, about 60 ng / mL, about 70 ng / mL, about 80 ng / mL, about 90 ng / mL, about 100 ng / mL, about 200 ng / mL, about 500 ng / mL, or about 1 μg / mL of the OKT-3 antibody. In some embodiments, the cell culture medium comprises OKT-3 antibody at concentrations of 0.1 ng / mL to 1 ng / mL, 1 ng / mL to 5 ng / mL, 5 ng / mL to 10 ng / mL, 10 ng / mL to 20 ng / mL, 20 ng / mL to 30 ng / mL, 30 ng / mL to 40 ng / mL, 40 ng / mL to 50 ng / mL, and 50 ng / mL to 100 ng / mL. In some embodiments, the cell culture medium does not contain OKT-3 antibody. In some embodiments, the OKT-3 antibody is muromonab. See, for example, Table 1.

[0483] In some embodiments, the cell culture medium comprises one or more TNFRSF agonists in the cell culture medium. In some embodiments, the TNFRSF agonist comprises a 4-1BB agonist. In some embodiments, the TNFRSF agonist is a 4-1BB agonist, and the 4-1BB agonist is selected from the group consisting of urelumab, utomilumab, EU-101, fusion proteins, and fragments, derivatives, variants, biosimilars, and combinations thereof. In some embodiments, the TNFRSF agonist is added at a concentration sufficient to achieve a cell culture medium concentration of 0.1 μg / mL to 100 μg / mL. In some embodiments, the TNFRSF agonist is added at a concentration sufficient to achieve a cell culture medium concentration of 20 μg / mL to 40 μg / mL.

[0484] In some embodiments, in addition to the one or more TNFRSF agonists, the cell culture medium further comprises IL-2 at an initial concentration of about 3000 IU / mL and OKT-3 at an initial concentration of about 30 ng / mL, and the one or more TNFRSF agonists comprise a 4-1BB agonist.

[0485] In some embodiments, the cell culture medium comprises one or more epigenetic reprogramming agents in the cell culture medium. In some embodiments, the epigenetic reprogramming agent is a DNA hypomethylating agent. In some embodiments, the DNA hypomethylating agent is selected from the group consisting of decitabine, azacitidine, GSK-3484862, RG-108, GSK-3685032, DHAC, SGI-1027, CM-272, zebularine, hinokitiol, guadecitabine, gamma-oryzanol, CM-579, DC-517, 5-fluoro-2′-deoxycytidine, 5-methyldeoxycytidine, DC-05, 6-methyl-5-azacytidine, procainamide, procaine, hydrazine, EGCG, FdCyd, CP-4200, nanomycin A, and pharmaceutically acceptable salts thereof. In some embodiments, the epigenetic reprogramming agent is decitabine.

[0486] In some embodiments, the epigenetic reprogramming agent is a MEK inhibitor. In some embodiments, the MEK inhibitor is selected from the group consisting of trametinib, cobimetinib, binimetinib, selumetinib, PD-325901, CI-1040, TAK-733, GDC-0623, pimasertinib, refametinib, BI-847325, and pharmaceutically acceptable salts thereof. In some embodiments, the epigenetic reprogramming agent is trametinib.

[0487] In some embodiments, the epigenetic reprogramming agent is an HDAC inhibitor. In some embodiments, the HDAC inhibitor is selected from the group consisting of rosirinostat, vorinostat, trichostatin A, belinostat, panabinostat, panobinostat, xinostat, divinostat, resminostat, abexinostat, xinostat, practinostat, CHR-3996, valproic acid, butyric acid, phenylbutyric acid, enethiostat, tacedinaline, mocetinostat, romidespin, nicotinamide, sirtinol, cambinol, EX-527, apicidin, depsipeptide, MS275, BML-210, splitomicin, RGFP966, and pharmaceutically acceptable salts thereof. In some embodiments, the epigenetic reprogramming agent is licorinistat.

[0488] In some embodiments, the epigenetic reprogramming agent is a bromodomain inhibitor. In some embodiments, the bromodomain inhibitor is selected from JQ1, ZEN-3694, I-BET762, OTX015, I-BET151, RVX-208, MS417, ABBV-075, ABBV-744, SJ432, AZD5153, INCB054329, INCB054329, FT-1101, CPI-0610, RO6870810, BAY1238097, RVX000222, and pharmaceutically acceptable salts thereof. In some embodiments, the epigenetic reprogramming agent is JQ1.

[0489] In some embodiments, the epigenetic reprogramming agent is an EZH2 inhibitor, hi some embodiments, the EZH2 inhibitor is selected from the group consisting of 3-deazaneplanocin A, tazemetostat, GSK343, GSK926, GSK126, EPZ005687, and pharmaceutically acceptable salts thereof.

[0490] In some embodiments, the AKT inhibitor is selected from the group consisting of ipatasertib, GSK690693, GSK2141795, GSK2110183, AZD5363, GDC-0068, AT7867, CCT128930, MK-2206, BAY1125976, perifosine, oridonin, herbacetin, teheranolide, isoliquirigenin, scutellarin, honokiol, and pharmaceutically acceptable salts thereof. In some embodiments, the epigenetic reprogramming agent is ipatasertib.

[0491] In some embodiments, the epigenetic reprogramming agent is a TET inhibitor. In some embodiments, the TET inhibitor is selected from the group consisting of C35, Bobcat339, D(R)-2-hydroxyglutarate (D2HG), D(R)-2-hydroxyglutarate (D2HG), and L-2-hydroxyglutarate (L2HG). In some embodiments, the epigenetic reprogramming agent is C35.

[0492] In some embodiments, the epigenetic reprogramming agent is a combination of a DNA hypomethylating agent and a MEK inhibitor. In some embodiments, the MEK inhibitor is selected from the group consisting of trametinib, cobimetinib, binimetinib, selumetinib, PD-325901, CI-1040, TAK-733, GDC-0623, pimasertinib, refametinib, BI-847325, and pharmaceutically acceptable salts thereof. In some embodiments, the MEK inhibitor is trametinib. In some embodiments, the DNA hypomethylating agent is selected from the group consisting of decitabine, azacitidine, GSK-3484862, RG-108, GSK-3685032, DHAC, SGI-1027, CM-272, zebularine, hinokitiol, guadecitabine, gamma-oryzanol, CM-579, DC-517, 5-fluoro-2′-deoxycytidine, 5-methyldeoxycytidine, DC-05, 6-methyl-5-azacytidine, procainamide, procaine, hydrazine, EGCG, FdCyd, CP-4200, nanomycin A, and pharmaceutically acceptable salts thereof. In some embodiments, the DNA hypomethylating agent is decitabine.

[0493] In some embodiments, the epigenetic reprogramming agent is a combination of a DNA hypomethylating agent and an HDAC inhibitor. In some embodiments, the HDAC inhibitor is selected from the group consisting of rosirinostat, vorinostat, trichostatin A, belinstat, panabinostat, panobinostat, xinostat, divinostat, resminostat, abexinostat, xinostat, practinostat, CHR-3996, valproic acid, butyric acid, phenylbutyric acid, entionstat, tacedinaline, mocetinostat, romidespin, nicotinamide, sirtinol, cambinol, EX-527, apicidin, depsipeptide, MS275, BML-210, splitomicin, RGFP966, and pharmaceutically acceptable salts thereof. In some embodiments, the HDAC inhibitor is rosirinostat. In some embodiments, the DNA hypomethylating agent is selected from the group consisting of decitabine, azacitidine, GSK-3484862, RG-108, GSK-3685032, DHAC, SGI-1027, CM-272, zebularine, hinokitiol, guadecitabine, gamma-oryzanol, CM-579, DC-517, 5-fluoro-2′-deoxycytidine, 5-methyldeoxycytidine, DC-05, 6-methyl-5-azacytidine, procainamide, procaine, hydrazine, EGCG, FdCyd, CP-4200, nanomycin A, and pharmaceutically acceptable salts thereof. In some embodiments, the DNA hypomethylating agent is decitabine.

[0494] In some embodiments, the epigenetic reprogramming agent is a combination of a DNA hypomethylating agent and an EZH2 inhibitor, hi some embodiments, the EZH2 inhibitor is selected from the group consisting of 3-deazaneplanocin A, tazemetostat, GSK343, GSK926, GSK126, EPZ005687, and pharmaceutically acceptable salts thereof. In some embodiments, the DNA hypomethylating agent is selected from the group consisting of decitabine, azacitidine, GSK-3484862, RG-108, GSK-3685032, DHAC, SGI-1027, CM-272, zebularine, hinokitiol, guadecitabine, gamma-oryzanol, CM-579, DC-517, 5-fluoro-2′-deoxycytidine, 5-methyldeoxycytidine, DC-05, 6-methyl-5-azacytidine, procainamide, procaine, hydrazine, EGCG, FdCyd, CP-4200, nanomycin A, and pharmaceutically acceptable salts thereof. In some embodiments, the DNA hypomethylating agent is decitabine.

[0495] In some embodiments, the epigenetic reprogramming agent is a combination of a DNA hypomethylating agent and a bromodomain inhibitor. In some embodiments, the bromodomain inhibitor is selected from JQ1, ZEN-3694, I-BET762, OTX015, I-BET151, RVX-208, MS417, ABBV-075, ABBV-744, SJ432, AZD5153, INCB054329, INCB054329, FT-1101, CPI-0610, RO6870810, BAY1238097, RVX000222, and pharmaceutically acceptable salts thereof. In some embodiments, the bromodomain inhibitor is JQ1. In some embodiments, the DNA hypomethylating agent is selected from the group consisting of decitabine, azacitidine, GSK-3484862, RG-108, GSK-3685032, DHAC, SGI-1027, CM-272, zebularine, hinokitiol, guadecitabine, gamma-oryzanol, CM-579, DC-517, 5-fluoro-2′-deoxycytidine, 5-methyldeoxycytidine, DC-05, 6-methyl-5-azacytidine, procainamide, procaine, hydrazine, EGCG, FdCyd, CP-4200, nanomycin A, and pharmaceutically acceptable salts thereof. In some embodiments, the DNA hypomethylating agent is decitabine.

[0496] In some embodiments, the epigenetic reprogramming agent is a combination of a DNA hypomethylating agent and an AKT inhibitor. In some embodiments, the AKT inhibitor is selected from the group consisting of ipatasertib, GSK690693, GSK2141795, GSK2110183, AZD5363, GDC-0068, AT7867, CCT128930, MK-2206, BAY1125976, perifosine, oridonin, herbacetin, teheranolide, isoliquirigenin, scutellarin, honokiol, and pharmaceutically acceptable salts thereof. In some embodiments, the AKT inhibitor is ipatasertib. In some embodiments, the DNA hypomethylating agent is selected from the group consisting of decitabine, azacitidine, GSK-3484862, RG-108, GSK-3685032, DHAC, SGI-1027, CM-272, zebularine, hinokitiol, guadecitabine, gamma-oryzanol, CM-579, DC-517, 5-fluoro-2′-deoxycytidine, 5-methyldeoxycytidine, DC-05, 6-methyl-5-azacytidine, procainamide, procaine, hydrazine, EGCG, FdCyd, CP-4200, nanomycin A, and pharmaceutically acceptable salts thereof. In some embodiments, the DNA hypomethylating agent is decitabine.

[0497] In some embodiments, the epigenetic reprogramming agent is a combination of a DNA hypomethylating agent and a TET inhibitor. In some embodiments, the TET inhibitor is selected from the group consisting of C35, Bobcat339, D(R)-2-hydroxyglutarate (D2HG), D(R)-2-hydroxyglutarate (D2HG), and L-2-hydroxyglutarate (L2HG). In some embodiments, the epigenetic reprogramming agent is C35. In some embodiments, the DNA hypomethylating agent is selected from the group consisting of decitabine, azacitidine, GSK-3484862, RG-108, GSK-3685032, DHAC, SGI-1027, CM-272, zebularine, hinokitiol, guadecitabine, gamma-oryzanol, CM-579, DC-517, 5-fluoro-2′-deoxycytidine, 5-methyldeoxycytidine, DC-05, 6-methyl-5-azacytidine, procainamide, procaine, hydrazine, EGCG, FdCyd, CP-4200, nanomycin A, and pharmaceutically acceptable salts thereof. In some embodiments, the DNA hypomethylating agent is decitabine.

[0498] In some embodiments, the epigenetic reprogramming agent is a combination of a MEK inhibitor and an HDAC inhibitor. In some embodiments, the HDAC inhibitor is selected from the group consisting of rosirinostat, vorinostat, trichostatin A, belinstat, panabinostat, panobinostat, xinostat, divinostat, resminostat, abexinostat, xinostat, practinostat, CHR-3996, valproic acid, butyric acid, phenylbutyric acid, entionstat, tacedinaline, mocetinostat, romidespin, nicotinamide, sirtinol, cambinol, EX-527, apicidin, depsipeptide, MS275, BML-210, splitomicin, RGFP966, and pharmaceutically acceptable salts thereof. In some embodiments, the HDAC inhibitor is rosirinostat. In some embodiments, the MEK inhibitor is selected from the group consisting of trametinib, cobimetinib, binimetinib, selumetinib, PD-325901, CI-1040, TAK-733, GDC-0623, pimasertinib, refametinib, BI-847325, and pharmaceutically acceptable salts thereof. In some embodiments, the MEK inhibitor is trametinib.

[0499] In some embodiments, the epigenetic reprogramming agent is a combination of a MEK inhibitor and an EZH2 inhibitor. In some embodiments, the EZH2 inhibitor is selected from the group consisting of 3-deazaneplanocin A, tazemetostat, GSK343, GSK926, GSK126, EPZ005687, and pharmaceutically acceptable salts thereof. In some embodiments, the MEK inhibitor is selected from the group consisting of trametinib, cobimetinib, binimetinib, selumetinib, PD-325901, CI-1040, TAK-733, GDC-0623, pimasertinib, refametinib, BI-847325, and pharmaceutically acceptable salts thereof. In some embodiments, the MEK inhibitor is trametinib.

[0500] In some embodiments, the epigenetic reprogramming agent is a combination of a MEK inhibitor and a bromodomain inhibitor. In some embodiments, the bromodomain inhibitor is selected from JQ1, ZEN-3694, I-BET762, OTX015, I-BET151, RVX-208, MS417, ABBV-075, ABBV-744, SJ432, AZD5153, INCB054329, INCB054329, FT-1101, CPI-0610, RO6870810, BAY1238097, RVX000222, and pharmaceutically acceptable salts thereof. In some embodiments, the bromodomain inhibitor is JQ1. In some embodiments, the MEK inhibitor is selected from the group consisting of trametinib, cobimetinib, binimetinib, selumetinib, PD-325901, CI-1040, TAK-733, GDC-0623, pimasertinib, refametinib, BI-847325, and pharmaceutically acceptable salts thereof. In some embodiments, the MEK inhibitor is trametinib.

[0501] In some embodiments, the epigenetic reprogramming agent is a combination of a MEK inhibitor and an AKT inhibitor. In some embodiments, the AKT inhibitor is selected from the group consisting of ipatasertib, GSK690693, GSK2141795, GSK2110183, AZD5363, GDC-0068, AT7867, CCT128930, MK-2206, BAY1125976, perifosine, oridonin, herbacetin, teheranolide, isoliquirigenin, scutellarin, honokiol, and pharmaceutically acceptable salts thereof. In some embodiments, the AKT inhibitor is ipatasertib. In some embodiments, the MEK inhibitor is selected from the group consisting of trametinib, cobimetinib, binimetinib, selumetinib, PD-325901, CI-1040, TAK-733, GDC-0623, pimasertinib, refametinib, BI-847325, and pharmaceutically acceptable salts thereof. In some embodiments, the MEK inhibitor is trametinib.

[0502] In some embodiments, the epigenetic reprogramming agent is a combination of a MEK inhibitor and a TET inhibitor. In some embodiments, the TET inhibitor is selected from the group consisting of C35, Bobcat339, D(R)-2-hydroxyglutarate (D2HG), D(R)-2-hydroxyglutarate (D2HG), and L-2-hydroxyglutarate (L2HG). In some embodiments, the epigenetic reprogramming agent is C35. In some embodiments, the MEK inhibitor is selected from the group consisting of trametinib, cobimetinib, binimetinib, selumetinib, PD-325901, CI-1040, TAK-733, GDC-0623, pimasertinib, refametinib, BI-847325, and pharmaceutically acceptable salts thereof. In some embodiments, the MEK inhibitor is trametinib.

[0503] In some embodiments, the epigenetic reprogramming agent is a combination of an HDAC inhibitor and an EZH2 inhibitor, hi some embodiments, the EZH2 inhibitor is selected from the group consisting of 3-deazaneplanocin A, tazemetostat, GSK343, GSK926, GSK126, EPZ005687, and pharmaceutically acceptable salts thereof. In some embodiments, the HDAC inhibitor is selected from the group consisting of rosirinostat, vorinostat, trichostatin A, belinstat, panabinostat, panobinostat, xinostat, divinostat, resminostat, abexinostat, xinostat, practinostat, CHR-3996, valproic acid, butyric acid, phenylbutyric acid, encethiostat, tacedinaline, mocetinostat, romidespin, nicotinamide, sirtinol, cambinol, EX-527, apicidin, depsipeptide, MS275, BML-210, splitomicin, RGFP966, and pharmaceutically acceptable salts thereof. In some embodiments, the HDAC inhibitor is rosirinostat.

[0504] In some embodiments, the epigenetic reprogramming agent is a combination of an HDAC inhibitor and a bromodomain inhibitor. In some embodiments, the bromodomain inhibitor is selected from JQ1, ZEN-3694, I-BET762, OTX015, I-BET151, RVX-208, MS417, ABBV-075, ABBV-744, SJ432, AZD5153, INCB054329, INCB054329, FT-1101, CPI-0610, RO6870810, BAY1238097, RVX000222, and pharmaceutically acceptable salts thereof. In some embodiments, the bromodomain inhibitor is JQ1. In some embodiments, the HDAC inhibitor is selected from the group consisting of rosirinostat, vorinostat, trichostatin A, belinstat, panabinostat, panobinostat, xinostat, divinostat, resminostat, abexinostat, xinostat, practinostat, CHR-3996, valproic acid, butyric acid, phenylbutyric acid, encethiostat, tacedinaline, mocetinostat, romidespin, nicotinamide, sirtinol, cambinol, EX-527, apicidin, depsipeptide, MS275, BML-210, splitomicin, RGFP966, and pharmaceutically acceptable salts thereof. In some embodiments, the HDAC inhibitor is rosirinostat.

[0505] In some embodiments, the epigenetic reprogramming agent is a combination of an HDAC inhibitor and an AKT inhibitor. In some embodiments, the AKT inhibitor is selected from the group consisting of ipatasertib, GSK690693, GSK2141795, GSK2110183, AZD5363, GDC-0068, AT7867, CCT128930, MK-2206, BAY1125976, perifosine, oridonin, herbacetin, teheranolide, isoliquirigenin, scutellarin, honokiol, and pharmaceutically acceptable salts thereof. In some embodiments, the AKT inhibitor is ipatasertib. In some embodiments, the HDAC inhibitor is selected from the group consisting of rosirinostat, vorinostat, trichostatin A, belinstat, panabinostat, panobinostat, xinostat, divinostat, resminostat, abexinostat, xinostat, practinostat, CHR-3996, valproic acid, butyric acid, phenylbutyric acid, encethiostat, tacedinaline, mocetinostat, romidespin, nicotinamide, sirtinol, cambinol, EX-527, apicidin, depsipeptide, MS275, BML-210, splitomicin, RGFP966, and pharmaceutically acceptable salts thereof. In some embodiments, the HDAC inhibitor is rosirinostat.

[0506] In some embodiments, the epigenetic reprogramming agent is a combination of an HDAC inhibitor and a TET inhibitor. In some embodiments, the TET inhibitor is selected from the group consisting of C35, Bobcat339, D(R)-2-hydroxyglutarate (D2HG), D(R)-2-hydroxyglutarate (D2HG), and L-2-hydroxyglutarate (L2HG). In some embodiments, the epigenetic reprogramming agent is C35. In some embodiments, the HDAC inhibitor is selected from the group consisting of rosirinostat, vorinostat, trichostatin A, belinstat, panabinostat, panobinostat, xinostat, divinostat, resminostat, abexinostat, xinostat, practinostat, CHR-3996, valproic acid, butyric acid, phenylbutyric acid, encethiostat, tacedinaline, mocetinostat, romidespin, nicotinamide, sirtinol, cambinol, EX-527, apicidin, depsipeptide, MS275, BML-210, splitomicin, RGFP966, and pharmaceutically acceptable salts thereof. In some embodiments, the HDAC inhibitor is rosirinostat.

[0507] In some embodiments, the epigenetic reprogramming agent is a combination of an EZH2 inhibitor and a bromodomain inhibitor. In some embodiments, the bromodomain inhibitor is selected from JQ1, ZEN-3694, I-BET762, OTX015, I-BET151, RVX-208, MS417, ABBV-075, ABBV-744, SJ432, AZD5153, INCB054329, INCB054329, FT-1101, CPI-0610, RO6870810, BAY1238097, RVX000222, and pharmaceutically acceptable salts thereof. In some embodiments, the bromodomain inhibitor is JQ1. In some embodiments, the EZH2 inhibitor is selected from the group consisting of 3-deazaneplanocin A, tazemetostat, GSK343, GSK926, GSK126, EPZ005687, and pharmaceutically acceptable salts thereof.

[0508] In some embodiments, the epigenetic reprogramming agent is a combination of an EZH2 inhibitor and an AKT inhibitor. In some embodiments, the AKT inhibitor is selected from the group consisting of ipatasertib, GSK690693, GSK2141795, GSK2110183, AZD5363, GDC-0068, AT7867, CCT128930, MK-2206, BAY1125976, perifosine, oridonin, herbacetin, teheranolide, isoliquirigenin, scutellarin, honokiol, and pharmaceutically acceptable salts thereof. In some embodiments, the AKT inhibitor is ipatasertib. In some embodiments, the EZH2 inhibitor is selected from the group consisting of 3-deazaneplanocin A, tazemetostat, GSK343, GSK926, GSK126, EPZ005687, and pharmaceutically acceptable salts thereof.

[0509] In some embodiments, the epigenetic reprogramming agent is a combination of an EZH2 inhibitor and a TET inhibitor. In some embodiments, the TET inhibitor is selected from the group consisting of C35, Bobcat339, D(R)-2-hydroxyglutarate (D2HG), D(R)-2-hydroxyglutarate (D2HG), and L-2-hydroxyglutarate (L2HG). In some embodiments, the epigenetic reprogramming agent is C35. In some embodiments, the EZH2 inhibitor is selected from the group consisting of 3-deazaneplanocin A, tazemetostat, GSK343, GSK926, GSK126, EPZ005687, and pharmaceutically acceptable salts thereof.

[0510] In some embodiments, the epigenetic reprogramming agent is a combination of a bromodomain inhibitor and an AKT inhibitor. In some embodiments, the AKT inhibitor is selected from the group consisting of ipatasertib, GSK690693, GSK2141795, GSK2110183, AZD5363, GDC-0068, AT7867, CCT128930, MK-2206, BAY1125976, perifosine, oridonin, herbacetin, teheranolide, isoliquirigenin, scutellarin, honokiol, and pharmaceutically acceptable salts thereof. In some embodiments, the AKT inhibitor is ipatasertib. In some embodiments, the bromodomain inhibitor is selected from JQ1, ZEN-3694, I-BET762, OTX015, I-BET151, RVX-208, MS417, ABBV-075, ABBV-744, SJ432, AZD5153, INCB054329, INCB054329, FT-1101, CPI-0610, RO6870810, BAY1238097, RVX000222, and pharmaceutically acceptable salts thereof. In some embodiments, the bromodomain inhibitor is JQ1.

[0511] In some embodiments, the epigenetic reprogramming agent is a combination of a bromodomain inhibitor and a TET inhibitor. In some embodiments, the TET inhibitor is selected from the group consisting of C35, Bobcat339, D(R)-2-hydroxyglutarate (D2HG), D(R)-2-hydroxyglutarate (D2HG), and L-2-hydroxyglutarate (L2HG). In some embodiments, the epigenetic reprogramming agent is C35. In some embodiments, the bromodomain inhibitor is selected from JQ1, ZEN-3694, I-BET762, OTX015, I-BET151, RVX-208, MS417, ABBV-075, ABBV-744, SJ432, AZD5153, INCB054329, INCB054329, FT-1101, CPI-0610, RO6870810, BAY1238097, RVX000222, and pharmaceutically acceptable salts thereof. In some embodiments, the bromodomain inhibitor is JQ1.

[0512] In some embodiments, the epigenetic reprogramming agent is a combination of an AKT inhibitor and a TET inhibitor. In some embodiments, the TET inhibitor is selected from the group consisting of C35, Bobcat339, D(R)-2-hydroxyglutarate (D2HG), D(R)-2-hydroxyglutarate (D2HG), and L-2-hydroxyglutarate (L2HG). In some embodiments, the epigenetic reprogramming agent is C35. In some embodiments, the AKT inhibitor is selected from the group consisting of ipatasertib, GSK690693, GSK2141795, GSK2110183, AZD5363, GDC-0068, AT7867, CCT128930, MK-2206, BAY1125976, perifosine, oridonin, herbacetin, teheranolide, isoliquirigenin, scutellarin, honokiol, and pharmaceutically acceptable salts thereof. In some embodiments, the AKT inhibitor is ipatasertib.

[0513] In some embodiments, the epigenetic reprogramming agent is a combination of three or more epigenetic reprogramming agents described herein.

[0514] In some embodiments, the epigenetic reprogramming agent may be added at a concentration ranging from about 5 nM to about 5 μM. For example, the concentration of the epigenetic reprogramming agent in the first cell culture medium may be about 5 nM, about 10 nM, about 15 nM, about 20 nM, about 25 nM, about 30 nM, about 35 nM, about 40 nM, about 45 nM, about 50 nM, about 60 nM, about 70 nM, about 80 nM, about 90 nM, about 100 nM, about 110 nM, about 120 nM, about 130 nM, about 140 nM, about 150 nM, about 160 nM, about 170 nM, about 180 nM, about 190 nM, about 200 nM, about 220 nM, about 240 nM, about 260 nM, about 280 nM, about The concentration may be 300 nM, about 320 nM, about 340 nM, about 360 nM, about 380 nM, about 400 nM, about 420 nM, about 440 nM, about 460 nM, about 480 nM, about 500 nM, about 550 nM, about 600 nM, about 650 nM, about 700 nM, about 750 nM, about 800 nM, about 850 nM, about 900 nM, about 950 nM, about 1 μM, about 1.5 μM, about 2 μM, about 2.5 μM, about 3 μM, about 3.5 μM, about 4 μM, about 4.5 μM, about 5 μM, or any other concentration between any two of these values.

[0515] In some embodiments, the epigenetic reprogramming agent may be a combination of two or more epigenetic reprogramming agents. In such embodiments, the two or more epigenetic reprogramming agents may be added at different concentrations. For example, a first epigenetic reprogramming agent may be added at a concentration of 5 nM, and a second epigenetic reprogramming agent may be added at a concentration of 50 nM. In another example, a first epigenetic reprogramming agent may be added at a concentration of 5 nM, a second epigenetic reprogramming agent may be added at a concentration of 50 nM, and a third epigenetic reprogramming agent may be added at a concentration of 100 nM. Other combinations of concentrations of one or more epigenetic reprogramming agents within the concentration ranges described herein are contemplated.

[0516] In some embodiments, the first expansion culture medium is referred to as "CM," an abbreviation for culture medium. In some embodiments, it is referred to as CM1 (culture medium 1). In some embodiments, the CM consists of RPMI 1640 with GlutaMAX, supplemented with 10% human AB serum, 25 mM Hepes, and 10 mg / mL gentamicin. The cultures are grown in a 40 mL volume and 10 cm 2 In embodiments initiated in gas-permeable flasks with gas-permeable silicon bottoms (e.g., G-REX10, Wilson Wolf Manufacturing, New Brighton, MN), 10-40 x 10 cells in 10-40 mL of CM containing IL-2 are cultured. 6Viable tumor digested cells or 5-30 tumor fragments were placed in each flask. Both the G-REX10 and 24-well plates were incubated in a humidified incubator at 37°C with 5% CO2. Five days after the start of culture, half of the medium was removed and replaced with fresh CM and IL-2. After five days, half of the medium was replaced every 2-3 days. In some embodiments, the CM is CM1 as described in the Examples (see Example 1). In some embodiments, the first expansion occurs in the initial cell culture medium or first cell culture medium. In some embodiments, the initial cell culture medium or first cell culture medium contains IL-2.

[0517] In some embodiments, the medium used in the expansion processes disclosed herein is a serum-free or synthetic medium. In some embodiments, the serum-free or synthetic medium comprises a basal cell culture medium and a serum supplement and / or serum replacement. In some embodiments, the serum-free or synthetic medium is used to prevent and / or reduce experimental variation due in part to lot-to-lot variation in serum-containing medium.

[0518] In some embodiments, the serum-free or defined medium comprises a basal cell culture medium and a serum supplement and / or serum replacement. In some embodiments, the basal cell culture medium includes, but is not limited to, CTS™ OpTmizer™ T-cell Expansion Basal Medium, CTS™ OpTmizer™ T-Cell Expansion SFM, CTS™ AIM-V Medium, CTS™ AIM-V SFM, LymphoONE™ T-Cell Expansion Xeno-Free Medium, Dulbecco's Modified Eagle's Medium (DMEM), Minimum Essential Medium (MEM), Basal Eagle's Medium (BME), RPMI 1640, F-10, F-12, Minimum Essential Medium (αMEM), Glasgow Minimum Essential Medium (G-MEM), RPMI Growth Medium, and Iscove's Modified Dulbecco's Medium.

[0519] In some embodiments, the serum supplement or serum replacement includes, but is not limited to, CTS™ OpTmizer T-Cell Expansion Serum Supplement, CTS™ Immune Cell Serum Replacement, one or more albumins or albumin substitutes, one or more amino acids, one or more vitamins, one or more transferrins or transferrin substitutes, one or more antioxidants, one or more insulins or insulin substitutes, one or more collagen precursors, one or more antibiotics, and one or more trace elements. In some embodiments, the synthetic medium contains albumin and one or more of glycine, L-histidine, L-isoleucine, L-methionine, L-phenylalanine, L-proline, L-hydroxyproline, L-serine, L-threonine, L-tryptophan, L-tyrosine, L-valine, thiamine, reduced glutathione, L-ascorbic acid-2-phosphate, iron-saturated transferrin, insulin, and trace element moieties. + , Al 3+ , Ba 2+ , Cd 2+ , Co 2+ , Cr 3+ , Ge 4+ , Se 4+ , Br, T, Mn 2+ , P, Si 4+ , V 5+ , Mo 6+ , Ni 2+ , Rb + , Sn 2+ , and Zr 4+ and one or more components selected from the group consisting of compounds containing: In some embodiments, the synthetic medium further comprises L-glutamine, sodium bicarbonate, and / or 2-mercaptoethanol.

[0520] In some embodiments, CTS™ OpTmizer™ T-cell Immune Cell Serum Replacement is used with conventional growth media, including, but not limited to, CTS™ OpTmizer™ T-cell Expansion Basal Medium, CTS™ OpTmizer™ T-cell Expansion SFM, CTS™ AIM-V Medium, CST™ AIM-V SFM, LymphoONE™ T-Cell Expansion Xeno-Free Medium, Dulbecco's Modified Eagle's Medium (DMEM), Minimum Essential Medium (MEM), Basal Eagle's Medium (BME), RPMI 1640, F-10, F-12, Minimum Essential Medium (αMEM), Glasgow Minimum Essential Medium (G-MEM), RPMI Growth Medium, and Iscove's Modified Dulbecco's Medium.

[0521] In some embodiments, the total serum replacement concentration (vol%) in the serum-free or synthetic medium is about 1%, 2%, 3%, 4%, 5%, 6%, 7%, 8%, 9%, 10%, 11%, 12%, 13%, 14%, 15%, 16%, 17%, 18%, 19%, or 20% by volume of the total serum-free or synthetic medium. In some embodiments, the total serum replacement concentration is about 3% of the total volume of the serum-free or synthetic medium. In some embodiments, the total serum replacement concentration is about 5% of the total volume of the serum-free or synthetic medium. In some embodiments, the total serum replacement concentration is about 10% of the total volume of the serum-free or synthetic medium.

[0522] In some embodiments, the serum-free or defined medium is CTS™ OpTmizer™ T-cell Expansion SFM (ThermoFisher Scientific). Any formulation of CTS™ OpTmizer™ is useful in the present invention. CTS™ OpTmizer™ T-cell Expansion SFM is a combination of 1 L of CTS™ OpTmizer™ T-cell Expansion Basal Medium and 26 mL of CTS™ OpTmizer™ T-Cell Expansion Supplement, which are mixed together before use. In some embodiments, CTS™ OpTmizer™ T-cell Expansion SFM is supplemented with approximately 3% CTS™ Immune Cell Serum Replacement (SR) (ThermoFisher Scientific). In some embodiments, CTS™ OpTmizer™ T-cell Expansion SFM is supplemented with about 3% CTS™ Immune Cell Serum Replacement (SR) (ThermoFisher Scientific) along with 55 mM 2-mercaptoethanol. In some embodiments, CTS™ OpTmizer™ T-cell Expansion SFM is supplemented with about 3% CTS™ Immune Cell Serum Replacement (SR) (ThermoFisher Scientific) along with 55 μM 2-mercaptoethanol.

[0523] In some embodiments, the defined medium is CTS™ OpTmizer™ T-cell Expansion SFM (ThermoFisher Scientific). Any formulation of CTS™ OpTmizer™ is useful in the present invention. CTS™ OpTmizer™ T-cell Expansion SFM is a combination of 1 L of CTS™ OpTmizer™ T-cell Expansion Basal Medium and 26 mL of CTS™ OpTmizer™ T-Cell Expansion Supplement, which are mixed together before use. In some embodiments, CTS™ OpTmizer™ T-cell Expansion SFM is supplemented with approximately 3% CTS™ Immune Cell Serum Replacement (SR) (ThermoFisher Scientific) along with 55 mM 2-mercaptoethanol. In some embodiments, CTS™ OpTmizer™ T-cell Expansion SFM is supplemented with about 3% CTS™ Immune Cell Serum Replacement (SR) (ThermoFisher Scientific), 55 mM 2-mercaptoethanol, and 2 mM L-glutamine. In some embodiments, CTS™ OpTmizer™ T-cell Expansion SFM is supplemented with about 3% CTS™ Immune Cell Serum Replacement (SR) (ThermoFisher Scientific), 55 mM 2-mercaptoethanol, and 2 mM L-glutamine, and further comprises about 1000 IU / mL to about 8000 IU / mL of IL-2. In some embodiments, CTS™ OpTmizer™ T-cell Expansion SFM is supplemented with about 3% CTS™ Immune Cell Serum Replacement (SR) (ThermoFisher Scientific), 55 mM 2-mercaptoethanol, and 2 mM L-glutamine, and further contains about 3000 IU / mL of IL-2.In some embodiments, CTS™ OpTmizer™ T-cell Expansion SFM is supplemented with about 3% CTS™ Immune Cell Serum Replacement (SR) (ThermoFisher Scientific), 55 mM 2-mercaptoethanol, and 2 mM L-glutamine, and further comprises about 6000 IU / mL of IL-2. In some embodiments, CTS™ OpTmizer™ T-cell Expansion SFM is supplemented with about 3% CTS™ Immune Cell Serum Replacement (SR) (ThermoFisher Scientific) and 55 mM 2-mercaptoethanol, and further comprises about 1000 IU / mL to about 8000 IU / mL of IL-2. In some embodiments, CTS™ OpTmizer™ T-cell Expansion SFM is supplemented with about 3% CTS™ Immune Cell Serum Replacement (SR) (ThermoFisher Scientific) and 55 mM 2-mercaptoethanol, and further comprises about 3000 IU / mL of IL-2. In some embodiments, CTS™ OpTmizer™ T-cell Expansion SFM is supplemented with about 3% CTS™ Immune Cell Serum Replacement (SR) (ThermoFisher Scientific) and 55 mM 2-mercaptoethanol, and further comprises about 1000 IU / mL to about 6000 IU / mL of IL-2. In some embodiments, CTS™ OpTmizer™ T-cell Expansion SFM is supplemented with about 3% CTS™ Immune Cell Serum Replacement (SR) (ThermoFisher Scientific) and about 2 mM glutamine, and further contains about 1000 IU / mL to about 8000 IU / mL of IL-2.In some embodiments, CTS™ OpTmizer™ T-cell Expansion SFM is supplemented with about 3% CTS™ Immune Cell Serum Replacement (SR) (ThermoFisher Scientific) and about 2 mM glutamine, and further comprises about 3000 IU / mL of IL-2. In some embodiments, CTS™ OpTmizer™ T-cell Expansion SFM is supplemented with about 3% CTS™ Immune Cell Serum Replacement (SR) (ThermoFisher Scientific) and about 2 mM glutamine, and further comprises about 6000 IU / mL of IL-2. In some embodiments, CTS™ OpTmizer™ T-cell Expansion SFM is supplemented with about 3% CTS™ Immune Cell Serum Replacement (SR) (ThermoFisher Scientific) with 55 μM 2-mercaptoethanol.

[0524] In some embodiments, serum-free or synthetic media is supplemented with glutamine (i.e., GlutaMAX®) at a concentration of about 0.1 mM to about 10 mM, 0.5 mM to about 9 mM, 1 mM to about 8 mM, 2 mM to about 7 mM, 3 mM to about 6 mM, or 4 mM to about 5 mM. In some embodiments, serum-free or synthetic media is supplemented with glutamine (i.e., GlutaMAX®) at a concentration of about 2 mM.

[0525] In some embodiments, the serum-free or synthetic medium is supplemented with 2-mercaptoethanol at a concentration of about 5 mM to about 150 mM, 10 mM to about 140 mM, 15 mM to about 130 mM, 20 mM to about 120 mM, 25 mM to about 110 mM, 30 mM to about 100 mM, 35 mM to about 95 mM, 40 mM to about 90 mM, 45 mM to about 85 mM, 50 mM to about 80 mM, 55 mM to about 75 mM, 60 mM to about 70 mM, or about 65 mM. In some embodiments, the serum-free or synthetic medium is supplemented with 2-mercaptoethanol at a concentration of about 55 mM.

[0526] In some embodiments, the defined media described in International Patent Application Publication No. WO 1998 / 030679 and U.S. Patent Application Publication No. US 2002 / 0076747 A1 (incorporated herein by reference) are useful in the present invention. These publications describe serum-free eukaryotic cell culture media. The serum-free eukaryotic cell culture media include basal cell culture media supplemented with serum-free supplements capable of supporting cell growth in serum-free culture. The serum-free eukaryotic cell culture medium supplement comprises, or is obtained by combining, one or more components selected from the group consisting of one or more albumins or albumin substitutes, one or more amino acids, one or more vitamins, one or more transferrins or transferrin substitutes, one or more antioxidants, one or more insulins or insulin substitutes, one or more collagen precursors, one or more trace elements, and one or more antibiotics. In some embodiments, the defined medium further comprises L-glutamine, sodium bicarbonate, and / or beta-mercaptoethanol. In some embodiments, the synthetic medium comprises albumin or an albumin substitute and one or more components selected from the group consisting of one or more amino acids, one or more vitamins, one or more transferrin or transferrin substitutes, one or more antioxidants, one or more insulin or insulin substitutes, one or more collagen precursors, and one or more trace elements. In some embodiments, the synthetic medium comprises albumin and one or more components selected from the group consisting of glycine, L-histidine, L-isoleucine, L-methionine, L-phenylalanine, L-proline, L-hydroxyproline, L-serine, L-threonine, L-tryptophan, L-tyrosine, L-valine, thiamine, reduced glutathione, L-ascorbic acid-2-phosphate, iron-saturated transferrin, insulin, and the trace element moiety Ag. + , Al 3+ , Ba 2+ , Cd 2+ , Co 2+ , Cr 3+ , Ge 4+ , Se 4+ , Br, T, Mn 2+ , P, Si 4+ , V 5+ , Mo6+ , Ni 2+ , Rb + , Sn 2+ , and Zr 4+ and one or more components selected from the group consisting of compounds containing: In some embodiments, the basal cell culture medium is selected from the group consisting of Dulbecco's Modified Eagle's Medium (DMEM), Minimum Essential Medium (MEM), Basal Eagle's Medium (BME), RPMI 1640, F-10, F-12, Minimum Essential Medium (αMEM), Glasgow Minimum Essential Medium (G-MEM), RPMI Growth Medium, and Iscove's Modified Dulbecco's Medium.

[0527] In some embodiments, the concentration of glycine in the synthetic medium ranges from about 5 to 200 mg / L, the concentration of L-histidine from about 5 to 250 mg / L, the concentration of L-isoleucine from about 5 to 300 mg / L, the concentration of L-methionine from about 5 to 200 mg / L, the concentration of L-phenylalanine from about 5 to 400 mg / L, the concentration of L-proline from about 1 to 1000 mg / L, the concentration of L-hydroxyproline from about 1 to 45 mg / L, the concentration of L-serine from about 1 to 250 mg / L, the concentration of L-threonine from about 10 to 500 mg / L, and the concentration of L-tryptophan from about 2 to 110 mg / L. / L, the concentration of L-tyrosine is about 3 to 175 mg / L, the concentration of L-valine is about 5 to 500 mg / L, the concentration of thiamine is about 1 to 20 mg / L, the concentration of reduced glutathione is about 1 to 20 mg / L, the concentration of L-ascorbic acid 2-phosphate is about 1 to 200 mg / L, the concentration of iron-saturated transferrin is about 1 to 50 mg / L, the concentration of insulin is about 1 to 100 mg / L, the concentration of sodium selenite is about 0.000001 to 0.0001 mg / L, and the concentration of albumin (e.g., AlbuMAX® I) is about 5,000 to 50,000 mg / L.

[0528] In some embodiments, the non-trace element components in the synthetic medium are present at the concentration ranges listed in the column under the heading "Concentration Ranges in 1X Medium" in Table 4 below. In other embodiments, the non-trace element components in the synthetic medium are present at the final concentrations listed in the column under the heading "Some Embodiments of 1X Medium" in Table 4 below. In other embodiments, the synthetic medium is a basal cell culture medium that includes a serum-free supplement. In some of these embodiments, the serum-free supplement includes non-trace element components of the type and concentration listed in the column under the heading "Some Embodiments in Supplement" in Table 4 below.

[0529] [Table 4]

[0530] In some embodiments, the osmolality of the synthetic medium is about 260-350 mOsmol. In some embodiments, the osmolality is about 280-310 mOsmol. In some embodiments, the synthetic medium is supplemented with sodium bicarbonate at up to about 3.7 g / L, or about 2.2 g / L. The synthetic medium may be further supplemented with L-glutamine (final concentration about 2 mM), one or more antibiotics, non-essential amino acids (NEAA, final concentration about 100 μM), and 2-mercaptoethanol (final concentration about 100 μM).

[0531] In some embodiments, the synthetic media described in Smith, et al., ClinTransl. Immunology, 2015, 4(1), e31 (the disclosure of which is incorporated herein by reference) are useful in the present invention. Briefly, RPMI or CTS™ OpTmizer™ was used as the basal cell culture medium, supplemented with either 0, 2%, 5%, or 10% CTS™ Immune Cell Serum Replacement.

[0532] In some embodiments, as discussed in the Examples and Figures, the first expansion (including processes such as those described in step B of FIG. 1, which may include what may be referred to as pre-REP) process is shortened to 3-14 days. In some embodiments, as discussed in the Examples and shown in FIGS. 4 and 5, the first expansion (including processes such as those described in step B of FIG. 1, which may include what may be referred to as pre-REP), including the expansion described in step B of FIG. 1, is shortened to 7-14 days. In some embodiments, the first expansion of step B is shortened to 10-14 days. In some embodiments, the first expansion is shortened to 11 days, as discussed in the expansion described in step B of FIG. 1, for example.

[0533] In some embodiments, the first TIL expansion can continue for 1 day, 2 days, 3 days, 4 days, 5 days, 6 days, 7 days, 8 days, 9 days, 10 days, 11 days, 12 days, 13 days, or 14 days. In some embodiments, the first TIL expansion can continue for 1 day to 14 days. In some embodiments, the first TIL expansion can continue for 2 days to 14 days. In some embodiments, the first TIL expansion can continue for 3 days to 14 days. In some embodiments, the first TIL expansion can continue for 4 days to 14 days. In some embodiments, the first TIL expansion can continue for 5 days to 14 days. In some embodiments, the first TIL expansion can continue for 6 days to 14 days. In some embodiments, the first TIL expansion can continue for 7 days to 14 days. In some embodiments, the first TIL expansion can continue for 8 days to 14 days. In some embodiments, the first TIL expansion can continue for 9 days to 14 days. In some embodiments, the first TIL expansion can continue for 10 to 14 days. In some embodiments, the first TIL expansion can continue for 11 to 14 days. In some embodiments, the first TIL expansion can continue for 12 to 14 days. In some embodiments, the first TIL expansion can continue for 13 to 14 days. In some embodiments, the first TIL expansion can continue for 14 days. In some embodiments, the first TIL expansion can continue for 1 to 11 days. In some embodiments, the first TIL expansion can continue for 2 to 11 days. In some embodiments, the first TIL expansion can continue for 3 to 11 days. In some embodiments, the first TIL expansion can continue for 4 to 11 days. In some embodiments, the first TIL expansion can continue for 5 to 11 days. In some embodiments, the first TIL expansion can continue for 6 to 11 days. In some embodiments, the first TIL expansion can continue for 7 to 11 days. In some embodiments, the first TIL expansion can continue for 8 to 11 days.In some embodiments, the first TIL expansion can continue for 9 to 11 days. In some embodiments, the first TIL expansion can continue for 10 to 11 days. In some embodiments, the first TIL expansion can continue for 11 days.

[0534] In some embodiments, a combination of IL-2, IL-7, IL-15, and / or IL-21 is used as a combination during the first expansion. In some embodiments, IL-2, IL-7, IL-15, and / or IL-21, and any combination thereof, may be included during the first expansion, including, for example, during step B of the process according to Figure 1 and described herein. In some embodiments, a combination of IL-2, IL-15, and IL-21 is used as a combination during the first expansion. In some embodiments, IL-2, IL-15, and IL-21, and any combination thereof, may be included during step B of the process according to Figure 1 and described herein.

[0535] In some embodiments, as discussed in the Examples and Figures, the first expansion (including the process referred to as Pre-REP, e.g., step B according to Figure 1) process is shortened to 3-14 days. In some embodiments, the first expansion of step B is shortened to 7-14 days. In some embodiments, the first expansion of step B is shortened to 10-14 days. In some embodiments, the first expansion is shortened to 11 days.

[0536] In some embodiments, the first expansion, e.g., step B according to FIG. 1, is performed in a closed system bioreactor. In some embodiments, a closed system is used for TIL expansion as described herein. In some embodiments, a single bioreactor is used. In some embodiments, the single bioreactor used is, for example, a G-REX-10 or G-REX-100. In some embodiments, the closed system bioreactor is a single bioreactor.

[0537] 1. Cytokines and other additives The expansion methods described herein generally use culture media containing high doses of cytokines, particularly IL-2, as known in the art.

[0538] Alternatively, the use of cytokine combinations for rapid and / or secondary expansion of TILs is additionally possible using combinations of two or more of IL-2, IL-15, and IL-21, as described in U.S. Patent Application Publication No. US2017 / 0107490A1 (the disclosure of which is incorporated herein by reference). Thus, possible combinations include IL-2 and IL-15, IL-2 and IL-21, IL-15 and IL-21, and IL-2 or IL-15 and IL-21, the latter finding particular use in many embodiments. The use of cytokine combinations is particularly advantageous for the generation of lymphocytes, particularly T cells as described therein.

[0539] In some embodiments, step B may also include the addition of an OKT-3 antibody or muromonab to the culture medium, as described elsewhere herein. In some embodiments, step B may also include the addition of a 4-1BB agonist to the culture medium, as described elsewhere herein. In some embodiments, step B may also include the addition of an OX-40 agonist to the culture medium, as described elsewhere herein. In other embodiments, additives such as peroxisome proliferator-activated receptor gamma coactivator I-alpha agonists, including proliferator-activated receptor (PPAR)-gamma agonists such as thiazolidinedione compounds, may be used in the culture medium during step B, as described in U.S. Patent Application Publication No. US2019 / 0307796 A1 (the disclosure of which is incorporated herein by reference).

[0540] C. Step C: Moving from the first expansion to the second expansion In some cases, the bulk TIL population obtained from the first expansion, including, for example, the TIL population obtained from step B shown in Figure 1, can be immediately cryopreserved using the protocols discussed below. Alternatively, the TIL population obtained from the first expansion, referred to as the second TIL population, can be subjected to a second expansion (which may include an expansion sometimes referred to as REP), as discussed below, and then cryopreserved. Similarly, when genetically modified TILs are used for therapy, the first TIL population (which may be referred to as the bulk TIL population) or the second TIL population (which in some embodiments may include a population referred to as the REP TIL population) can be subjected to genetic modification for the appropriate therapy before expansion or after the first expansion and before the second expansion.

[0541] In some embodiments, TILs obtained from the first expansion (e.g., from step B shown in FIG. 1 ) are stored until phenotyping for selection. In some embodiments, TILs obtained from the first expansion (e.g., from step B shown in FIG. 1 ) are not stored and proceed directly to the second expansion. In some embodiments, TILs obtained from the first expansion are not cryopreserved after the first expansion and before the second expansion. In some embodiments, the transition from the first expansion to the second expansion occurs about 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, or 14 days after fragmentation occurs. In some embodiments, the transition from the first expansion to the second expansion occurs about 3-14 days after fragmentation occurs. In some embodiments, the transition from the first expansion to the second expansion occurs about 4-14 days after fragmentation occurs. In some embodiments, the transition from the first expansion to the second expansion occurs about 4-10 days after fragmentation occurs. In some embodiments, the transition from the first expansion to the second expansion occurs about 7-14 days after fragmentation occurs. In some embodiments, the transition from the first expansion to the second expansion occurs about 14 days after fragmentation occurs.

[0542] In some embodiments, the transition from the first expansion to the second expansion occurs 1 day, 2 days, 3 days, 4 days, 5 days, 6 days, 7 days, 8 days, 9 days, 10 days, 11 days, 12 days, 13 days, or 14 days after fragmentation occurs. In some embodiments, the transition from the first expansion to the second expansion occurs 1 day to 14 days after fragmentation occurs. In some embodiments, the first TIL expansion can continue for 2 days to 14 days. In some embodiments, the transition from the first expansion to the third expansion occurs 2 days to 14 days after fragmentation occurs. In some embodiments, the transition from the first expansion to the second expansion occurs 4 days to 14 days after fragmentation occurs. In some embodiments, the transition from the first expansion to the second expansion occurs 5 days to 14 days after fragmentation occurs. In some embodiments, the transition from the first expansion to the second expansion occurs 6 days to 14 days after fragmentation occurs. In some embodiments, the transition from the first expansion to the second expansion occurs 7 to 14 days after fragmentation occurs. In some embodiments, the transition from the first expansion to the second expansion occurs 8 to 14 days after fragmentation occurs. In some embodiments, the transition from the first expansion to the second expansion occurs 9 to 14 days after fragmentation occurs. In some embodiments, the transition from the first expansion to the second expansion occurs 10 to 14 days after fragmentation occurs. In some embodiments, the transition from the first expansion to the second expansion occurs 11 to 14 days after fragmentation occurs. In some embodiments, the transition from the first expansion to the second expansion occurs 12 to 14 days after fragmentation occurs. In some embodiments, the transition from the first expansion to the second expansion occurs 13 to 14 days after fragmentation occurs. In some embodiments, the transition from the first expansion to the second expansion occurs 14 days after fragmentation occurs. In some embodiments, the transition from the first expansion to the second expansion occurs 1 to 11 days after fragmentation occurs. In some embodiments, the transition from the first expansion to the second expansion occurs 2 to 11 days after fragmentation occurs. In some embodiments, the transition from the first expansion to the second expansion occurs 3 to 11 days after fragmentation occurs.In some embodiments, the transition from the first expansion to the second expansion occurs 4 to 11 days after fragmentation occurs. In some embodiments, the transition from the first expansion to the second expansion occurs 5 to 11 days after fragmentation occurs. In some embodiments, the transition from the first expansion to the second expansion occurs 6 to 11 days after fragmentation occurs. In some embodiments, the transition from the first expansion to the second expansion occurs 7 to 11 days after fragmentation occurs. In some embodiments, the transition from the first expansion to the second expansion occurs 8 to 11 days after fragmentation occurs. In some embodiments, the transition from the first expansion to the second expansion occurs 9 to 11 days after fragmentation occurs. In some embodiments, the transition from the first expansion to the second expansion occurs 10 to 11 days after fragmentation occurs. In some embodiments, the transition from the first expansion to the second expansion occurs 11 days after fragmentation occurs.

[0543] In some embodiments, the second cell population is at least 5 times more numerous than the first TIL population, and the first cell culture medium comprises IL-2. For example, the second cell population can be about 5 times, about 6 times, about 7 times, about 8 times, about 9 times, about 10 times, about 11 times, about 12 times, about 13 times, about 14 times, about 15 times, about 16 times, about 17 times, about 18 times, about 19 times, about 20 times more numerous than the first TIL population.

[0544] In some embodiments, the second TIL population has an increased frequency of CD8 TILs when compared to a corresponding TIL population expanded in cell culture medium without the epigenetic reprogramming agent. Additionally or alternatively, the second TIL population has an increased ratio of CD4 TILs to CD8 TILs when compared to a corresponding TIL population expanded in cell culture medium without the epigenetic reprogramming agent.

[0545] In some embodiments, the TILs are not stored after the first expansion and before the second expansion, and the TILs proceed directly to the second expansion (e.g., in some embodiments, they are not stored during the transition from step B to step D shown in FIG. 1). In some embodiments, the transfer occurs in a closed system as described herein. In some embodiments, the second TIL population, the TILs from the first expansion, proceed directly to the second expansion without a transfer period.

[0546] In some embodiments, the transition from the first expansion to the second expansion, e.g., step C according to FIG. 1, is performed in a closed system bioreactor. In some embodiments, a closed system is used for TIL expansion as described herein. In some embodiments, a single bioreactor is used. In some embodiments, the single bioreactor used is, for example, a G-REX-10 or G-REX-100. In some embodiments, the closed system bioreactor is a single bioreactor.

[0547] D. Step D: Second Expansion In some embodiments, the TIL cell population is expanded in number after harvesting and initial bulk processing, e.g., after steps A and B, and the transition referred to as step C, as shown in Figure 1. This further expansion is referred to herein as secondary expansion, which may include an expansion process commonly referred to in the art as a rapid expansion process (REP), as well as the process shown in Figure 1 at step D. Secondary expansion is generally accomplished in a gas-permeable container using culture medium containing several components, including feeder cells, a cytokine source, and an anti-CD3 antibody.

[0548] In some embodiments, the second expansion or second TIL expansion (which may include expansion sometimes referred to as REP, and the process shown in step D of FIG. 1) may be performed using any TIL flask or vessel known to one of skill in the art. In some embodiments, the second TIL expansion may proceed for 7, 8, 9, 10, 11, 12, 13, or 14 days. In some embodiments, the second TIL expansion may proceed for about 7 days to about 14 days. In some embodiments, the second TIL expansion may proceed for about 8 days to about 14 days. In some embodiments, the second TIL expansion may proceed for about 9 days to about 14 days. In some embodiments, the second TIL expansion may proceed for about 10 days to about 14 days. In some embodiments, the second TIL expansion may proceed for about 11 days to about 14 days. In some embodiments, the second TIL expansion may proceed for about 12 days to about 14 days. In some embodiments, the second TIL expansion can proceed for about 13 to about 14 days. In some embodiments, the second TIL expansion can proceed for about 14 days.

[0549] In some embodiments, the second expansion can be performed in a gas-permeable container using the methods of the present disclosure (e.g., expansion referred to as REP, including the process shown in step D of FIG. 1). For example, TILs can be rapidly expanded using nonspecific T cell receptor stimulation in the presence of interleukin-2 (IL-2) or interleukin-15 (IL-15). Nonspecific T cell receptor stimulation can include, for example, an anti-CD3 antibody, such as about 30 ng / ml OKT3, a mouse monoclonal anti-CD3 antibody (commercially available from Ortho-McNeil, Raritan, NJ or Miltenyi Biotech, Auburn, CA), or UHCT-1 (commercially available from BioLegend, San Diego, CA, USA). TILs can be expanded in vitro to induce further stimulation of TILs by including one or more antigens during the second expansion, including an antigenic moiety, such as a cancer epitope(s), which can be expressed from a vector, e.g., a human leukocyte antigen A2 (HLA-A2)-binding peptide, e.g., 0.3 μM MART-1:26-35(27L) or gpl00:209-217(210M), optionally in the presence of a T cell growth factor, such as 300 IU / mL IL-2 or IL-15. Other suitable antigens can include, e.g., NY-ESO-1, TRP-1, TRP-2, tyrosinase cancer antigen, MAGE-A3, SSX-2, and VEGFR2, or antigenic moieties thereof. TILs can also be rapidly expanded by restimulating with the same cancer antigen(s) pulsed onto antigen-presenting cells expressing HLA-A2. Alternatively, the TILs can be further restimulated, for example, with irradiated autologous lymphocytes or irradiated HLA-A2+ allogeneic lymphocytes and IL-2. In some embodiments, the restimulation occurs as part of a second expansion. In some embodiments, the second expansion occurs in the presence of irradiated autologous lymphocytes or with irradiated HLA-A2+ allogeneic lymphocytes and IL-2.

[0550] In some embodiments, the cell culture medium further comprises IL-2. In some embodiments, the cell culture medium comprises about 3000 IU / mL of IL-2. In some embodiments, the cell culture medium comprises about 1000 IU / mL, about 1500 IU / mL, about 2000 IU / mL, about 2500 IU / mL, about 3000 IU / mL, about 3500 IU / mL, about 4000 IU / mL, about 4500 IU / mL, about 5000 IU / mL, about 5500 IU / mL, about 6000 IU / mL, about 6500 IU / mL, about 7000 IU / mL, about 7500 IU / mL, or about 8000 IU / mL of IL-2. In some embodiments, the cell culture medium contains 1000-2000 IU / mL, 2000-3000 IU / mL, 3000-4000 IU / mL, 4000-5000 IU / mL, 5000-6000 IU / mL, 6000-7000 IU / mL, 7000-8000 IU / mL, or 8000 IU / mL of IL-2.

[0551] In some embodiments, the cell culture medium comprises an OKT-3 antibody. In some embodiments, the cell culture medium comprises about 30 ng / mL of the OKT-3 antibody. In some embodiments, the cell culture medium comprises about 0.1 ng / mL, about 0.5 ng / mL, about 1 ng / mL, about 2.5 ng / mL, about 5 ng / mL, about 7.5 ng / mL, about 10 ng / mL, about 15 ng / mL, about 20 ng / mL, about 25 ng / mL, about 30 ng / mL, about 35 ng / mL, about 40 ng / mL, about 50 ng / mL, about 60 ng / mL, about 70 ng / mL, about 80 ng / mL, about 90 ng / mL, about 100 ng / mL, about 200 ng / mL, about 500 ng / mL, or about 1 μg / mL of the OKT-3 antibody. In some embodiments, the cell culture medium comprises an OKT-3 antibody at a concentration of 0.1 ng / mL to 1 ng / mL, 1 ng / mL to 5 ng / mL, 5 ng / mL to 10 ng / mL, 10 ng / mL to 20 ng / mL, 20 ng / mL to 30 ng / mL, 30 ng / mL to 40 ng / mL, 40 ng / mL to 50 ng / mL, or 50 ng / mL to 100 ng / mL. In some embodiments, the cell culture medium does not contain an OKT-3 antibody. In some embodiments, the OKT-3 antibody is muromonab.

[0552] In some embodiments, the cell culture medium comprises one or more TNFRSF agonists in the cell culture medium. In some embodiments, the TNFRSF agonist comprises a 4-1BB agonist. In some embodiments, the TNFRSF agonist is a 4-1BB agonist, and the 4-1BB agonist is selected from the group consisting of urelumab, utomilumab, EU-101, fusion proteins, and fragments, derivatives, variants, biosimilars, and combinations thereof. In some embodiments, the TNFRSF agonist is added at a concentration sufficient to achieve a cell culture medium concentration of 0.1 μg / mL to 100 μg / mL. In some embodiments, the TNFRSF agonist is added at a concentration sufficient to achieve a cell culture medium concentration of 20 μg / mL to 40 μg / mL.

[0553] In some embodiments, in addition to the one or more TNFRSF agonists, the cell culture medium further comprises IL-2 at an initial concentration of about 3000 IU / mL and OKT-3 at an initial concentration of about 30 ng / mL, and the one or more TNFRSF agonists comprise a 4-1BB agonist.

[0554] In some embodiments, a combination of IL-2, IL-7, IL-15, and / or IL-21 is used as a combination during the second expansion. In some embodiments, IL-2, IL-7, IL-15, and / or IL-21, and any combination thereof, may be included during the second expansion, including, for example, during step D of the process according to Figure 1 and described herein. In some embodiments, a combination of IL-2, IL-15, and IL-21 is used as a combination during the second expansion. In some embodiments, IL-2, IL-15, and IL-21, and any combination thereof, may be included during the process according to Figure 1 and described herein.

[0555] In some embodiments, the second cell culture medium may comprise one or more epigenetic reprogramming agents. In some embodiments, the epigenetic reprogramming agent is a DNA hypomethylating agent. In some embodiments, the DNA hypomethylating agent is selected from the group consisting of decitabine, azacitidine, GSK-3484862, RG-108, GSK-3685032, DHAC, SGI-1027, CM-272, zebularine, hinokitiol, guadecitabine, gamma-oryzanol, CM-579, DC-517, 5-fluoro-2′-deoxycytidine, 5-methyldeoxycytidine, DC-05, 6-methyl-5-azacytidine, procainamide, procaine, hydrazine, EGCG, FdCyd, CP-4200, nanomycin A, and pharmaceutically acceptable salts thereof. In some embodiments, the epigenetic reprogramming agent is decitabine.

[0556] In some embodiments, the epigenetic reprogramming agent is a MEK inhibitor. In some embodiments, the MEK inhibitor is selected from the group consisting of trametinib, cobimetinib, binimetinib, selumetinib, PD-325901, CI-1040, TAK-733, GDC-0623, pimasertinib, refametinib, BI-847325, and pharmaceutically acceptable salts thereof. In some embodiments, the epigenetic reprogramming agent is trametinib.

[0557] In some embodiments, the epigenetic reprogramming agent is an HDAC inhibitor. In some embodiments, the HDAC inhibitor is selected from the group consisting of rosirinostat, vorinostat, trichostatin A, belinostat, panabinostat, panobinostat, xinostat, divinostat, resminostat, abexinostat, xinostat, practinostat, CHR-3996, valproic acid, butyric acid, phenylbutyric acid, enethiostat, tacedinaline, mocetinostat, romidespin, nicotinamide, sirtinol, cambinol, EX-527, apicidin, depsipeptide, MS275, BML-210, splitomicin, RGFP966, and pharmaceutically acceptable salts thereof. In some embodiments, the epigenetic reprogramming agent is licorinistat.

[0558] In some embodiments, the epigenetic reprogramming agent is a bromodomain inhibitor. In some embodiments, the bromodomain inhibitor is selected from JQ1, ZEN-3694, I-BET762, OTX015, I-BET151, RVX-208, MS417, ABBV-075, ABBV-744, SJ432, AZD5153, INCB054329, INCB054329, FT-1101, CPI-0610, RO6870810, BAY1238097, RVX000222, and pharmaceutically acceptable salts thereof. In some embodiments, the epigenetic reprogramming agent is JQ1.

[0559] In some embodiments, the epigenetic reprogramming agent is an EZH2 inhibitor, hi some embodiments, the EZH2 inhibitor is selected from the group consisting of 3-deazaneplanocin A, tazemetostat, GSK343, GSK926, GSK126, EPZ005687, and pharmaceutically acceptable salts thereof.

[0560] In some embodiments, the AKT inhibitor is selected from the group consisting of ipatasertib, GSK690693, GSK2141795, GSK2110183, AZD5363, GDC-0068, AT7867, CCT128930, MK-2206, BAY1125976, perifosine, oridonin, herbacetin, teheranolide, isoliquirigenin, scutellarin, honokiol, and pharmaceutically acceptable salts thereof. In some embodiments, the epigenetic reprogramming agent is ipatasertib.

[0561] In some embodiments, the epigenetic reprogramming agent is a TET inhibitor. In some embodiments, the TET inhibitor is selected from the group consisting of C35, Bobcat339, D(R)-2-hydroxyglutarate (D2HG), D(R)-2-hydroxyglutarate (D2HG), and L-2-hydroxyglutarate (L2HG). In some embodiments, the epigenetic reprogramming agent is C35.

[0562] In some embodiments, the epigenetic reprogramming agent is a combination of a DNA hypomethylating agent and a MEK inhibitor. In some embodiments, the MEK inhibitor is selected from the group consisting of trametinib, cobimetinib, binimetinib, selumetinib, PD-325901, CI-1040, TAK-733, GDC-0623, pimasertinib, refametinib, BI-847325, and pharmaceutically acceptable salts thereof. In some embodiments, the MEK inhibitor is trametinib. In some embodiments, the DNA hypomethylating agent is selected from the group consisting of decitabine, azacitidine, GSK-3484862, RG-108, GSK-3685032, DHAC, SGI-1027, CM-272, zebularine, hinokitiol, guadecitabine, gamma-oryzanol, CM-579, DC-517, 5-fluoro-2′-deoxycytidine, 5-methyldeoxycytidine, DC-05, 6-methyl-5-azacytidine, procainamide, procaine, hydrazine, EGCG, FdCyd, CP-4200, nanomycin A, and pharmaceutically acceptable salts thereof. In some embodiments, the DNA hypomethylating agent is decitabine.

[0563] In some embodiments, the epigenetic reprogramming agent is a combination of a DNA hypomethylating agent and an HDAC inhibitor. In some embodiments, the HDAC inhibitor is selected from the group consisting of rosirinostat, vorinostat, trichostatin A, belinstat, panabinostat, panobinostat, xinostat, divinostat, resminostat, abexinostat, xinostat, practinostat, CHR-3996, valproic acid, butyric acid, phenylbutyric acid, entionstat, tacedinaline, mocetinostat, romidespin, nicotinamide, sirtinol, cambinol, EX-527, apicidin, depsipeptide, MS275, BML-210, splitomicin, RGFP966, and pharmaceutically acceptable salts thereof. In some embodiments, the HDAC inhibitor is rosirinostat. In some embodiments, the DNA hypomethylating agent is selected from the group consisting of decitabine, azacitidine, GSK-3484862, RG-108, GSK-3685032, DHAC, SGI-1027, CM-272, zebularine, hinokitiol, guadecitabine, gamma-oryzanol, CM-579, DC-517, 5-fluoro-2′-deoxycytidine, 5-methyldeoxycytidine, DC-05, 6-methyl-5-azacytidine, procainamide, procaine, hydrazine, EGCG, FdCyd, CP-4200, nanomycin A, and pharmaceutically acceptable salts thereof. In some embodiments, the DNA hypomethylating agent is decitabine.

[0564] In some embodiments, the epigenetic reprogramming agent is a combination of a DNA hypomethylating agent and an EZH2 inhibitor, hi some embodiments, the EZH2 inhibitor is selected from the group consisting of 3-deazaneplanocin A, tazemetostat, GSK343, GSK926, GSK126, EPZ005687, and pharmaceutically acceptable salts thereof. In some embodiments, the DNA hypomethylating agent is selected from the group consisting of decitabine, azacitidine, GSK-3484862, RG-108, GSK-3685032, DHAC, SGI-1027, CM-272, zebularine, hinokitiol, guadecitabine, gamma-oryzanol, CM-579, DC-517, 5-fluoro-2′-deoxycytidine, 5-methyldeoxycytidine, DC-05, 6-methyl-5-azacytidine, procainamide, procaine, hydrazine, EGCG, FdCyd, CP-4200, nanomycin A, and pharmaceutically acceptable salts thereof. In some embodiments, the DNA hypomethylating agent is decitabine.

[0565] In some embodiments, the epigenetic reprogramming agent is a combination of a DNA hypomethylating agent and a bromodomain inhibitor. In some embodiments, the bromodomain inhibitor is selected from JQ1, ZEN-3694, I-BET762, OTX015, I-BET151, RVX-208, MS417, ABBV-075, ABBV-744, SJ432, AZD5153, INCB054329, INCB054329, FT-1101, CPI-0610, RO6870810, BAY1238097, RVX000222, and pharmaceutically acceptable salts thereof. In some embodiments, the bromodomain inhibitor is JQ1. In some embodiments, the DNA hypomethylating agent is selected from the group consisting of decitabine, azacitidine, GSK-3484862, RG-108, GSK-3685032, DHAC, SGI-1027, CM-272, zebularine, hinokitiol, guadecitabine, gamma-oryzanol, CM-579, DC-517, 5-fluoro-2′-deoxycytidine, 5-methyldeoxycytidine, DC-05, 6-methyl-5-azacytidine, procainamide, procaine, hydrazine, EGCG, FdCyd, CP-4200, nanomycin A, and pharmaceutically acceptable salts thereof. In some embodiments, the DNA hypomethylating agent is decitabine.

[0566] In some embodiments, the epigenetic reprogramming agent is a combination of a DNA hypomethylating agent and an AKT inhibitor. In some embodiments, the AKT inhibitor is selected from the group consisting of ipatasertib, GSK690693, GSK2141795, GSK2110183, AZD5363, GDC-0068, AT7867, CCT128930, MK-2206, BAY1125976, perifosine, oridonin, herbacetin, teheranolide, isoliquirigenin, scutellarin, honokiol, and pharmaceutically acceptable salts thereof. In some embodiments, the AKT inhibitor is ipatasertib. In some embodiments, the DNA hypomethylating agent is selected from the group consisting of decitabine, azacitidine, GSK-3484862, RG-108, GSK-3685032, DHAC, SGI-1027, CM-272, zebularine, hinokitiol, guadecitabine, gamma-oryzanol, CM-579, DC-517, 5-fluoro-2′-deoxycytidine, 5-methyldeoxycytidine, DC-05, 6-methyl-5-azacytidine, procainamide, procaine, hydrazine, EGCG, FdCyd, CP-4200, nanomycin A, and pharmaceutically acceptable salts thereof. In some embodiments, the DNA hypomethylating agent is decitabine.

[0567] In some embodiments, the epigenetic reprogramming agent is a combination of a DNA hypomethylating agent and a TET inhibitor. In some embodiments, the TET inhibitor is selected from the group consisting of C35, Bobcat339, D(R)-2-hydroxyglutarate (D2HG), D(R)-2-hydroxyglutarate (D2HG), and L-2-hydroxyglutarate (L2HG). In some embodiments, the epigenetic reprogramming agent is C35. In some embodiments, the DNA hypomethylating agent is selected from the group consisting of decitabine, azacitidine, GSK-3484862, RG-108, GSK-3685032, DHAC, SGI-1027, CM-272, zebularine, hinokitiol, guadecitabine, gamma-oryzanol, CM-579, DC-517, 5-fluoro-2′-deoxycytidine, 5-methyldeoxycytidine, DC-05, 6-methyl-5-azacytidine, procainamide, procaine, hydrazine, EGCG, FdCyd, CP-4200, nanomycin A, and pharmaceutically acceptable salts thereof. In some embodiments, the DNA hypomethylating agent is decitabine.

[0568] In some embodiments, the epigenetic reprogramming agent is a combination of a MEK inhibitor and an HDAC inhibitor. In some embodiments, the HDAC inhibitor is selected from the group consisting of rosirinostat, vorinostat, trichostatin A, belinstat, panabinostat, panobinostat, xinostat, divinostat, resminostat, abexinostat, xinostat, practinostat, CHR-3996, valproic acid, butyric acid, phenylbutyric acid, entionstat, tacedinaline, mocetinostat, romidespin, nicotinamide, sirtinol, cambinol, EX-527, apicidin, depsipeptide, MS275, BML-210, splitomicin, RGFP966, and pharmaceutically acceptable salts thereof. In some embodiments, the HDAC inhibitor is rosirinostat. In some embodiments, the MEK inhibitor is selected from the group consisting of trametinib, cobimetinib, binimetinib, selumetinib, PD-325901, CI-1040, TAK-733, GDC-0623, pimasertinib, refametinib, BI-847325, and pharmaceutically acceptable salts thereof. In some embodiments, the MEK inhibitor is trametinib.

[0569] In some embodiments, the epigenetic reprogramming agent is a combination of a MEK inhibitor and an EZH2 inhibitor. In some embodiments, the EZH2 inhibitor is selected from the group consisting of 3-deazaneplanocin A, tazemetostat, GSK343, GSK926, GSK126, EPZ005687, and pharmaceutically acceptable salts thereof. In some embodiments, the MEK inhibitor is selected from the group consisting of trametinib, cobimetinib, binimetinib, selumetinib, PD-325901, CI-1040, TAK-733, GDC-0623, pimasertinib, refametinib, BI-847325, and pharmaceutically acceptable salts thereof. In some embodiments, the MEK inhibitor is trametinib.

[0570] In some embodiments, the epigenetic reprogramming agent is a combination of a MEK inhibitor and a bromodomain inhibitor. In some embodiments, the bromodomain inhibitor is selected from JQ1, ZEN-3694, I-BET762, OTX015, I-BET151, RVX-208, MS417, ABBV-075, ABBV-744, SJ432, AZD5153, INCB054329, INCB054329, FT-1101, CPI-0610, RO6870810, BAY1238097, RVX000222, and pharmaceutically acceptable salts thereof. In some embodiments, the bromodomain inhibitor is JQ1. In some embodiments, the MEK inhibitor is selected from the group consisting of trametinib, cobimetinib, binimetinib, selumetinib, PD-325901, CI-1040, TAK-733, GDC-0623, pimasertinib, refametinib, BI-847325, and pharmaceutically acceptable salts thereof. In some embodiments, the MEK inhibitor is trametinib.

[0571] In some embodiments, the epigenetic reprogramming agent is a combination of a MEK inhibitor and an AKT inhibitor. In some embodiments, the AKT inhibitor is selected from the group consisting of ipatasertib, GSK690693, GSK2141795, GSK2110183, AZD5363, GDC-0068, AT7867, CCT128930, MK-2206, BAY1125976, perifosine, oridonin, herbacetin, teheranolide, isoliquirigenin, scutellarin, honokiol, and pharmaceutically acceptable salts thereof. In some embodiments, the AKT inhibitor is ipatasertib. In some embodiments, the MEK inhibitor is selected from the group consisting of trametinib, cobimetinib, binimetinib, selumetinib, PD-325901, CI-1040, TAK-733, GDC-0623, pimasertinib, r...

Claims

1. A population of modified tumor-infiltrating lymphocytes (TILs) for use in a method of treating cancer in a subject in need thereof, said method comprising providing for administration to said subject a population of TILs, said method comprising: a. obtaining and / or receiving a first population of TILs from a tumor excised from said subject or patient by processing a tumor sample obtained from said subject into a plurality of tumor fragments or by processing a tumor sample obtained from said subject into a tumor digest; b. 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 optionally performed in a sealed container providing a first gas permeable surface area, and the first expansion is performed for about 3-14 days to obtain the second TIL population; c. 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, and the second expansion is optionally performed in a sealed container that provides a second gas-permeable surface area, and the transition from step (b) to step (c) optionally occurs without opening the system to produce the third TIL population; d. harvesting the third population of TILs obtained from step (c), wherein the transition from step (c) to step (d) occurs without opening the system; e. providing a therapeutically effective dose of the third population of TILs obtained in step (d) for administration to the subject; f. adding an epigenetic reprogramming agent to the cell culture medium in step (b) and / or step (c), and optionally adding a cell permeabilization agent.

2. A population of tumor-infiltrating lymphocytes (TILs) for use in a method of treating cancer in a patient or subject in need thereof, said method comprising providing a population of TILs for administration to said patient or subject, said method comprising: (a) obtaining a first population of TILs from a tumor excised from the subject by processing a tumor sample obtained from the subject into a plurality of tumor fragments or by processing a tumor sample obtained from the subject into a tumor digest; (b) performing a first expansion by culturing the first TIL population in a cell culture medium comprising IL-2 to produce a second TIL population, wherein the first expansion is performed in a sealed container providing a first gas permeable surface area, and the first expansion is performed for about 3 to 11 days to obtain the second TIL population; (c) 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 7-11 days to obtain the third TIL population, and the second expansion is optionally performed in a sealed container that provides a second gas-permeable surface area, and the transition from step (b) to step (c) optionally occurs without opening the system to produce the third TIL population; (d) harvesting the third population of TILs obtained from step (c), wherein the transition from step (c) to step (d) optionally occurs without opening the system; (e) transferring the harvested third population of TILs from step (d) to an infusion bag, wherein the transition from step (d) to (e) optionally occurs without opening the system; (f) cryopreserving the infusion bag containing the harvested TIL population from step (e) using a cryopreservation process; (g) providing a therapeutically effective dose of the third population of TILs from the infusion bag in step (f) for administration to the subject; (h) in step (b) and / or step (c), adding an epigenetic reprogramming agent to the cell culture medium, and optionally adding a cell permeation agent.

3. 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 or by processing a tumor sample obtained from the subject into a tumor digest; (b) performing a first expansion by culturing the first TIL population in a cell culture medium comprising IL-2 to produce a second TIL population, wherein the first expansion is performed in a sealed container providing a first gas permeable surface area, and the first expansion is performed for about 3 to 14 days to obtain the second TIL population; (c) 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 (b) to step (c) optionally occurs without opening the system; (d) harvesting the third population of TILs obtained from step (c), wherein the transition from step (c) to step (d) optionally occurs without opening the system; (e) transferring the harvested third population of TILs from step (d) to an infusion bag, wherein the transition from step (d) to (e) optionally occurs without opening the system; (h) in step (b) and / or step (c), adding an epigenetic reprogramming agent to the cell culture medium, and optionally adding a cell permeation agent.

4. 1. A method for expanding tumor infiltrating lymphocytes (TILs) into a therapeutic TIL population, said 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 or by processing a tumor sample obtained from the subject into a tumor digest; (b) performing a first expansion by culturing the first TIL population in a cell culture medium comprising IL-2 to produce a second TIL population, wherein the first expansion is performed in a sealed container providing a first gas permeable surface area, and the first expansion is performed for about 3 to 11 days to obtain the second TIL population; (c) 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 7-11 days to obtain the third TIL population, and the second expansion is optionally performed in a sealed container that provides a second gas-permeable surface area, and the transition from step (b) to step (c) optionally occurs without opening the system to produce the third TIL population; (d) harvesting the third population of TILs obtained from step (c), wherein the transition from step (c) to step (d) optionally occurs without opening the system; (e) transferring the harvested third population of TILs from step (d) to an infusion bag, wherein the transition from step (d) to (e) optionally occurs without opening the system; (h) in step (b) and / or step (c), adding an epigenetic reprogramming agent to the cell culture medium, and optionally adding a cell permeation agent.

5. 1. A method for expanding tumor infiltrating lymphocytes (TILs) into a therapeutic TIL population, said method comprising: (a) providing a first population of TILs obtained and / or received 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) performing a first expansion by culturing the first TIL population in a cell culture medium comprising IL-2 to produce a second TIL population, wherein the first expansion is performed in a sealed container providing a first gas permeable surface area, and the first expansion is performed for about 3 to 11 days to obtain the second TIL population; (c) 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 7-11 days to obtain the third TIL population, and the second expansion is optionally performed in a sealed container that provides a second gas-permeable surface area, and the transition from step (b) to step (c) optionally occurs without opening the system to produce the third TIL population; (d) harvesting the third population of TILs obtained from step (c), wherein the transition from step (c) to step (d) optionally occurs without opening the system; (e) transferring the harvested third population of TILs from step (d) to an infusion bag, wherein the transition from step (d) to (e) optionally occurs without opening the system; (f) in step (b) and / or step (c), adding an epigenetic reprogramming agent to the cell culture medium, and optionally adding a cell permeation agent.

6. 1. 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 or by processing a tumor sample obtained from the subject into a tumor digest; (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 11 days to obtain the second TIL population, and producing the second TIL population, wherein the second TIL population is more numerous 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 the third TIL population, wherein the third TIL population is a therapeutic TIL population; (d) harvesting the therapeutic TIL population obtained from step (c); (g) in step (b) and / or step (c), adding an epigenetic reprogramming agent to the cell culture medium, and optionally adding a cell permeabilization agent.

7. 7. The method of claim 6, wherein in step (c), the cell culture medium further comprises antigen-presenting cells (APCs), and the number of APCs in the culture medium in step (d) is greater than the number of APCs in the culture medium in step (c).

8. 3. The TIL population for use according to claim 1 or 2, 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. The method according to any one of claims 3 to 7.

9. 1. A method for expanding T cells, comprising: (a) performing a first priming expansion of a first TIL population obtained from a donor by culturing the first TIL population to result in growth and prime activation of the first T cell population; (b) after activation of the first TIL population primed in step (a) begins to decay, performing a rapid second expansion of the first TIL population by culturing the first TIL population to result in growth and promote activation of the first T cell population to obtain a second T cell population; (c) harvesting the second population of T cells; (d) in step (a) and / or step (b), adding an epigenetic reprogramming agent to the cell culture medium, and optionally adding a cell permeabilization agent.

10. 10. The TIL population for use of any one of claims 1, 2, or 8, or the method of any of claims 3 to 9, wherein the second TIL population and / or the third TIL population have an increased frequency of CD8 TILs and / or an increased ratio of CD4 TILs to CD8 TILs compared to a corresponding TIL population expanded in cell culture medium without the epigenetic reprogramming agent.

11. the second TIL population is at least 5-fold more numerous than the first TIL population, and the first cell culture medium comprises IL-2; or the third population of TILs is at least 50-fold more numerous than the second population of TILs 7-8 days after the onset of the rapid expansion. A TIL population for use according to claim 1, 2, 8 or 10, or a method according to any of claims 3 to 10.

12. the epigenetic reprogramming agent comprises one or more of a DNA hypomethylating agent, a MEK inhibitor, an HDAC inhibitor, an EZH2 inhibitor, a bromodomain inhibitor, an AKT inhibitor, and / or a TET inhibitor, and optionally, the epigenetic reprogramming agent is: (i) a combination of a DNA hypomethylating agent with a MEK inhibitor, HDAC inhibitor, EZH2 inhibitor, bromodomain inhibitor, AKT inhibitor, or TET inhibitor; (ii) a combination of a MEK inhibitor with an HDAC inhibitor, an EZH2 inhibitor, a bromodomain inhibitor, an AKT inhibitor, or a TET inhibitor; (iii) a combination of an HDAC inhibitor with an EZH2 inhibitor, a bromodomain inhibitor, an AKT inhibitor, or a TET inhibitor; (iv) a combination of an EZH2 inhibitor with a bromodomain inhibitor, an AKT inhibitor, or a TET inhibitor; (v) a combination of a bromodomain inhibitor with an AKT inhibitor or a TET inhibitor; (vi) a combination of an AKT inhibitor and a TET inhibitor; A TIL population for use according to any one of claims 1, 2, 8, 10 or 11, or a method according to any one of claims 3 to 11, selected from:

13. (i) The DNA hypomethylating agent is selected from the group consisting of decitabine, azacitidine, GSK-3484862, RG-108, GSK-3685032, DHAC, SGI-1027, CM-272, zebularine, hinokitiol, guadecitabine, gamma-oryzanol, CM-579, DC-517, 5-fluoro-2'-deoxycytidine, 5-methyldeoxycytidine, DC-05, 6-methyl-5-azacytidine, procainamide, procaine, hydrazine, EGCG, FdCyd, CP-4200, nanomycin A, and pharmaceutically acceptable salts thereof; (ii) the HDAC inhibitor is selected from the group consisting of rosirinostat, vorinostat, trichostatin A, belinostat, panabinostat, panobinostat, xinostat, divinostat, resminostat, abexinostat, xinostat, practinostat, CHR-3996, valproic acid, butyric acid, phenylbutyric acid, entionstat, tacedinaline, mocetinostat, romidespin, nicotinamide, sirtinol, cambinol, EX-527, apicidin, depsipeptide, MS275, BML-210, splitomicin, RGFP966, and pharmaceutically acceptable salts thereof; (iii) the TET inhibitor is selected from the group consisting of C35, Bobcat 339, D(R)-2-hydroxyglutarate (D2HG) and L-2-hydroxyglutarate (L2HG); (iv) the bromodomain inhibitor is one or more selected from JQ1, ZEN-3694, I-BET762, OTX015, I-BET151, RVX-208, MS417, ABBV-075, ABBV-744, SJ432, AZD5153, INCB054329, INCB054329, FT-1101, CPI-0610, RO6870810, BAY1238097, RVX000222, and pharmaceutically acceptable salts thereof; (v) the EZH2 inhibitor is selected from the group consisting of 3-deazaneplanocin A, tazemetostat, GSK343, GSK926, GSK126, EPZ005687, and pharmaceutically acceptable salts thereof; (vi) the AKT inhibitor is selected from the group consisting of ipatasertib, GSK690693, GSK2141795, GSK2110183, AZD5363, GDC-0068, AT7867, CCT128930, MK-2206, BAY1125976, perifosine, oridonin, herbacetin, teheranolide, isoliquirigenin, scutellarin, honokiol, and pharmaceutically acceptable salts thereof; (vii) the MEK inhibitor inhibits MEK1 and / or MEK2, and optionally the MEK inhibitor is selected from the group consisting of trametinib, cobimetinib, binimetinib, selumetinib, PD-325901, CI-1040, TAK-733, GDC-0623, pimasertinib, refametinib, BI-847325, and pharmaceutically acceptable salts thereof, preferably the MEK inhibitor is trametinib; A TIL population for use according to claim 12, or a method according to claim 12.

14. (i) the second TIL population and / or the third TIL population have increased expression of IL-7 receptor compared to a corresponding TIL population expanded in cell culture medium that does not contain the epigenetic reprogramming agent; (ii) the second TIL population and / or the third TIL population have increased expression of at least one of CD25, CD28, ICOS, Ki-67, and GZMB compared to a corresponding TIL population expanded in cell culture medium without the epigenetic reprogramming agent; (iii) the second TIL population and / or the third TIL population have increased expression of at least one of PD-I and TIGIT compared to a corresponding TIL population expanded in cell culture medium without the epigenetic reprogramming agent; (iv) the second TIL population and / or the third TIL population have increased expression of at least one of TCFI, EOMES, and KLF2 compared to a corresponding TIL population expanded in cell culture medium without the epigenetic reprogramming agent. A TIL population for use according to any one of claims 1, 2, 8 or 10 to 13, or a method according to any one of claims 3 to 13.

15. 10. The TIL population for use according to any one of claims 1, 2 or 8, or the method according to any one of claims 3 to 9, wherein the cell permeation agent comprises an octyl ester or a disodium salt, and optionally the octyl ester is selected from (2S)-octyl-a-hydroxyglutarate and (2R)-octyl-a-hydroxyglutarate, or the disodium salt is selected from R-2-hydroxyglutaric acid disodium salt and S-2-hydroxyglutaric acid disodium salt.

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