Treatment of cancer patients by tumor-infiltrating lymphocyte therapy in combination with an IL-15R agonist

Abstract

The present invention provides an improved and / or shortened process and method for preparing tumor-infiltrating lymphocytes (TILs) to prepare a therapeutically effective TIL population with increased therapeutic effect for the treatment of cancer by the TILs described herein in combination with an IL-15R agonist. In some embodiments, the IL-15R agonist is selected from the group consisting of NIZ985 (recombinant heterodimer of IL-15 / IL-15Rα; Novartis), NKTR-255 (polymer conjugate of IL-15; Nektar), N-803 (IL-15 / IL-15Rα-Fc; ImmunityBio), XmAb306 (attenuated IL15 / IL15Rα-Fc fusion protein; Xencor), BJ-001 (tumor-targeted IL-15 / IL-15Rα-Fc; BJ Bioscience), CYP0150 (Cytune), and combinations thereof.

Description

[Technical field]

[0001] CROSS-REFERENCE TO RELATED APPLICATIONS This application claims priority to U.S. Provisional Patent No. 63 / 387,919, filed December 16, 2022, U.S. Provisional Patent No. 63 / 353,832, filed June 20, 2022, and U.S. Provisional Patent No. 63 / 340,446, filed May 10, 2022, all of which are incorporated by reference in their entireties herein. [Background technology]

[0002] The treatment of bulky refractory cancers using adoptive autologous transfer of tumor infiltrating lymphocytes (TILs) represents a powerful approach to the treatment of patients with poor prognosis. Gattinoni, et al., Nat. Rev. Immunol. 2006, 6, 383-393. TILs are dominated by T cells, and IL-2-based TIL expansion followed by the "rapid expansion process" (REP) has become the preferred method of TIL expansion due to its speed and efficiency. Dudley, et al., Science 2002, 298, 850-54; Dudley, et al., J. Clin. Oncol. 2005, 23, 2346-57; Dudley, et al., J. Clin. Oncol. 2008, 26, 5233-39; Riddell, et al., Science 1992, 257, 238-41; Dudley, et al., J. Immunother. 2003, 26, 332-42. Many approaches to improve response to TIL therapy in melanoma and extend TIL therapy to other tumor types have met with limited success, and the field remains challenging. Goff, et al., J.Clin.Oncol.2016,34,2389-97; Dudley, et al., J.Clin.Oncol.2008,26,5233-39; Rosenberg,et al., Clin.Cancer Res.2011,17,4550-57. Combination studies with single immune checkpoint inhibitors have also been described, but further studies are ongoing and additional treatment approaches are needed (Kverneland,et al.,Oncotarget,2020,11(22),2092-2105).

[0003] The current TIL regimen platform relies on the administration of nonmyeloablative lymphodepletion (NMALD) followed by TIL cell therapy and then a short course of high-dose IL-2 (aldesleukin).

[0004] Pre-TIL NMALD serves to i) reduce lymphocyte populations and provide an environment supportive of the incoming TIL cell therapy product for optimal expansion / survival, and ii) optimize the tumor microenvironment by reducing Tregs and MDSCs that may contribute to the inhibitory signaling of neoantigen-reactive TILs.

[0005] IL-2 (aldesleukin) following TIL serves to further support the survival and proliferation of the infused TIL cell product.

[0006] Given the current relatively high doses of cyclophosphamide in NMALD and the safety profile of high-dose IL-2 (aldesleukin), improved methods of using TILs to treat cancer while reducing patient exposure to chemotherapeutic agents during NMALD with high-dose IL-2 before and / or after TIL infusion are needed. Summary of the Invention

[0007] Provided herein are methods of treating cancer in patients using expanded populations of TILs and IL-15R agonists, as well as methods of producing therapeutic populations of TILs from patients or subjects pretreated with at least one ICI.

[0008] The present invention provides a method of treating cancer in a patient in need thereof, comprising administering a population of tumor infiltrating lymphocytes (TILs) and an IL-15R agonist.

[0009] In some embodiments, the IL-15R agonist is selected from the group consisting of NIZ985 (recombinant heterodimer of IL-15 / IL-15Rα; Novartis), NKTR-255 (polymer-conjugated IL-15; Nektar), N-803 (IL-15 / IL-15Rα-Fc; ImmunityBio), XmAb306 (reduced potency IL15 / IL15Rα-Fc fusion protein; Xencor), BJ-001 (tumor-targeted IL-15 / IL-15Rα-Fc; BJ Bioscience), CYP0150 (Cytune), and combinations thereof.

[0010] In some embodiments, the IL-15R agonist is NIZ985 (recombinant heterodimer of IL-15 / IL-15Rα; Novartis).

[0011] In some embodiments, the IL-15R agonist is NKTR-255 (polymer conjugated IL-15; Nektar).

[0012] In some embodiments, the IL-15R agonist is N-803 (IL-15 / IL-15Rα-Fc; ImmunityBio).

[0013] In some embodiments, the IL-15R agonist is XmAb306 (reduced potency IL15 / IL15Rα-Fc fusion protein; Xencor).

[0014] In some embodiments, the IL-15R agonist is administered to the patient on the same day that the population of TILs is administered.

[0015] In some embodiments, the IL-15R agonist is administered to the patient about 1 to about 10 days after administration of the population of TILs.

[0016] In some embodiments, the IL-15R agonist is administered once a day, once every two days, once every three days, once every four days, once every five days, once a week, once every two weeks, once every three weeks, or once a month.

[0017] In some embodiments, the IL-15R agonist is administered in a total of about 1 to about 28 doses.

[0018] In some embodiments, the IL-15R agonist is administered at a dosage of about 1 μg / kg to about 100 μg / kg.

[0019] In some embodiments, the IL-15R agonist is administered at a dosage of 20 μg / kg, once every 5 days for up to 3 total doses.

[0020] In some embodiments, the method further comprises treating the patient with a non-myeloablative lymphodepletion regimen prior to administering the TILs to the patient.

[0021] In some embodiments, the non-myeloablative lymphodepleting regimen comprises cyclophosphamide at a dose of 60 mg / kg / day for 2 days, followed by fludarabine at 25 mg / m 2 / day for 5 days.

[0022] In some embodiments, the non-myeloablative lymphodepleting regimen comprises cyclophosphamide at a dose of 60 mg / kg / day and fludarabine at a dose of 25 mg / m 2 / day for 2 days, followed by fludarabine at 25 mg / m 2 / day for three days.

[0023] In some embodiments, cyclophosphamide is administered with mesna.

[0024] In some embodiments, the patient undergoes a reduced intensity non-myeloablative lymphodepleting regimen.

[0025] In some embodiments, the reduced intensity non-myeloablative lymphodepleting regimen comprises cyclophosphamide at 750 mg / m 2 / day for 4 days, followed by fludarabine at 30 mg / m 2 / day for four days, optionally with cyclophosphamide administered along with mesna.

[0026] In some embodiments, the patient does not receive a non-myeloablative lymphodepleting regimen.

[0027] In some embodiments, the method further comprises treating the patient with an IL-2 regimen beginning the day after the population of TILs is administered to the patient.

[0028] In some embodiments, the method further comprises treating the patient with an IL-2 regimen beginning on the same day that the population of TILs is administered to the patient.

[0029] In some embodiments, the IL-2 regimen is a high-dose IL-2 regimen including 600,000 or 720,000 IU / kg aldesleukin, or a biosimilar or variant thereof, administered as a 15-minute bolus intravenous infusion every 8 hours until tolerated.

[0030] In some embodiments, the IL-2 regimen is a reduced dose IL-2 regimen comprising a reduced number, e.g., 1, 2, 3, 4, or 5 doses of 600,000 or 720,000 IU / kg aldesleukin, or a biosimilar or variant thereof, administered as a 15-minute bolus intravenous infusion every 8 hours.

[0031] In some embodiments, the patient does not receive an IL-2 regimen.

[0032] In some embodiments, the IL-15R agonist results in increased survival and / or expansion of TIL populations.

[0033] In some embodiments, the IL-15R agonist results in the persistence of TILs at 14 days, 28 days, and / or 42 days after administration of the TILs.

[0034] In some embodiments, the IL-15R agonist is administered at a dosage of about 0.5 μg / kg, 1.0 μg / kg, about 1.5 μg / kg, about 2.0 μg / kg, about 2.5 μg / kg, about 3.0 μg / kg, about 3.5 μg / kg, about 4.0 μg / kg, about 4.5 μg / kg, about 5.0 μg / kg, about 10 μg / kg, about 15 μg / kg, about 20 μg / kg, about 30 μg / kg, about 40 μg / kg, about 50 μg / kg, or about 100 μg / kg.

[0035] In some embodiments, the method further comprises administering to the patient an immune checkpoint inhibitor (ICI).

[0036] In some embodiments, the method further comprises administering to the patient a PD-1 inhibitor or a biosimilar thereof.

[0037] In some embodiments, the PD-1 inhibitor is selected from the group consisting of nivolumab, pembrolizumab, and biosimilars thereof.

[0038] In some embodiments, the method further comprises administering to the patient a PD-L1 inhibitor or a biosimilar thereof.

[0039] In some embodiments, the PD-L1 inhibitor is selected from the group consisting of avelumab, atezolizumab, durvalumab, and biosimilars thereof.

[0040] In some embodiments, the method further comprises administering to the patient a CTLA-4 inhibitor or a biosimilar thereof.

[0041] In some embodiments, the CTLA-4 inhibitor is selected from the group consisting of ipilumumab, tremelimumab, and biosimilars thereof.

[0042] In some embodiments, the method further comprises administering a chemotherapeutic agent to the patient.

[0043] In some embodiments, the TIL population comprises: (a) obtaining and / or receiving a first population of TILs from a tumor excised from a patient by processing a tumor sample obtained from the patient into a plurality of tumor fragments or tumor digests before the patient receives an ICI or chemotherapy agent; (b) cryopreserving the tumor fragment or tumor digest comprising the first population of TILs from step (a) to produce a cryopreserved tumor fragment or tumor digest; If the patient exhibits progressive disease during or after treatment with an ICI or chemotherapeutic agent, the first TIL population is expanded into a TIL population.

[0044] In some embodiments, the TIL population comprises: (a) optionally removing a tumor from a patient by surgical resection, needle biopsy, core biopsy, mini-biopsy, or other means to obtain a sample containing a mixture of tumor cells and TIL cells from the tumor, and fragmenting the tumor into tumor fragments, before the patient receives an ICI or chemotherapy agent; (b) cryopreserving the tumor fragment or tumor digest comprising the first population of TILs from step (a) to produce a cryopreserved tumor fragment or tumor digest; If the patient exhibits progressive disease during or after treatment with an ICI or chemotherapeutic agent, the first TIL population is expanded into a TIL population.

[0045] In some embodiments, the expansion of the first TIL population comprises: (c) thawing the cryopreserved tumor fragment or tumor digest and adding the first population of TILs to the closed system; (d) performing a first expansion by culturing the first TIL population in a first cell culture medium containing IL-2 to produce a second TIL population, wherein the first expansion is performed in a closed vessel providing a first gas permeable surface area, and the first expansion is performed for about 3-11 days to obtain the second TIL population, and the transition from step (c) to step (d) occurs without opening the system; (e) performing a second expansion by supplementing the second 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 a third TIL population, the third TIL population being a therapeutic TIL population, and wherein the second expansion is performed in a closed vessel providing a second gas permeable surface area, and the transition from step (d) to step (e) occurs without opening the system. (f) harvesting the therapeutic TIL population obtained from step (e), wherein the transition from step (e) to step (f) occurs without opening the system; (g) transferring the harvested TIL population from step (f) into an infusion bag, wherein the transition from step (f) to (g) occurs without opening the system; (h) cryopreserving the infusion bag containing the harvested TIL population from step (g) using a cryopreservation process.

[0046] In some embodiments, the expansion of the first TIL population comprises: (c) thawing the cryopreserved tumor fragment or tumor digest and adding the first population of TILs to the closed system; (d) performing a first expansion by culturing the first TIL population in a first cell culture medium comprising IL-2 to produce a second TIL population, wherein the first expansion is performed in a closed vessel providing a first gas permeable surface area, and wherein the first expansion is performed for about 3-14 days to obtain the second TIL population, and wherein the transition from step (c) to step (d) occurs without opening the system; (e) performing a second expansion by supplementing the second 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 a third TIL population, the third TIL population being a therapeutic TIL population, and wherein the second expansion is performed in a closed vessel providing a second gas permeable surface area, and wherein the transition from step (d) to step (e) occurs without opening the system. (f) harvesting a therapeutic population of TILs obtained from step (e), wherein the transition from step (e) to step (f) occurs without opening the system; (g) transferring the harvested TIL population from step (f) into an infusion bag, wherein the transition from step (f) to (g) occurs without opening the system; (h) cryopreserving the infusion bag containing the harvested TIL population from step (g) using a cryopreservation process.

[0047] In some embodiments, the expansion of the first TIL population comprises: (c) thawing the cryopreserved tumor fragment or tumor digest and adding the first population of TILs to the closed system; (d) 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 1-7 / 8 days to obtain a second TIL population, wherein the second TIL population is more numerous than the first TIL population to produce a second TIL population; (e) performing a second rapid expansion by supplementing a second cell culture medium of the second TIL population with additional IL-2, OKT-3, and APCs to produce a third TIL population, wherein the number of APCs added to the 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 1-11 days to obtain a third TIL population, wherein the third TIL population is a therapeutic TIL population, and wherein the rapid second expansion is performed in a container comprising a second gas permeable surface area; (f) harvesting a therapeutic population of TILs obtained from step (e), wherein the transition from step (e) to step (f) occurs without opening the system; (g) transferring the harvested TIL population from step (f) into an infusion bag, wherein the transition from step (f) to (g) occurs without opening the system; (h) cryopreserving the infusion bag containing the harvested TIL population from step (g) using a cryopreservation process.

[0048] In some embodiments, the patient exhibits progressive disease at least about 1 week, 2 weeks, 3 weeks, 1 month, 2 months, 3 months, 4 months, 5 months, 6 months, 7 months, 8 months, 9 months, 10 months, 11 months, 12 months, 13 months, 14 months, 15 months, 16 months, 17 months, 18 months, 19 months, 20 months, 21 months, 22 months, 23 months, 24 months, 25 months, 26 months, 27 months, 28 months, 29 months, 30 months, 31 months, 32 months, 33 months, 34 months, 35 months, 36 months after the cryopreservation step (b).

[0049] In some embodiments, step (b) comprises flash freezing of tumor fragments or tumor digests.

[0050] In some embodiments, the quick freezing comprises: i) incubating the tumor fragments or tumor digests in a cryopreservation medium, optionally for about 30 minutes to about 60 minutes at about 2° C. to about 8° C., in a cryopreservation medium containing 10% v / v DMSO; ii) freezing the tumor, wherein the freezing is rapid freezing using the vapor phase of liquid nitrogen.

[0051] In some embodiments, step (b) comprises controlled rate freezing of tumor fragments or tumor digests.

[0052] In some embodiments, the controlled rate freezing comprises: i) adding a cryopreservation medium to a closable container; ii) pre-cooling the closeable container in a controlled rate freezer; iii) placing the tumor in a closable container containing a cryopreservation medium and closing the container; iv) incubating the closed container containing the tumor and the cryopreservation medium for about 30-60 minutes at a temperature of about 2-8° C.; v) slow freezing the container in a controlled rate freezer.

[0053] In some embodiments, the TIL population comprises: (a) obtaining a first population of TILs from a tumor excised from a patient by processing a tumor sample obtained from the patient into a plurality of tumor fragments or tumor digests before the patient receives an ICI or chemotherapy agent; (b) adding the first population of TILs to the closed system; (c) performing a first expansion by culturing the first TIL population in a first cell culture medium containing IL-2 to produce a second TIL population, wherein the first expansion is performed in a closed vessel providing a first gas permeable surface area, and the first expansion is performed for about 3-11 days to obtain the second TIL population, and wherein the transition from step (b) to step (c) occurs without opening the system; (d) performing a second expansion by supplementing the second 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 wherein the second expansion is performed in a closed vessel providing a second gas permeable surface area, and the transition from step (c) to step (d) occurs without opening the system to produce a third TIL population; (e) harvesting the third population of TILs obtained from step (d), wherein the transition from step (d) to step (e) occurs without opening the system; (f) transferring the harvested third population of TILs from step (e) to an infusion bag, wherein the transition from step (e) to (f) occurs without opening the system; (g) cryopreserving the infusion bag containing the harvested TIL population from step (f) using a cryopreservation process.

[0054] In some embodiments, the TIL population comprises: (a) removing a tumor from a patient, optionally by surgical resection, needle biopsy, core biopsy, mini-biopsy, or other means to obtain a sample containing a mixture of tumor cells and TIL cells from the tumor, before the patient receives an ICI or chemotherapy agent; (b) fragmenting the tumor into tumor fragments; (c) contacting the tumor fragment with a first cell culture medium; (d) performing an initial expansion (or a first expansion by priming) of the first TIL population in a first cell culture medium to obtain a second TIL population, the first cell culture medium comprising IL-2, and optionally the first expansion by priming occurs over a period of 1 to 8 days; (e) performing 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 (anti-CD3 antibody), and optionally irradiated allogeneic peripheral blood mononuclear cells (PBMCs), and the rapid expansion is performed over a period of no more than 14 days, and optionally the second TIL expansion can proceed for 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 days after initiation of the rapid second expansion; (f) harvesting a third population of TILs; (g) transferring the harvested third population of TILs from step (f) to an infusion bag; (h) cryopreserving the infusion bag containing the harvested TIL population from step (g) using a cryopreservation process.

[0055] In some embodiments, the patient is naïve to treatment with an ICI and / or a chemotherapeutic agent.

[0056] In some embodiments, the TIL population comprises: (a) obtaining and / or receiving a first population of TILs from a tumor excised from a subject or patient by processing a tumor sample obtained from the subject into a plurality of tumor fragments; (b) adding the first population of TILs to the closed system; (c) performing a first expansion by culturing the first TIL population in a first cell culture medium containing IL-2 to produce a second TIL population, wherein the first expansion is performed in a closed vessel providing a first gas permeable surface area, and the first expansion is performed for about 3-14 days to obtain the second TIL population, and wherein the transition from step (b) to step (c) occurs without opening the system; (d) performing a second expansion by supplementing the second 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 a third TIL population, the third TIL population being a therapeutic TIL population, and wherein the second expansion is performed in a closed container providing a second gas permeable surface area, and wherein the transition from step (c) to step (d) occurs without opening the system. (e) harvesting the therapeutic TIL population obtained from step (d), wherein the transition from step (d) to step (e) occurs without opening the system; (f) transferring the harvested TIL population from step (e) into an infusion bag, wherein the transition from step (e) to (f) occurs without opening the system; (g) cryopreserving the infusion bag containing the harvested TIL population from step (f) using a cryopreservation process.

[0057] In some embodiments, the TIL population comprises: (a) obtaining a first population of TILs from a tumor excised from a subject by processing a tumor sample obtained from the subject into a plurality of tumor fragments; (b) adding the tumor fragment to the closed system; (c) performing a first expansion by culturing the first TIL population in a first cell culture medium containing IL-2 to produce a second TIL population, wherein the first expansion is performed in a closed vessel providing a first gas permeable surface area, and the first expansion is performed for about 3-11 days to obtain the second TIL population, and wherein the transition from step (b) to step (c) occurs without opening the system; (d) performing a second expansion by supplementing the second 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 wherein the second expansion is performed in a closed vessel providing a second gas permeable surface area, and the transition from step (c) to step (d) occurs without opening the system to produce a third TIL population; (e) harvesting the third population of TILs obtained from step (d), wherein the transition from step (d) to step (e) occurs without opening the system; (f) transferring the harvested third population of TILs from step (e) to an infusion bag, wherein the transfer from step (e) to (f) occurs without opening the system; (g) cryopreserving the infusion bag containing the harvested TIL population from step (f) using a cryopreservation process.

[0058] In some embodiments, the second population of TILs is at least 50-fold more numerous than the first population of TILs.

[0059] In some embodiments, the TIL population comprises: (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 said patient or said subject; (b) adding the first population of TILs to the closed system; (c) performing a first expansion by culturing the first TIL population in a first cell culture medium containing IL-2 to produce a second TIL population, wherein the first expansion is performed in a closed vessel providing a first gas permeable surface area, and the first expansion is performed for about 3-11 days to obtain the second TIL population, and wherein the transition from step (b) to step (c) occurs without opening the system; (d) performing a second expansion by supplementing the second 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 wherein the second expansion is performed in a closed vessel providing a second gas permeable surface area, and the transition from step (c) to step (d) occurs without opening the system to produce a third TIL population; (e) harvesting the third population of TILs obtained from step (d), wherein the transition from step (d) to step (e) occurs without opening the system; (f) transferring the harvested third population of TILs from step (e) to an infusion bag, wherein the transition from step (e) to (f) occurs without opening the system; (g) cryopreserving the infusion bag containing the harvested TIL population from step (f) using a cryopreservation process.

[0060] In some embodiments, the TIL population comprises: (a) optionally, resecting a tumor from a subject or patient by 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 tumor, wherein the tumor comprises a first TIL population; (b) adding the tumor fragment to the closed system; (c) performing a first expansion by culturing the first TIL population in a first cell culture medium containing IL-2 to produce a second TIL population, wherein the first expansion is performed in a closed vessel providing a first gas permeable surface area, and the first expansion is performed for about 3-11 days to obtain the second TIL population, and wherein the transition from step (b) to step (c) occurs without opening the system; (d) performing a second expansion by supplementing the second 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 wherein the second expansion is performed in a closed vessel providing a second gas permeable surface area, and the transition from step (c) to step (d) occurs without opening the system to produce a third TIL population; (e) harvesting the third population of TILs obtained from step (d), wherein the transition from step (d) to step (e) occurs without opening the system; (f) transferring the harvested third population of TILs from step (e) to an infusion bag, wherein the transfer from step (e) to (f) occurs without opening the system; (g) cryopreserving the infusion bag containing the harvested TIL population from step (f) using a cryopreservation process.

[0061] In some embodiments, the TIL population comprises: (a) obtaining and / or receiving a first population of TILs from a surgical resection, needle biopsy, core biopsy, mini-biopsy, or other means for obtaining a sample containing a mixture of tumor cells and TIL cells from a subject or patient; (c) contacting the first population of TILs with a first cell culture medium; (d) performing an initial expansion (or a first expansion by priming) of the first TIL population in a first cell culture medium to obtain a second TIL population, the first cell culture medium comprising IL-2, and optionally the first expansion by priming occurs over a period of 1 to 8 days; (e) performing 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 (anti-CD3 antibody), and optionally irradiated allogeneic peripheral blood mononuclear cells (PBMCs), and the rapid expansion is performed over a period of no more than 14 days, and optionally the second TIL expansion can proceed for 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 days after initiation of the rapid second expansion; (f) harvesting a third population of TILs.

[0062] In some embodiments, the TIL population comprises: (a) optionally removing a tumor from a patient by surgical resection, needle biopsy, core biopsy, mini biopsy, or other means to obtain a sample containing a mixture of tumor cells and TIL cells from the tumor; (b) fragmenting the tumor into tumor fragments; (c) contacting the tumor fragment with a first cell culture medium; (d) performing an initial expansion (or a first expansion by priming) of the first TIL population in a first cell culture medium to obtain a second TIL population, the first cell culture medium comprising IL-2, and optionally the first expansion by priming occurs over a period of 1 to 8 days; (e) performing 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 (anti-CD3 antibody), and optionally irradiated allogeneic peripheral blood mononuclear cells (PBMCs), and the rapid expansion is performed over a period of no more than 14 days, and optionally the second TIL expansion can proceed for 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 days after initiation of the rapid second expansion; (f) harvesting a third population of TILs.

[0063] In some embodiments, the third population of TILs is at least 50-fold more numerous than the second population of TILs 7-8 days after the start of rapid expansion.

[0064] In some embodiments, the first cell culture medium further comprises an IL-15R agonist.

[0065] In some embodiments, the second cell culture medium further comprises an IL-15R agonist.

[0066] In some embodiments, the IL-15R agonist is selected from the group consisting of NIZ985, NKTR-255, N-803, XmAb306, BJ-001, CYP0150 (Cytune), and combinations thereof.

[0067] In some embodiments, the IL-15R agonist is NIZ985.

[0068] In some embodiments, the IL-15R agonist is NKTR-255.

[0069] In some embodiments, the IL-15R agonist is N-803.

[0070] In some embodiments, the IL-15R agonist is XmAb306.

[0071] In some embodiments, the IL-15R agonist is supplemented in the first cell culture medium at a concentration of about 0.1 ng / mL, about 0.5 ng / mL, about 1 ng / mL, about 5 ng / mL, about 10 ng / mL, about 50 ng / mL, about 100 ng / mL, about 150 ng / mL, or about 200 ng / mL.

[0072] In some embodiments, the IL-15R agonist is supplemented in the second cell culture medium at a concentration of about 0.1 ng / mL, about 0.5 ng / mL, about 1 ng / mL, about 5 ng / mL, about 10 ng / mL, about 50 ng / mL, about 100 ng / mL, about 150 ng / mL, or about 200 ng / mL.

[0073] In some embodiments, the expression of one or more genes of a TIL population is modulated.

[0074] In some embodiments, the one or more genes are selected from the group consisting of PD-1, CTLA-4, LAG-3, CISH, TIGIT, and CBL-B.

[0075] In some embodiments, expression of PD-1 and CTLA-4 is modulated in the TIL population.

[0076] In some embodiments, expression of PD-1 and LAG-3 is regulated in a TIL population.

[0077] In some embodiments, expression of PD-1 and CISH is regulated in the TIL population.

[0078] In some embodiments, expression of PD-1 and CBL-B is regulated in a TIL population.

[0079] In some embodiments, expression of PD-1 and TIGIT is regulated in a TIL population.

[0080] In some embodiments, expression of CTLA-4 and LAG-3 is regulated in a TIL population.

[0081] In some embodiments, expression of CTLA-4 and CISH is modulated in the TIL population.

[0082] In some embodiments, expression of CTLA-4 and CBL-B is regulated in a TIL population.

[0083] In some embodiments, expression of LAG-3 and CISH is regulated in a TIL population.

[0084] In some embodiments, expression of LAG-3 and CBL-B is regulated in a TIL population.

[0085] In some embodiments, expression of CISH and CBL-B is regulated in a TIL population.

[0086] In some embodiments, expression of PD-1 is regulated in a TIL population.

[0087] In some embodiments, expression of CTLA-4 is regulated in a TIL population.

[0088] In some embodiments, expression of LAG-3 is regulated in a TIL population.

[0089] In some embodiments, expression of CISH is regulated in a TIL population.

[0090] In some embodiments, expression of CBL-B is regulated in a TIL population.

[0091] In some embodiments, expression of TIGIT is regulated in a TIL population.

[0092] In some embodiments, the cancer has been previously treated with a PD-1 inhibitor and / or a PD-L1 inhibitor or a biosimilar thereof.

[0093] In some embodiments, the cancer has been previously treated with a PD-1 inhibitor or a biosimilar thereof.

[0094] In some embodiments, the PD-1 inhibitor is selected from the group consisting of nivolumab, pembrolizumab, and biosimilars thereof.

[0095] In some embodiments, the patient has been previously treated with a PD-L1 inhibitor or a biosimilar thereof.

[0096] In some embodiments, the PD-L1 inhibitor is selected from the group consisting of avelumab, atezolizumab, durvalumab, and biosimilars thereof.

[0097] In some embodiments, the cancer has been previously treated with a CTLA-4 inhibitor or a biosimilar thereof.

[0098] In some embodiments, the CTLA-4 inhibitor is selected from the group consisting of ipilumumab, tremelimumab, and biosimilars thereof.

[0099] 81. The method of any one of claims 1-80, wherein the cancer has been previously treated with a chemotherapy regimen.

[0100] In some embodiments, the chemotherapy regimen includes dacarbazine or temozolimide.

[0101] In some embodiments, the first expansion is performed for a period of about 11 days.

[0102] In some embodiments, IL-2 is present in the cell culture medium at an initial concentration of 1000 IU / mL to 6000 IU / mL in the first expansion.

[0103] In some embodiments, IL-2 is present in the cell culture medium at an initial concentration of between 1000 IU / mL and 6000 IU / mL during initial expansion.

[0104] In some embodiments, in the second expansion step, IL-2 is present at an initial concentration of 1000 IU / mL to 6000 IU / mL and the OKT-3 antibody is present at an initial concentration of about 30 ng / mL.

[0105] In some embodiments, in the rapid-expansion step, IL-2 is present at an initial concentration of 1000 IU / mL to 6000 IU / mL and the OKT-3 antibody is present at an initial concentration of about 30 ng / mL.

[0106] In some embodiments, the first expansion is performed using a gas permeable container.

[0107] In some embodiments, the initial expansion is performed using a gas permeable container.

[0108] In some embodiments, the second expansion is performed using a gas permeable container.

[0109] In some embodiments, the rapid expansion is accomplished using a gas permeable container.

[0110] In some embodiments, the first cell culture medium further comprises a cytokine selected from the group consisting of IL-4, IL-7, IL-15, IL-21, and combinations thereof.

[0111] In some embodiments, the cell culture medium of the first expansion further comprises a cytokine selected from the group consisting of IL-4, IL-7, IL-15, IL-21, and combinations thereof.

[0112] In some embodiments, the second cell culture medium further comprises a cytokine selected from the group consisting of IL-4, IL-7, IL-15, IL-21, and combinations thereof.

[0113] In some embodiments, the cell culture medium of the second expansion further comprises a cytokine selected from the group consisting of IL-4, IL-7, IL-15, IL-21, and combinations thereof.

[0114] In some embodiments, a therapeutically effective population of TILs is administered and is about 2.3×10 10 ~Approx. 13.7×10 10 Contains TILs.

[0115] In some embodiments, the initial expansion is performed for a period of 21 days or less.

[0116] In some embodiments, the initial expansion is performed for a period of 7 days or less.

[0117] In some embodiments, the rapid expansion occurs over a period of 7 days or less.

[0118] In some embodiments, the first expansion in step (c) and the second expansion in step (d) are each performed separately within a period of 11 days.

[0119] In some embodiments, steps (a)-(f) are carried out in about 10 days to about 22 days.

[0120] In some embodiments, the cancer is selected from the group consisting of glioblastoma (GBM), gastrointestinal cancer, melanoma, ovarian cancer, endometrial cancer, thyroid cancer, colorectal cancer, cervical cancer, non-small cell lung cancer (NSCLC), lung cancer, bladder cancer, breast cancer, endometrial cancer, cholangiocarcinoma, cancer caused by human papillomavirus, head and neck cancer (including head and neck squamous cell carcinoma (HNSCC)), renal cancer, renal cell carcinoma, multiple myeloma, chronic lymphocytic leukemia, acute lymphoblastic leukemia, diffuse large B-cell lymphoma, non-Hodgkin's lymphoma, Hodgkin's lymphoma, follicular lymphoma, and mantle cell lymphoma.

[0121] In some embodiments, the cancer is selected from the group consisting of cutaneous melanoma, ocular melanoma, uveal melanoma, and conjunctival melanoma.

[0122] In some embodiments, the cancer is selected from the group consisting of polymorphic xanthoastrocytoma, dysembryoplastic neuroepithelial tumor, ganglioglioma, and pilocytic astrocytoma.

[0123] In some embodiments, the cancer is endometrial adenocarcinoma with non-small cell lung cancer (NSCLC).

[0124] In some embodiments, the cancer is endometrial adenocarcinoma with prominent mucinous differentiation (ECMD).

[0125] In some embodiments, the cancer is papillary thyroid cancer.

[0126] In some embodiments, the cancer is low-grade serous or borderline ovarian cancer.

[0127] In some embodiments, the cancer is hairy cell leukemia.

[0128] In some embodiments, the cancer is Langerhans cell histiocytosis. [Brief description of the drawings]

[0129] [Figure 1]FIG. 1 is an exemplary Gen2 (Process 2A) chart providing an overview of steps A-F. [Figure 2A] FIG. 1 is a process flow diagram of an embodiment of Gen2 (Process 2A) for TIL fabrication. [Figure 2B] FIG. 1 is a process flow diagram of an embodiment of Gen2 (Process 2A) for TIL fabrication. [Figure 2C] FIG. 1 is a process flow diagram of an embodiment of Gen2 (Process 2A) for TIL fabrication. [Diagram 3] 1 shows a diagram of an embodiment of an exemplary manufacturing process (approximately 22 days) for cryopreserved TILs. [Figure 4] 1 shows a diagram of an embodiment of Gen2 (Process 2A), a 22 day process for TIL manufacturing. [Diagram 5] Comparison table of steps A-F from exemplary embodiments of Process 1C and Gen2 (Process 2A) for TIL fabrication. [Figure 6] Detailed comparison of Process 1C and Gen2 (Process 2A) embodiments for TIL fabrication. [Figure 7] 1 is an exemplary Gen3 TIL manufacturing process. [Figure 8A] 1 shows a comparison between an embodiment of a 2A process (approximately a 22 day process) and an embodiment of a Gen3 process (approximately a 14-16 day process) for TIL fabrication. [Figure 8B] An exemplary process Gen3 chart providing an overview of steps A-F (approximately a 14-16 day process). [Figure 8C] A chart providing three exemplary Gen3 processes with an overview of steps A-F (approximately 14-16 day processes) for each of the three process variations. [Figure 8D] FIG. 1 provides an overview of steps A through F of an exemplary modified Gen2-like process (approximately a 22 day process). [Figure 8E]FIG. 1 provides an overview of steps A-F comprising an exemplary embodiment of a KO TIL TALEN process; an exemplary modified Gen 2-like process (approximately 22 day process). [Figure 8F] FIG. 1 provides an overview of steps A-F comprising an exemplary embodiment of a KO TIL TALEN process; an exemplary modified Gen 2-like process (approximately 22 day process). [Figure 8G] FIG. 1 provides an overview of steps A-F comprising an exemplary embodiment of a KO TIL TALEN process; an exemplary modified Gen 2-like process (approximately 22 day process). [Figure 8H] FIG. 1 provides an overview of steps A-F comprising an exemplary embodiment of a KO TIL TALEN process; an exemplary modified Gen 2-like process (approximately 22 day process). [Figure 8I] FIG. 1 provides an overview of steps A-F comprising an exemplary embodiment of a KO TIL TALEN process; an exemplary modified Gen 2-like process (approximately 22 day process). [Figure 8J] FIG. 1 provides an overview of steps A-F comprising an exemplary embodiment of a KO TIL TALEN process; an exemplary modified Gen 2-like process (approximately 22 day process). [Figure 8K] FIG. 1 provides an overview of steps A-F comprising an exemplary embodiment of a KO TIL TALEN process; an exemplary modified Gen 2-like process (approximately 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 an embodiment of the Gen3 process, designated Gen3.1. [Figure 13] 1 is a table illustrating various features of embodiments of the Gen2, Gen2.1 and Gen3.0 processes. [Figure 14] 1 is a table comparing various features of embodiments of the Gen2 and Gen3.0 processes. [Figure 15] 1 is a table providing media use in various embodiments of the described expansion process. [Figure 16] FIG. 1 is a schematic diagram 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 the individual polypeptide binding domains. Structures IA and IB include three linearly linked TNFRSF binding domains, e.g., from an antibody that binds 4-1BBL or 4-1BB, that fold together to form a trivalent protein, which is then linked to a second trivalent protein by IgG1-Fc (containing CH3 and CH2 domains), which is then used to link two of the trivalent proteins together by disulfide bonds (small, elongated ovals), 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, including VH and VL chains linked by a linker that can include hydrophilic residues and Gly and Ser sequences for flexibility, and Glu and Lys for solubility. [Figure 19] FIG. 1 is a schematic diagram 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] FIG. 1 is a schematic diagram of an exemplary embodiment of the Gen3.1 testing process (16-17 day process). [Figure 22] FIG. 1 is a schematic diagram of an exemplary embodiment of the Gen3 process (16-day process). [Figure 23A]1 is a comparison table of an exemplary Gen2 process and an exemplary Gen3 process. [Figure 23B] 1 is a comparison table of an exemplary Gen2 process and an exemplary Gen3 process. [Figure 24] FIG. 1 is a schematic diagram of an exemplary embodiment of the preparation timeline for the Gen3 process (16-17 day process). [Diagram 25] FIG. 1 is a schematic diagram of an exemplary embodiment of the Gen3 process (14-16 day process). [Figure 26A] FIG. 1 is a schematic diagram of an exemplary embodiment of the Gen3 process (16-day process). [Figure 26B] FIG. 1 is a schematic diagram of an exemplary embodiment of the Gen3 process (16-day process). [Figure 27] FIG. 1 is a schematic diagram of an exemplary embodiment of the Gen3 process (16-day process). [Figure 28] Comparison of Gen2, Gen2.1, and Gen3 process (16 day process) embodiments. [Figure 29] Comparison of Gen2, Gen2.1, and Gen3 process (16 day process) embodiments. [Diagram 30] Components of the Gen3 embodiment. [Diagram 31] Flowchart comparison of Gen3 embodiments (Gen3.0, Gen3.1 Control, Gen3.1 Test). [Diagram 32] Components of an exemplary embodiment of the Gen3 process (a 16-17 day process) are shown. [Diagram 33] Approval criteria table. [Diagram 34] Schematic diagram of the TIL-based immunotherapy manufacturing process relevant to the study described in Example 19. Abbreviations: CMO = contract manufacturing organization, GMP = good manufacturing practice, IL-2 = interleukin-2, OKT3 = monoclonal antibody against CD3, TIL = tumor infiltrating lymphocytes. [Diagram 35] Experimental flow diagram of the full-scale PD-1 KO TIL TALEN process. [Diagram 36] Experimental flow diagram of the full-scale PD-1 KO TIL TALEN process. [Figure 37A] Exemplary membrane-anchored immunomodulatory fusion proteins that may be included in the TILs described herein. [Figure 37B] Exemplary membrane-anchored immunomodulatory fusion proteins that may be included in the TILs described herein. [Figure 37C] Exemplary membrane-anchored immunomodulatory fusion proteins that may be included in the TILs described herein. [Figure 37D] Exemplary membrane-anchored immunomodulatory fusion proteins that may be included in the TILs described herein. [Figure 37E] Exemplary membrane-anchored immunomodulatory fusion proteins that may be included in the TILs described herein. [Figure 37F] Exemplary membrane-anchored immunomodulatory fusion proteins that may be included in the TILs described herein. [Figure 37G] Exemplary membrane-anchored immunomodulatory fusion proteins that may be included in the TILs described herein. [Fig. 37H] Exemplary membrane-anchored immunomodulatory fusion proteins that may be included in the TILs described herein. [Fig. 37I] Exemplary membrane-anchored immunomodulatory fusion proteins that may be included in the TILs described herein. [Figure 37J] Exemplary membrane-anchored immunomodulatory fusion proteins that may be included in the TILs described herein. [Figure 38A] Exemplary membrane-anchored immunomodulatory fusion proteins that may be included in the TILs described herein. [Figure 38B] Exemplary membrane-anchored immunomodulatory fusion proteins that may be included in the TILs described herein. [Figure 38C] Exemplary membrane-anchored immunomodulatory fusion proteins that may be included in the TILs described herein. [Figure 38D] Exemplary membrane-anchored immunomodulatory fusion proteins that may be included in the TILs described herein. [Figure 39]Exemplary IL-15 agents used to treat cancer (Figure from Waldmann, TA, et. al., Frontiers in Immunology, 11:1-10 (2020)). [Diagram 40] Diagram of exemplary IL-15 agent ALT-803 (Diagram from Chu, Y., et al., J Immunother Cancer, 8(2):1-14, supplementary content (2020)). [Diagram 41] An illustration of the clinical trial design disclosed in Example 13.

[0130] Brief Description of the Sequence Listing SEQ ID NO:1 is the amino acid sequence of the heavy chain of muromonab.

[0131] SEQ ID NO:2 is the amino acid sequence of the light chain of muromonab.

[0132] SEQ ID NO:3 is the amino acid sequence of recombinant human IL-2 protein.

[0133] SEQ ID NO:4 is the amino acid sequence of aldesleukin.

[0134] SEQ ID NO:5 is an IL-2 form.

[0135] SEQ ID NO:6 is the amino acid sequence of nembareukin alpha.

[0136] SEQ ID NO:7 is an IL-2 form.

[0137] SEQ ID NO:8 is a mucin domain polypeptide.

[0138] SEQ ID NO:9 is the amino acid sequence of recombinant human IL-4 protein.

[0139] SEQ ID NO:10 is the amino acid sequence of recombinant human IL-7 protein.

[0140] SEQ ID NO:11 is the amino acid sequence of recombinant human IL-15 protein.

[0141] SEQ ID NO:12 is the amino acid sequence of recombinant human IL-21 protein.

[0142] SEQ ID NO:13 is the IL-2 sequence.

[0143] SEQ ID NO: 14 is an IL-2 mutein sequence.

[0144] SEQ ID NO: 15 is an IL-2 mutein sequence.

[0145] SEQ ID NO: 16 is HCDR1_IL-2 of IgG.IL2R67A.H1.

[0146] SEQ ID NO: 17 is HCDR2 of IgG.IL2R67A.H1.

[0147] SEQ ID NO: 18 is the HCDR3 of IgG.IL2R67A.H1.

[0148] SEQ ID NO: 19 is HCDR1_IL-2 Kabat of IgG.IL2R67A.H1.

[0149] SEQ ID NO:20 is the HCDR2 Kabat of IgG.IL2R67A.H1.

[0150] SEQ ID NO: 21 is the HCDR3 Kabat of IgG.IL2R67A.H1.

[0151] SEQ ID NO: 22 is the HCDR1_IL-2 clotia of IgG.IL2R67A.H1.

[0152] SEQ ID NO:23 is the HCDR2 clone of IgG.IL2R67A.H1.

[0153] SEQ ID NO:24 is the HCDR3 clone of IgG.IL2R67A.H1.

[0154] SEQ ID NO: 25 is HCDR1_IL-2 IMGT of IgG.IL2R67A.H1.

[0155] SEQ ID NO: 26 is the HCDR2 IMGT of IgG.IL2R67A.H1.

[0156] SEQ ID NO: 27 is the HCDR3 IMGT of IgG.IL2R67A.H1.

[0157] SEQ ID NO:28 is the V H It's a chain.

[0158] SEQ ID NO:29 is the heavy chain of IgG.IL2R67A.H1.

[0159] SEQ ID NO:30 is the LCDR1 Kabat of IgG.IL2R67A.H1.

[0160] SEQ ID NO:31 is the LCDR2 Kabat of IgG.IL2R67A.H1.

[0161] SEQ ID NO: 32 is the LCDR3 Kabat of IgG.IL2R67A.H1.

[0162] SEQ ID NO: 33 is the LCDR1 clotia of IgG.IL2R67A.H1.

[0163] SEQ ID NO:34 is the LCDR2 clone of IgG.IL2R67A.H1.

[0164] SEQ ID NO:35 is the LCDR3 clotting domain of IgG.IL2R67A.H1.

[0165] SEQ ID NO: 36 is V L It's a chain.

[0166] SEQ ID NO:37 is the light chain.

[0167] SEQ ID NO:38 is the light chain.

[0168] SEQ ID NO:39 is the light chain.

[0169] SEQ ID NO:40 is the amino acid sequence of human 4-1BB.

[0170] SEQ ID NO:41 is the amino acid sequence of mouse 4-1BB.

[0171] SEQ ID NO: 42 is the heavy chain of the 4-1BB agonist monoclonal antibody utomirumab (PF-05082566).

[0172] SEQ ID NO: 43 is the light chain of the 4-1BB agonist monoclonal antibody utomirumab (PF-05082566).

[0173] SEQ ID NO: 44 represents the heavy chain variable region (V H ).

[0174] SEQ ID NO: 45 represents the light chain variable region (V) of the 4-1BB agonist monoclonal antibody utomirumab (PF-05082566). L ).

[0175] SEQ ID NO: 46 is the heavy chain CDR1 of the 4-1BB agonist monoclonal antibody utomirumab (PF-05082566).

[0176] SEQ ID NO: 47 is the heavy chain CDR2 of the 4-1BB agonist monoclonal antibody utomirumab (PF-05082566).

[0177] SEQ ID NO: 48 is the heavy chain CDR3 of the 4-1BB agonist monoclonal antibody utomirumab (PF-05082566).

[0178] SEQ ID NO: 49 is the light chain CDR1 of the 4-1BB agonist monoclonal antibody utomirumab (PF-05082566).

[0179] SEQ ID NO:50 is the light chain CDR2 of the 4-1BB agonist monoclonal antibody utomirumab (PF-05082566).

[0180] SEQ ID NO:51 is the light chain CDR3 of the 4-1BB agonist monoclonal antibody utomirumab (PF-05082566).

[0181] SEQ ID NO:52 is the heavy chain of the 4-1BB agonist monoclonal antibody urelumab (BMS-663513).

[0182] SEQ ID NO:53 is the light chain of the 4-1BB agonist monoclonal antibody urelumab (BMS-663513).

[0183] SEQ ID NO:54 is the heavy chain variable region (VH) of the 4-1BB agonist monoclonal antibody urelumab (BMS-663513).

[0184] SEQ ID NO:55 is the light chain variable region (VL) of the 4-1BB agonist monoclonal antibody urelumab (BMS-663513).

[0185] SEQ ID NO:56 is the heavy chain CDR1 of the 4-1BB agonist monoclonal antibody urelumab (BMS-663513).

[0186] SEQ ID NO:57 is the heavy chain CDR2 of the 4-1BB agonist monoclonal antibody urelumab (BMS-663513).

[0187] SEQ ID NO:58 is the heavy chain CDR3 of the 4-1BB agonist monoclonal antibody urelumab (BMS-663513).

[0188] SEQ ID NO:59 is the light chain CDR1 of the 4-1BB agonist monoclonal antibody urelumab (BMS-663513).

[0189] SEQ ID NO: 60 is the light chain CDR2 of the 4-1BB agonist monoclonal antibody urelumab (BMS-663513).

[0190] SEQ ID NO: 61 is the light chain CDR3 of the 4-1BB agonist monoclonal antibody urelumab (BMS-663513).

[0191] SEQ ID NO:62 is the Fc domain of the TNFRSF agonist fusion protein.

[0192] SEQ ID NO:63 is the linker for the TNFRSF agonist fusion protein.

[0193] SEQ ID NO:64 is the linker for the TNFRSF agonist fusion protein.

[0194] SEQ ID NO:65 is the linker for the TNFRSF agonist fusion protein.

[0195] SEQ ID NO:66 is the linker for the TNFRSF agonist fusion protein.

[0196] SEQ ID NO:67 is the linker for the TNFRSF agonist fusion protein.

[0197] SEQ ID NO:68 is the linker for the TNFRSF agonist fusion protein.

[0198] SEQ ID NO:69 is the linker for the TNFRSF agonist fusion protein.

[0199] SEQ ID NO:70 is the linker for the TNFRSF agonist fusion protein.

[0200] SEQ ID NO:71 is the linker for the TNFRSF agonist fusion protein.

[0201] SEQ ID NO:72 is the linker for the TNFRSF agonist fusion protein.

[0202] SEQ ID NO:73 is the Fc domain of the TNFRSF agonist fusion protein.

[0203] SEQ ID NO:74 is the linker for the TNFRSF agonist fusion protein.

[0204] SEQ ID NO:75 is the linker for the TNFRSF agonist fusion protein.

[0205] SEQ ID NO:76 is the linker for the TNFRSF agonist fusion protein.

[0206] SEQ ID NO:77 is the 4-1BB ligand (4-1BBL) amino acid sequence.

[0207] SEQ ID NO:78 is a soluble portion of the 4-1BBL polypeptide.

[0208] SEQ ID NO: 79 represents the heavy chain variable region (V H ).

[0209] SEQ ID NO: 80 represents the light chain variable region (V L ).

[0210] SEQ ID NO: 81 represents the heavy chain variable region (V H ).

[0211] SEQ ID NO: 82 represents the light chain variable region (V L ).

[0212] SEQ ID NO: 83 represents the heavy chain variable region (V H ).

[0213] SEQ ID NO: 84 represents the light chain variable region (V L ).

[0214] SEQ ID NO:85 is the amino acid sequence of human OX40.

[0215] SEQ ID NO: 86 is the amino acid sequence of mouse OX40.

[0216] SEQ ID NO:87 is the heavy chain of the OX40 agonist monoclonal antibody tabolixizumab (MEDI-0562).

[0217] SEQ ID NO:88 is the light chain of the OX40 agonist monoclonal antibody tabolixizumab (MEDI-0562).

[0218] SEQ ID NO:89 is the heavy chain variable region (V) of the OX40 agonist monoclonal antibody tabolixizumab (MEDI-0562). H ).

[0219] SEQ ID NO: 90 represents the light chain variable region (V) of the OX40 agonist monoclonal antibody tabolixizumab (MEDI-0562). L ).

[0220] SEQ ID NO: 91 is the heavy chain CDR1 of the OX40 agonist monoclonal antibody tabolixizumab (MEDI-0562).

[0221] SEQ ID NO: 92 is the heavy chain CDR2 of the OX40 agonist monoclonal antibody tabolixizumab (MEDI-0562).

[0222] SEQ ID NO: 93 is the heavy chain CDR3 of the OX40 agonist monoclonal antibody tabolixizumab (MEDI-0562).

[0223] SEQ ID NO: 94 is the light chain CDR1 of the OX40 agonist monoclonal antibody tabolixizumab (MEDI-0562).

[0224] SEQ ID NO: 95 is the light chain CDR2 of the OX40 agonist monoclonal antibody tabolixizumab (MEDI-0562).

[0225] SEQ ID NO: 96 is the light chain CDR3 of the OX40 agonist monoclonal antibody tabolixizumab (MEDI-0562).

[0226] SEQ ID NO: 97 is the heavy chain of the OX40 agonist monoclonal antibody 11D4.

[0227] SEQ ID NO: 98 is the light chain of the OX40 agonist monoclonal antibody 11D4.

[0228] SEQ ID NO: 99 is the heavy chain variable region (V H ).

[0229] SEQ ID NO:100 represents the light chain variable region (V L ).

[0230] SEQ ID NO: 101 is the heavy chain CDR1 of the OX40 agonist monoclonal antibody 11D4.

[0231] SEQ ID NO: 102 is the heavy chain CDR2 of the OX40 agonist monoclonal antibody 11D4.

[0232] SEQ ID NO: 103 is the heavy chain CDR3 of the OX40 agonist monoclonal antibody 11D4.

[0233] SEQ ID NO: 104 is the light chain CDR1 of the OX40 agonist monoclonal antibody 11D4.

[0234] SEQ ID NO: 105 is the light chain CDR2 of the OX40 agonist monoclonal antibody 11D4.

[0235] SEQ ID NO: 106 is the light chain CDR3 of the OX40 agonist monoclonal antibody 11D4.

[0236] SEQ ID NO: 107 is the heavy chain of the OX40 agonist monoclonal antibody 18D8.

[0237] SEQ ID NO: 108 is the light chain of the OX40 agonist monoclonal antibody 18D8.

[0238] SEQ ID NO: 109 is the heavy chain variable region (V H ).

[0239] SEQ ID NO:110 is the light chain variable region (V L ).

[0240] SEQ ID NO:111 is the heavy chain CDR1 of the OX40 agonist monoclonal antibody 18D8.

[0241] SEQ ID NO: 112 is the heavy chain CDR2 of the OX40 agonist monoclonal antibody 18D8.

[0242] SEQ ID NO: 113 is the heavy chain CDR3 of the OX40 agonist monoclonal antibody 18D8.

[0243] SEQ ID NO: 114 is the light chain CDR1 of the OX40 agonist monoclonal antibody 18D8.

[0244] SEQ ID NO: 115 is the light chain CDR2 of the OX40 agonist monoclonal antibody 18D8.

[0245] SEQ ID NO: 116 is the light chain CDR3 of the OX40 agonist monoclonal antibody 18D8.

[0246] SEQ ID NO:117 is the heavy chain variable region (V H ).

[0247] SEQ ID NO:118 is the light chain variable region (V L ).

[0248] SEQ ID NO:119 is the heavy chain CDR1 of the OX40 agonist monoclonal antibody Hu119-122.

[0249] SEQ ID NO:120 is the heavy chain CDR2 of the OX40 agonist monoclonal antibody Hu119-122.

[0250] SEQ ID NO: 121 is the heavy chain CDR3 of the OX40 agonist monoclonal antibody Hu119-122.

[0251] SEQ ID NO: 122 is the light chain CDR1 of the OX40 agonist monoclonal antibody Hu119-122.

[0252] SEQ ID NO: 123 is the light chain CDR2 of the OX40 agonist monoclonal antibody Hu119-122.

[0253] SEQ ID NO: 124 is the light chain CDR3 of the OX40 agonist monoclonal antibody Hu119-122.

[0254] SEQ ID NO: 125 is the heavy chain variable region (V H ).

[0255] SEQ ID NO: 126 is the light chain variable region (V L ).

[0256] SEQ ID NO: 127 is the heavy chain CDR1 of the OX40 agonist monoclonal antibody Hu106-222.

[0257] SEQ ID NO: 128 is the heavy chain CDR2 of the OX40 agonist monoclonal antibody Hu106-222.

[0258] SEQ ID NO: 129 is the heavy chain CDR3 of the OX40 agonist monoclonal antibody Hu106-222.

[0259] SEQ ID NO: 130 is the light chain CDR1 of the OX40 agonist monoclonal antibody Hu106-222.

[0260] SEQ ID NO: 131 is the light chain CDR2 of the OX40 agonist monoclonal antibody Hu106-222.

[0261] SEQ ID NO: 132 is the light chain CDR3 of the OX40 agonist monoclonal antibody Hu106-222.

[0262] SEQ ID NO: 133 is the OX40 ligand (OX40L) amino acid sequence.

[0263] SEQ ID NO:134 is a soluble portion of the OX40L polypeptide.

[0264] SEQ ID NO:135 is an alternative soluble portion of the OX40L polypeptide.

[0265] SEQ ID NO: 136 is the heavy chain variable region (V H ).

[0266] SEQ ID NO: 137 is the light chain variable region (V L ).

[0267] SEQ ID NO: 138 is the heavy chain variable region (V H ).

[0268] SEQ ID NO: 139 is the light chain variable region (V L ).

[0269] SEQ ID NO: 140 represents the heavy chain variable region (V H ).

[0270] SEQ ID NO: 141 represents the light chain variable region (V L).

[0271] SEQ ID NO: 142 is the heavy chain variable region (V H ).

[0272] SEQ ID NO: 143 is the light chain variable region (V L ).

[0273] SEQ ID NO: 144 is the heavy chain variable region (V H ).

[0274] SEQ ID NO: 145 is the light chain variable region (V L ).

[0275] SEQ ID NO: 146 is the heavy chain variable region (V H ).

[0276] SEQ ID NO: 147 is the light chain variable region (V L ).

[0277] SEQ ID NO: 148 is the heavy chain variable region (V H ).

[0278] SEQ ID NO: 149 is the heavy chain variable region (V H ).

[0279] SEQ ID NO: 150 is the light chain variable region (V L ).

[0280] SEQ ID NO: 151 is the light chain variable region (V L ).

[0281] SEQ ID NO: 152 is the heavy chain variable region (V H ).

[0282] SEQ ID NO: 153 is the heavy chain variable region (V H ).

[0283] SEQ ID NO: 154 is the light chain variable region (V L ).

[0284] SEQ ID NO: 155 is the light chain variable region (V L ).

[0285] SEQ ID NO: 156 is the heavy chain variable region (V H ).

[0286] SEQ ID NO: 157 is the light chain variable region (V L ).

[0287] SEQ ID NO: 158 is the heavy chain amino acid sequence of the PD-1 inhibitor nivolumab.

[0288] SEQ ID NO: 159 is the light chain amino acid sequence of the PD-1 inhibitor nivolumab.

[0289] SEQ ID NO: 160 represents the heavy chain variable region (V H ) amino acid sequence.

[0290] SEQ ID NO: 161 represents the light chain variable region (V L ) amino acid sequence.

[0291] SEQ ID NO: 162 is the heavy chain CDR1 amino acid sequence of the PD-1 inhibitor nivolumab.

[0292] SEQ ID NO: 163 is the heavy chain CDR2 amino acid sequence of the PD-1 inhibitor nivolumab.

[0293] SEQ ID NO: 164 is the heavy chain CDR3 amino acid sequence of the PD-1 inhibitor nivolumab.

[0294] SEQ ID NO: 165 is the light chain CDR1 amino acid sequence of the PD-1 inhibitor nivolumab.

[0295] SEQ ID NO: 166 is the light chain CDR2 amino acid sequence of the PD-1 inhibitor nivolumab.

[0296] SEQ ID NO: 167 is the light chain CDR3 amino acid sequence of the PD-1 inhibitor nivolumab.

[0297] SEQ ID NO: 168 is the heavy chain amino acid sequence of the PD-1 inhibitor pembrolizumab.

[0298] SEQ ID NO: 169 is the light chain amino acid sequence of the PD-1 inhibitor pembrolizumab.

[0299] SEQ ID NO: 170 represents the heavy chain variable region (V H ) amino acid sequence.

[0300] SEQ ID NO: 171 represents the light chain variable region (V L ) amino acid sequence.

[0301] SEQ ID NO: 172 is the heavy chain CDR1 amino acid sequence of the PD-1 inhibitor pembrolizumab.

[0302] SEQ ID NO: 173 is the heavy chain CDR2 amino acid sequence of the PD-1 inhibitor pembrolizumab.

[0303] SEQ ID NO: 174 is the heavy chain CDR3 amino acid sequence of the PD-1 inhibitor pembrolizumab.

[0304] SEQ ID NO: 175 is the light chain CDR1 amino acid sequence of the PD-1 inhibitor pembrolizumab.

[0305] SEQ ID NO: 176 is the light chain CDR2 amino acid sequence of the PD-1 inhibitor pembrolizumab.

[0306] SEQ ID NO: 177 is the light chain CDR3 amino acid sequence of the PD-1 inhibitor pembrolizumab.

[0307] SEQ ID NO: 178 is the heavy chain amino acid sequence of the PD-L1 inhibitor durvalumab.

[0308] SEQ ID NO: 179 is the light chain amino acid sequence of the PD-L1 inhibitor durvalumab.

[0309] SEQ ID NO: 180 represents the heavy chain variable region (V H ) amino acid sequence.

[0310] SEQ ID NO: 181 represents the light chain variable region (V L ) amino acid sequence.

[0311] SEQ ID NO: 182 is the heavy chain CDR1 amino acid sequence of the PD-L1 inhibitor durvalumab.

[0312] SEQ ID NO: 183 is the heavy chain CDR2 amino acid sequence of the PD-L1 inhibitor durvalumab.

[0313] SEQ ID NO: 184 is the heavy chain CDR3 amino acid sequence of the PD-L1 inhibitor durvalumab.

[0314] SEQ ID NO: 185 is the light chain CDR1 amino acid sequence of the PD-L1 inhibitor durvalumab.

[0315] SEQ ID NO: 186 is the light chain CDR2 amino acid sequence of the PD-L1 inhibitor durvalumab.

[0316] SEQ ID NO: 187 is the light chain CDR3 amino acid sequence of the PD-L1 inhibitor durvalumab.

[0317] SEQ ID NO: 188 is the heavy chain amino acid sequence of the PD-L1 inhibitor avelumab.

[0318] SEQ ID NO: 189 is the light chain amino acid sequence of the PD-L1 inhibitor avelumab.

[0319] SEQ ID NO: 190 represents the heavy chain variable region (V H ) amino acid sequence.

[0320] SEQ ID NO: 191 represents the light chain variable region (V L ) amino acid sequence.

[0321] SEQ ID NO: 192 is the heavy chain CDR1 amino acid sequence of the PD-L1 inhibitor avelumab.

[0322] SEQ ID NO: 193 is the heavy chain CDR2 amino acid sequence of the PD-L1 inhibitor avelumab.

[0323] SEQ ID NO: 194 is the heavy chain CDR3 amino acid sequence of the PD-L1 inhibitor avelumab.

[0324] SEQ ID NO: 195 is the light chain CDR1 amino acid sequence of the PD-L1 inhibitor avelumab.

[0325] SEQ ID NO: 196 is the light chain CDR2 amino acid sequence of the PD-L1 inhibitor avelumab.

[0326] SEQ ID NO: 197 is the light chain CDR3 amino acid sequence of the PD-L1 inhibitor avelumab.

[0327] SEQ ID NO: 198 is the heavy chain amino acid sequence of the PD-L1 inhibitor atezolizumab.

[0328] SEQ ID NO: 199 is the light chain amino acid sequence of the PD-L1 inhibitor atezolizumab.

[0329] SEQ ID NO: 200 represents the heavy chain variable region (V H ) amino acid sequence.

[0330] SEQ ID NO: 201 represents the light chain variable region (V L ) amino acid sequence.

[0331] SEQ ID NO: 202 is the heavy chain CDR1 amino acid sequence of the PD-L1 inhibitor atezolizumab.

[0332] SEQ ID NO: 203 is the heavy chain CDR2 amino acid sequence of the PD-L1 inhibitor atezolizumab.

[0333] SEQ ID NO: 204 is the heavy chain CDR3 amino acid sequence of the PD-L1 inhibitor atezolizumab.

[0334] SEQ ID NO: 205 is the light chain CDR1 amino acid sequence of the PD-L1 inhibitor atezolizumab.

[0335] SEQ ID NO: 206 is the light chain CDR2 amino acid sequence of the PD-L1 inhibitor atezolizumab.

[0336] SEQ ID NO: 207 is the light chain CDR3 amino acid sequence of the PD-L1 inhibitor atezolizumab.

[0337] SEQ ID NO: 208 is the heavy chain amino acid sequence of the CTLA-4 inhibitor ipilimumab.

[0338] SEQ ID NO: 209 is the light chain amino acid sequence of the CTLA-4 inhibitor ipilimumab.

[0339] SEQ ID NO: 210 represents the heavy chain variable region (V H ) amino acid sequence.

[0340] SEQ ID NO: 211 represents the light chain variable region (V L) amino acid sequence.

[0341] SEQ ID NO: 212 is the heavy chain CDR1 amino acid sequence of the CTLA-4 inhibitor ipilimumab.

[0342] SEQ ID NO: 213 is the heavy chain CDR2 amino acid sequence of the CTLA-4 inhibitor ipilimumab.

[0343] SEQ ID NO: 214 is the heavy chain CDR3 amino acid sequence of the CTLA-4 inhibitor ipilimumab.

[0344] SEQ ID NO: 215 is the light chain CDR1 amino acid sequence of the CTLA-4 inhibitor ipilimumab.

[0345] SEQ ID NO: 216 is the light chain CDR2 amino acid sequence of the CTLA-4 inhibitor ipilimumab.

[0346] SEQ ID NO: 217 is the light chain CDR3 amino acid sequence of the CTLA-4 inhibitor ipilimumab.

[0347] SEQ ID NO: 218 is the heavy chain amino acid sequence of the CTLA-4 inhibitor tremelimumab.

[0348] SEQ ID NO: 219 is the light chain amino acid sequence of the CTLA-4 inhibitor tremelimumab.

[0349] SEQ ID NO: 220 represents the heavy chain variable region (V H ) amino acid sequence.

[0350] SEQ ID NO: 221 represents the light chain variable region (V L ) amino acid sequence.

[0351] SEQ ID NO: 222 is the heavy chain CDR1 amino acid sequence of the CTLA-4 inhibitor tremelimumab.

[0352] SEQ ID NO: 223 is the heavy chain CDR2 amino acid sequence of the CTLA-4 inhibitor tremelimumab.

[0353] SEQ ID NO: 224 is the heavy chain CDR3 amino acid sequence of the CTLA-4 inhibitor tremelimumab.

[0354] SEQ ID NO: 225 is the light chain CDR1 amino acid sequence of the CTLA-4 inhibitor tremelimumab.

[0355] SEQ ID NO: 226 is the light chain CDR2 amino acid sequence of the CTLA-4 inhibitor tremelimumab.

[0356] SEQ ID NO: 227 is the light chain CDR3 amino acid sequence of the CTLA-4 inhibitor tremelimumab.

[0357] SEQ ID NO: 228 is the heavy chain amino acid sequence of the CTLA-4 inhibitor zalifrelimab.

[0358] SEQ ID NO: 229 is the light chain amino acid sequence of the CTLA-4 inhibitor zalifrelimab.

[0359] SEQ ID NO:230 represents the heavy chain variable region (V H ) amino acid sequence.

[0360] SEQ ID NO: 231 represents the light chain variable region (V L ) amino acid sequence.

[0361] SEQ ID NO: 232 is the heavy chain CDR1 amino acid sequence of the CTLA-4 inhibitor zalifrelimab.

[0362] SEQ ID NO: 233 is the heavy chain CDR2 amino acid sequence of the CTLA-4 inhibitor zalifrelimab.

[0363] SEQ ID NO: 234 is the heavy chain CDR3 amino acid sequence of the CTLA-4 inhibitor zalifrelimab.

[0364] SEQ ID NO: 235 is the light chain CDR1 amino acid sequence of the CTLA-4 inhibitor zalifrelimab.

[0365] SEQ ID NO: 236 is the light chain CDR2 amino acid sequence of the CTLA-4 inhibitor zalifrelimab.

[0366] SEQ ID NO: 237 is the light chain CDR3 amino acid sequence of the CTLA-4 inhibitor zalifrelimab.

[0367] SEQ ID NO: 238 is the CD8a transmembrane domain.

[0368] SEQ ID NO:239 is the B7-1 transmembrane intracellular domain.

[0369] SEQ ID NOs:240-245 are exemplary glycine-serine linkers that are useful in the immunomodulatory fusion proteins described herein.

[0370] SEQ ID NO:246 is an exemplary linker that is useful in the immunomodulatory fusion proteins described herein.

[0371] SEQ ID NO:247 is the 2A peptide C-terminal sequence.

[0372] SEQ ID NO:248 is the porcine teschovirus-1 2A peptide.

[0373] SEQ ID NO:249 is the Equine rhinitis A virus 2A peptide.

[0374] SEQ ID NO:250 is the foot and mouth disease virus 2A peptide.

[0375] SEQ ID NO:251 is an exemplary furin-cleavable 2A peptide.

[0376] SEQ ID NOs: 252 and 253 are human IgE signal peptide sequences.

[0377] SEQ ID NO:254 is the human IL-2 signal peptide sequence.

[0378] SEQ ID NO:255 is the 6xNFAT IL-2 minimal promoter.

[0379] SEQ ID NO:256 is a NFAT responsive element.

[0380] SEQ ID NO: 257 is the human IL-2 promoter sequence.

[0381] SEQ ID NO: 258 is human IL-15 (N72D mutant).

[0382] SEQ ID NO: 259 is human IL-15R-alpha-Su / Fc domain.

[0383] SEQ ID NO: 260 is human IL-15R-alpha-Su (65aa truncated extracellular domain).

[0384] SEQ ID NO: 261 is human IL-15 isoform 2.

[0385] SEQ ID NO: 262 is human IL-15 isoform 1.

[0386] SEQ ID NO:263 is human IL-15 (without the signal peptide).

[0387] SEQ ID NO: 264 is human IL-15R-alpha (85aa truncated extracellular domain).

[0388] SEQ ID NO: 265 is human IL-15R-alpha (182aa truncated extracellular domain).

[0389] SEQ ID NO: 266 is human IL-15R-alpha.

[0390] SEQ ID NO:267 is the human IL-12 p35 subunit.

[0391] SEQ ID NO:268 is the human IL-12p40 subunit.

[0392] SEQ ID NO: 269 is human IL-18.

[0393] SEQ ID NO: 270 is a human IL-18 variant.

[0394] SEQ ID NO: 271 is human IL-21.

[0395] SEQ ID NO: 272 is human IL-2.

[0396] SEQ ID NO: 273 is human CD40L.

[0397] SEQ ID NO: 274 is an agonistic anti-human CD40 VH (sotigaliimab).

[0398] SEQ ID NO: 275 is an agonist anti-human CD40 VL (sotigaliimab).

[0399] SEQ ID NO: 276 is an agonistic anti-human CD40 scFv (sotigaliimab).

[0400] SEQ ID NO: 277 is an agonist anti-human CD40 VH (dacetuzumab).

[0401] SEQ ID NO: 278 is an agonist anti-human CD40 VL (dacetuzumab).

[0402] SEQ ID NO: 279 is an agonistic anti-human CD40 scFv (dacetuzumab).

[0403] SEQ ID NO: 280 is an agonistic anti-human CD40 VH (lucatuzumab).

[0404] SEQ ID NO: 281 is an agonist anti-human CD40 VL (lucatuzumab).

[0405] SEQ ID NO: 282 is an agonistic anti-human CD40 scFv (lucatuzumab).

[0406] SEQ ID NO: 283 is an agonistic anti-human CD40 VH (celiclerumab).

[0407] SEQ ID NO: 284 is an agonist anti-human CD40 VL (celiclerumab).

[0408] SEQ ID NO: 285 is an agonistic anti-human CD40 scFv (celiclerumab).

[0409] SEQ ID NO:286 is the target PD-1 sequence.

[0410] SEQ ID NO:287 is the target PD-1 sequence.

[0411] SEQ ID NO:288 is a repeated PD-1 left repeat sequence.

[0412] SEQ ID NO:289 is a repeated PD-1 right repeat sequence.

[0413] SEQ ID NO:290 is a repeated PD-1 left repeat sequence.

[0414] SEQ ID NO:291 is a repeated PD-1 right repeat sequence.

[0415] SEQ ID NO: 292 is the PD-1 left TALEN nuclease sequence.

[0416] SEQ ID NO: 293 is the PD-1 right TALEN nuclease sequence.

[0417] SEQ ID NO: 294 is the PD-1 left TALEN nuclease sequence.

[0418] SEQ ID NO: 295 is the PD-1 right TALEN nuclease sequence.

[0419] SEQ ID NO:296 is a nucleic acid sequence encoding the tethered IL-15 of SEQ ID NO:328.

[0420] SEQ ID NO:297 is a nucleic acid sequence encoding the tethered IL-21 of SEQ ID NO:331.

[0421] SEQ ID NO:298 is a nucleic acid sequence encoding the tethered IL-15 fusion protein of SEQ ID NO:328 and the tethered IL-21 fusion protein of SEQ ID NO:331.

[0422] SEQ ID NO: 299 is a nucleic acid sequence encoding the tethered IL-12 fusion protein of SEQ ID NO: 303. The nucleic acid sequence includes the NFAT promoter.

[0423] SEQ ID NO:300 is a nucleic acid sequence encoding the tethered IL-15 fusion protein of SEQ ID NO: 328. The nucleic acid sequence includes the NFAT promoter.

[0424] SEQ ID NO:301 is a nucleic acid sequence encoding the tethered IL-21 fusion protein of SEQ ID NO:331. The nucleic acid sequence includes the NFAT promoter.

[0425] SEQ ID NO:302 is a nucleic acid sequence encoding a tethered IL-15 fusion protein of SEQ ID NO:328 and a tethered IL-21 fusion protein of SEQ ID NO:331. The nucleic acid sequence includes a NFAT promoter.

[0426] SEQ ID NO:303 is the amino acid sequence of an exemplary tethered IL-12 (tethered IL-12-Lr1-Ar2).

[0427] SEQ ID NO:304 is a nucleic acid sequence encoding the tethered IL-12 of SEQ ID NO:303.

[0428] SEQ ID NO:305 is the amino acid sequence of an exemplary tethered IL-18 (tethered IL-18-Lr1-Ar2).

[0429] SEQ ID NO:306 is a nucleic acid sequence encoding the tethered IL-18 of SEQ ID NO:305.

[0430] SEQ ID NO:307 is the amino acid sequence of an exemplary tethered variant IL-18 (tethered DR-IL-18(6-27 variant)-Lr1-Ar2).

[0431] SEQ ID NO:308 is a nucleic acid sequence encoding the tethered variant IL-18 of SEQ ID NO:307.

[0432] SEQ ID NO:309 is the amino acid sequence of an exemplary tethered IL-12 / IL-15.

[0433] SEQ ID NO:310 is a nucleic acid sequence encoding the tethered IL-12 / IL-15 of SEQ ID NO:309.

[0434] SEQ ID NO:311 is the amino acid sequence of an exemplary tethered IL-18 / IL-15.

[0435] SEQ ID NO:312 is a nucleic acid sequence encoding the tethered IL-18 / IL-15 of SEQ ID NO:311.

[0436] SEQ ID NO:313 is the amino acid sequence of an exemplary tethered anti-CD40scFV (APX005M).

[0437] SEQ ID NO:314 is a nucleic acid sequence encoding the tethered anti-CD40scFV (APX005M) of SEQ ID NO:313.

[0438] SEQ ID NO:315 is the amino acid sequence of an exemplary tethered anti-CD40scFV (dacetuzumab).

[0439] SEQ ID NO:316 is a nucleic acid sequence encoding the tethered anti-CD40scFV (dacetuzumab) of SEQ ID NO:315.

[0440] SEQ ID NO:317 is the amino acid sequence of an exemplary tethered anti-CD40 scFV (lucatuzumab).

[0441] SEQ ID NO:318 is a nucleic acid sequence encoding the tethered anti-CD40 scFV (lucatuzumab) of SEQ ID NO:317.

[0442] SEQ ID NO:319 is the amino acid sequence of an exemplary tethered anti-CD40 scFV (celiclerumab).

[0443] SEQ ID NO:320 is a nucleic acid sequence encoding the tethered anti-CD40 scFV (celiclerumab) of SEQ ID NO:319.

[0444] SEQ ID NO:321 is a nucleic acid sequence encoding CD40L of SEQ ID NO:273.

[0445] SEQ ID NO:322 is the amino acid sequence of an exemplary tethered CD40L / IL-15.

[0446] SEQ ID NO:323 is a nucleic acid sequence encoding the tethered CD40L / IL-15 of SEQ ID NO:311.

[0447] SEQ ID NO:324 is the amino acid sequence of an exemplary tethered IL-2.

[0448] SEQ ID NO:325 is a nucleic acid sequence encoding the tethered IL-2 of SEQ ID NO:313.

[0449] SEQ ID NO:326 is the amino acid sequence of an exemplary tethered IL-12.

[0450] SEQ ID NO:327 is a nucleic acid sequence encoding the tethered IL-12 of SEQ ID NO:315.

[0451] SEQ ID NO:328 is the amino acid sequence of an exemplary tethered IL-15.

[0452] SEQ ID NO:329 is a nucleic acid sequence encoding the tethered IL-15 of SEQ ID NO:317.

[0453] SEQ ID NO:330 is a nucleic acid sequence encoding GFP.

[0454] SEQ ID NO:331 is the amino acid sequence of an exemplary tethered IL-21.

[0455] SEQ ID NO: 332 is the amino acid sequence of human IL-15 of NIZ985.

[0456] SEQ ID NO: 333 is the amino acid sequence of human soluble IL-15Rα of NIZ985.

[0457] SEQ ID NO: 334 is the amino acid sequence of chain 1 of XmAb306.

[0458] SEQ ID NO: 335 is the amino acid sequence of chain 2 of XmAb306.

[0459] SEQ ID NO: 336 is the amino acid sequence of IL-15N72D of N-803.

[0460] SEQ ID NO: 337 is the amino acid sequence of IL-15RαSu / Fc of N-803.

[0461] SEQ ID NO:338 is the amino acid sequence of human IL-15 of CYP0150.

[0462] SEQ ID NO:339 is the amino acid sequence of human IL-15 of CYP0150.

[0463] SEQ ID NO:340 is the amino acid sequence of the human IL-15Rα sushi and hinge domain of CYP0150.

[0464] SEQ ID NO: 341 is the amino acid sequence of the tumor-targeted IL-15 / IL-15Rα-Fc of BJ-001.

[0465] I. 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.

[0466] As used herein, the terms "co-administration," "co-administering," "administered in combination," "administering in combination," "simultaneous," and "concurrent" encompass administration of two or more active pharmaceutical ingredients (e.g., multiple TILs in preferred embodiments of the invention) 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.

[0467] The term "in vivo" refers to events that take place inside a subject's body.

[0468] 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.

[0469] 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 for a surgical or therapeutic procedure.

[0470] The term "rapid expansion" refers to an increase in the number of antigen-specific TILs of at least about 3-fold (or 4, 5, 6, 7, 8, or 9-fold) over a one-week period, more preferably at least about 10-fold (or 20, 30, 40, 50, 60, 70, 80, or 90-fold) over a one-week period, or most preferably at least about 100-fold over a one-week period. Several rapid expansion protocols are described herein. Several rapid expansion protocols are described herein.

[0471] 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 can include genetically modified TILs.

[0472] As used herein, a "population of cells" (including TILs) refers to a number of cells that share a common trait. Generally, a population is generally on the order of 1×10 6 ~1×10 10 The number of TILs ranges from 1×10 to 1×10, with different TIL populations containing different numbers. For example, the initial growth of primary TILs in the presence of IL-2 ranges from approximately 1×10 to 1×10 8 REP expansion typically results in a bulk TIL population of 1.5 x 10 cells for injection. 9 ~1.5×10 10 This is done to provide a population of cells.

[0473] By "cryopreserved TILs" herein is meant that TILs, either primary, bulk, or expanded (REP TILs), are processed and stored at temperatures ranging from about -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.

[0474] 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.

[0475] TILs can generally be either biochemically defined using cell surface markers or functionally defined 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 the patient.

[0476] The term "cryopreservation media" or "cryopreservation medium" refers to any medium that can be used for cryopreservation of cells. Such media can include media containing 7%-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. In some embodiments, CS10 medium contains 10% DMSO.

[0477] The term "central memory T cells" refers to T cells that are CD45R0+ and CCR7 (CCR7 高 ) and CD62L (CD62 高 ) constitutively expressing CD3, CD127 (IL-7R), and IL-15R. Transcription factors of central memory T cells include BCL-6, BCL-6B, MBD2, and BMI1. Central memory T cells primarily secrete IL-2 and CD40L as effector molecules after TCR triggering. Central memory T cells predominate in the CD4 compartment in the blood and are proportionally enriched in lymph nodes and tonsils in humans.

[0478] 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 antigenic 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.

[0479] The term "closed system" refers to a system that is closed to the outside environment. Any closed system suitable for cell culture methods can be used in the method of the present invention. Closed systems include, but are not limited to, closed G-vessels. Once tumor segments are added to the closed system, the system is not opened to the outside environment until the TILs are ready to be administered to the patient.

[0480] The terms "fragmenting," "fragment," and "fragmented" as used herein to describe processes for destroying tumors include mechanical fragmentation methods such as crushing, slicing, splitting, and mincing tumor tissue, as well as any other method for disrupting the physical structure of tumor tissue.

[0481] 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 cells), the peripheral blood mononuclear cells are preferably irradiated allogeneic peripheral blood mononuclear cells.

[0482] 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.

[0483] 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 against the CD3 receptor in the T cell antigen receptor of mature T cells. Anti-CD3 antibodies include OKT-3, also known as muromonab. Anti-CD3 antibodies also include UHCT1 clones, also known as T3 and CD3ε. Other anti-CD3 antibodies include, for example, otelixizumab, teplizumab, and visilizumab.

[0484] The term "OKT-3" (also referred to herein as "OKT3") refers to a monoclonal antibody or biosimilar or variant thereof, including a human, humanized, chimeric, or murine antibody against the CD3 receptor in the T cell antigen receptor of mature T cells, 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 at the American Type Culture Collection and assigned ATCC accession number CRL8001. A hybridoma capable of producing OKT-3 has also been deposited at the European Collection of Authenticated Cell Cultures (ECACC) and assigned catalog number 86022706.

[0485] [Table 1]

[0486] 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 recombinant IL-2 forms marketed by CellGenix, Inc., Portsmouth, NH, USA (CELLGRO GMP) or ProSpec-Tany TechnoGene Ltd., East Brunswick, NJ, USA (catalog number CYT-209-b) 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 IL2 prodrug benpegaldesleukin (NKTR-214, an N-terminal lysine derivative in which an average of six lysine residues have been replaced with [(2,7-bis{[methylpoly(oxyethylene)]carbamoyl}-9H-fluoren-9-yl)methoxy]carbonyl). 6Also encompassed are pegylated forms of IL-2 as described herein, including pegylated human recombinant IL-2, such as SEQ ID NO: 4, which is available from Nektar Therapeutics (South San Francisco, Calif., USA) or can be prepared by methods known in the art, such as the methods described in Example 19 of International Patent Application Publication No. WO2018 / 132496A1, or Example 1 of U.S. Patent Application Publication No. US2019 / 0275133A1, 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 / 0328791A1 and International Patent Application Publication No. WO2012 / 065086A1, the disclosures of which are incorporated herein by reference. Alternative forms of conjugated IL-2 suitable for use in the present invention are described in U.S. Patent Nos. 4,766,106, 5,206,344, 5,089,261, and 4,902,502, the disclosures of which are incorporated herein by reference. Formulations of IL-2 suitable for use in the present invention are described in U.S. Patent No. 6,706,289, the disclosure of which is incorporated herein by reference.

[0487] In some embodiments, a suitable IL-2 form for use in the present invention is THOR-707 available from Synthorx, Inc. The preparation and properties of THOR-707 and additional alternative forms of IL-2 suitable for use in the present invention are described in U.S. Patent Application Publication Nos. US2020 / 0181220A1 and US2020 / 0330601A1, the disclosures of which are incorporated herein by reference. In some embodiments, a suitable IL-2 form for use in the present invention is an interleukin 2 (IL-2) complex comprising an isolated and purified IL-2 polypeptide and a conjugation moiety that binds to the isolated and purified IL-2 polypeptide at an amino acid position selected from K35, T37, R38, T41, F42, K43, F44, Y45, E61, E62, E68, K64, P65, V69, L72, and Y107, where 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 further mutated to a non-natural amino acid. In some embodiments, the non-natural 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-oxooctanoic acid, p-acetyl-L-phenylalanine ... -Oxononanoic acid, p-propargyloxyphenylalanine, p-propargyl-phenylalanine, 3-methyl-phenylalanine, L-dopa, fluorinated phenylalanine, isopropyl-L-phenylalanine, p-azido-L-phenylalanine, p-acyl-L-phenylalanine, p-benzoyl-L-phenylalanine, p-bromophenylalanine, p-amino-L-phenylalanine, isopropyl-L-phenylalanine, O-allyl tyrosine, O-methyl-L-tyrosine, O-4-allyl-L-tyrosine, 4- propyl-L-tyrosine, phosphonotyrosine, tri-O-acetyl-GlcNAcp-serine, L-phosphoserine, phosphonoserine, L-3-(2-naphthyl)alanine, 2-amino-3-((2-((3-(benzyloxy)-3-oxopropyl)amino)ethyl)selanyl)propanoic acid, 2-amino-3-(phenylselanyl)propanoic acid, or selenocysteine. In some embodiments, the IL-2 complex has a decreased affinity for the IL-2 receptor alpha (IL-2Rα) subunit compared to a wild-type IL-2 polypeptide. In some embodiments, the decreased 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 decreased affinity is about 1 fold, 2 fold, 3 fold, 4 fold, 5 fold, 6 fold, 7 fold, 8 fold, 9 fold, 10 fold, 30 fold, 50 fold, 100 fold, 200 fold, 300 fold, 500 fold,1000-fold or more. In some embodiments, the conjugated moiety impairs or blocks the binding of IL-2 to IL-2Rα. In some embodiments, the conjugated moiety comprises a water-soluble polymer. In some embodiments, the additional conjugated moiety comprises a water-soluble polymer. In some embodiments, each of the water-soluble polymers independently comprises polyethylene glycol (PEG), poly(propylene glycol) (PPG), copolymers of ethylene glycol and propylene glycol, poly(oxyethylated polyol), poly(olefinic alcohol), poly(vinylpyrrolidone), poly(hydroxyalkyl methacrylamide), poly(hydroxyalkyl methacrylate), poly(saccharide), poly(α-hydroxy acid), poly(vinyl alcohol), polyphosphazene, polyoxazoline (POZ), poly(N-acryloylmorpholine), or combinations thereof. In some embodiments, each of the water-soluble polymers independently comprises PEG. In some embodiments, the PEG is a linear PEG or a branched PEG. In some embodiments, each of the water-soluble polymers independently comprises a polysaccharide. In some embodiments, the polysaccharide comprises dextran, polysialic acid (PSA), hyaluronic acid (HA), amylose, heparin, heparan sulfate (HS), dextrin, or hydroxyethyl starch (HES). In some embodiments, each of the water soluble polymers independently comprises a glycan. In some embodiments, each of the water soluble polymers independently comprises a polyamine. In some embodiments, the conjugation moiety comprises a protein. In some embodiments, the additional conjugation 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 conjugation moiety comprises a polypeptide. In some embodiments, the additional conjugation moiety comprises a polypeptide. In some embodiments, each of the proteins independently comprises an XTEN peptide, a glycine-rich homoamino acid polymer (HAP),The conjugate moiety may comprise a PAS polypeptide, an elastin-like polypeptide (ELP), a CTP peptide, or a gelatin-like protein (GLK) polymer. In some embodiments, the isolated and purified IL-2 polypeptide is modified by glutamylation. In some embodiments, the 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 a homobifunctional linker such as the Romant reagent dithiobis(succinimidyl propionate) DSP, 3'3'-dithiobis(sulfosuccinimidyl propionate) (DTSSP), disuccinimidyl suberate (DSS), bis(sulfosuccinimidyl) suberate (BS), disuccinimidyl tartrate (DST), disulfosuccinimidyl tartrate (sulfoDS T), ethylene glycobis(succinimidyl succinate) (EGS), disuccinimidyl glutarate (DSG), N,N′-disuccinimidyl carbonate (DSC), dimethyl adipimidate (DMA), dimethyl pimelimidate (DMP), dimethyl suberimidate (DMS), dimethyl-3,3′-dithiobispropionimidate (DTBP), 1,4-di-(3′-(2′-pyridyldithio) (e) propionamido) butane (DPDPB), bismaleimidohexane (BMH), halogenated aryl-containing compounds (DFDNB), such as 1,5-difluoro-2,4-dinitrobenzene or 1,3-difluoro-4,6-dinitrobenzene, 4,4′-difluoro-3,3′-dinitrophenyl sulfone (DFDNPS), bis-[β-(4-azidosalicylamido)ethyl] disulfide (BASED), formaldehyde, glutaraldehyde, 1,4-butanediol diglycidyl ether, adipic acid dihydrazide, carbohydrazide, o-toluidine, 3,3′-dimethylbenzidine, benzidine, α,α′-p-diaminodiphenyl, diiodo-p-xylenesulfonic acid, N,N′-ethylene-bis(iodoacetamide), or N,N′-hexamethylenebis(iodoacetamide). In some embodiments, the linker isContains 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)toluamide]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 (M2C2H), 3-(2-pyridyldithio)propionyl hydrazide (PDPH), N-hydroxysuccinimidyl-4-azidosalicylate (NHs-AsA), N-hydroxysulfosuccinimidyl-4-azidosalicylate (sulfo-NHs-AsA), sulfosuccinimide, 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) sulfosuccinimidyl 4-(ρ-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-azidobenzoylhydrazide (ABH), 4-(ρ-azidosalicylamido)butylamine (AsBA), or p-azidophenylglyoxal (APG). In some embodiments, the linker optionally comprises a non-cleavable linker, including a dipeptide linker. In some embodiments, the dipeptide linker comprises Val-Cit, Phe-Lys, Val-Ala, or Val-Lys.In some embodiments, the linker comprises a non-cleavable linker. In some embodiments, the linker optionally comprises a maleimide group, including maleimidocaproyl (mc), succinimidyl-4-(N-maleimidomethyl)cyclohexane-1-carboxylate (sMCC), or sulfosuccinimidyl-4-(N-maleimidomethyl)cyclohexane-1-carboxylate (sulfo-sMCC). In some embodiments, the linker further comprises a spacer. In some embodiments, the spacer comprises p-aminobenzyl alcohol (PAB), p-aminobenzyloxycarbonyl (PABC), derivatives, or analogs thereof. In some embodiments, the conjugated moiety can extend the serum half-life of the IL-2 conjugate. In some embodiments, the additional conjugated moiety can extend the serum half-life of the IL-2 conjugate. In some embodiments, the IL-2 form suitable for use in the present invention is a fragment of any of the IL-2 forms described herein. In some embodiments, IL-2 forms suitable for use in the present invention are pegylated as disclosed in US Patent Application Publication No. US2020 / 0181220A1 and US Patent Application Publication No. US2020 / 0330601A1. In some embodiments, IL-2 forms suitable for use in the present invention are IL-2 conjugates comprising an IL-2 polypeptide comprising N6-azidoethoxy-L-lysine (AzK) covalently attached to a conjugation moiety comprising polyethylene glycol (PEG), wherein the IL-2 polypeptide comprises an amino acid sequence having at least 80% sequence identity to SEQ ID NO:5, and wherein AzK substitutes an amino acid at position K35, F42, F44, K43, E62, P65, R38, T41, E68, Y45, V69, or L72 with respect to amino acid positions in SEQ ID NO:5. In some embodiments, the IL-2 polypeptide comprises a one-residue N-terminal deletion with respect to SEQ ID NO:5. In some embodiments, forms of IL-2 suitable for use in the present invention lack IL-2R alpha chain association but retain normal binding to the intermediate affinity IL-2R beta-gamma signaling complex.In some embodiments, 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 conjugation moiety comprising polyethylene glycol (PEG), wherein the IL-2 polypeptide comprises an amino acid sequence having at least 90% sequence identity to SEQ ID NO:5, and wherein AzK substitutes an amino acid at position K35, F42, F44, K43, E62, P65, R38, T41, E68, Y45, V69, or L72 with respect to amino acid positions 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 conjugation moiety comprising polyethylene glycol (PEG), wherein the IL-2 polypeptide comprises an amino acid sequence having at least 95% sequence identity to SEQ ID NO:5, and wherein AzK substitutes an amino acid at position K35, F42, F44, K43, E62, P65, R38, T41, E68, Y45, V69, or L72 with respect to amino acid positions 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 conjugation moiety comprising polyethylene glycol (PEG), wherein the IL-2 polypeptide comprises an amino acid sequence having at least 98% sequence identity to SEQ ID NO:5, and wherein AzK substitutes an amino acid at position K35, F42, F44, K43, E62, P65, R38, T41, E68, Y45, V69, or L72 with respect to amino acid positions in SEQ ID NO:5.

[0488] In some embodiments, a form of IL-2 suitable for use in the present invention is nembareukin alpha, also known as ALKS-4230 (SEQ ID NO: 6), available from Alkermes, Inc. Nembareukin alpha 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 138Human interleukin-2 fragment (1-59), variant (Cys), fused to human interleukin-2 receptor α chain fragment (139-303) via 5'-Cyclohexyl 1'-amino-1'-propanediol (AAV) and produced in Chinese hamster ovary (CHO) cells, is glycosylated. 125 >Ser 51 );G 2 The peptide was fused to human interleukin 2 (IL-2) (4-74)-peptide (62-132) via a peptide linker (60-61), and GSG 3 Human interleukin-2 (IL-2) (75-133)-peptide [Cys 125(51)>Ser]-mutant (1-59). The amino acid sequence of nembareukin alpha is set forth in SEQ ID NO: 6. In some embodiments, nembareukin alpha 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 nembareukin alpha, 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 / 0038684A1 and U.S. Patent No. 10,183,979, the disclosures of which are incorporated herein by reference. In some embodiments, an IL-2 form suitable for use in the present invention is a protein having at least 80%, at least 90%, at least 95%, or at least 90% sequence identity to SEQ ID NO:6. In some embodiments, an IL-2 form suitable for use in the present invention has the amino acid sequence set forth in SEQ ID NO:6, or a conservative amino acid substitution 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, an IL-2 form 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 of 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.

[0489] [Table 2]

[0490] In some embodiments, IL-2 forms suitable for use in the present invention comprise a heavy chain variable region (V H ) and the 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, wherein the antibody cytokine graft protein preferentially expands T effector cells over regulatory T cells. In some embodiments, the antibody cytokine graft protein comprises a heavy chain variable region (V) comprising complementarity determining regions HCDR1, HCDR2, and HCDR3. H ) and the 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 graft 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 / 0270334A1, the disclosure of which is incorporated herein by reference. In some embodiments, the antibody cytokine graft protein comprises a heavy chain variable region (VV) comprising complementarity determining regions HCDR1, HCDR2, HCDR3, H ) and the light chain variable region (V L ) and an IL-2 molecule or a fragment thereof grafted into the CDR of VH or VL, wherein the IL-2 molecule is a mutein, and the antibody cytokine graft 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.

[0491] In some embodiments, the IL-2 molecule or fragment thereof is H In some embodiments, the IL-2 molecule or a fragment thereof is grafted into the HCDR1 of V H In some embodiments, the IL-2 molecule or fragment thereof is grafted onto the HCDR2 of V H In some embodiments, the IL-2 molecule or fragment thereof is grafted onto the HCDR3 of V L In some embodiments, the IL-2 molecule or a fragment thereof is grafted onto the LCDR1 of V LIn some embodiments, the IL-2 molecule or fragment thereof is grafted onto the LCDR2 of V L The IL-2 molecule is a mutein.

[0492] 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, and 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, and 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.

[0493] 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 and with one or more additional amino acids between the CDR sequence and the IL-2 sequence.

[0494] In some embodiments, the IL-2 molecule described herein is an IL-2 mutein. In some cases, the IL-2 mutein comprises an R67A substitution. In some embodiments, the IL-2 mutein comprises the amino acid sequence of SEQ ID NO: 14 or SEQ ID NO: 15. In some embodiments, the IL-2 mutein comprises the amino acid sequence of Table 1 of U.S. Patent Application Publication No. US2020 / 0270334A1, the disclosure of which is incorporated herein by reference.

[0495] In some embodiments, the antibody cytokine transplant protein comprises an HCDR1 selected from the group consisting of SEQ ID NO:16, SEQ ID NO:19, SEQ ID NO:22, and SEQ ID NO:25. In some embodiments, the antibody cytokine transplant protein comprises an HCDR1 selected from the group consisting of SEQ ID NO:7, SEQ ID NO:10, SEQ ID NO:13, and SEQ ID NO:16. In some embodiments, the antibody cytokine transplant protein comprises an HCDR2 selected from the group consisting of SEQ ID NO:17, SEQ ID NO:20, SEQ ID NO:23, and SEQ ID NO:26. In some embodiments, the antibody cytokine transplant protein comprises an HCDR3 selected from the group consisting of SEQ ID NO:18, SEQ ID NO:21, SEQ ID NO:24, and SEQ ID NO:27. In some embodiments, the antibody cytokine transplant protein comprises an HCDR3 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 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 an IgG.IL2F71A.H1 or IgG.IL2R67A.H1 of U.S. Patent Application Publication No. 2020 / 0270334A1, or a variant, derivative, or fragment thereof, or a conservative amino acid substitution 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 as set forth in Table 3.

[0496] [Table 3-1] [Table 3-2]

[0497] The term "IL-4" (also referred to herein as "IL4") refers to the cytokine known as interleukin 4, which is produced by Th2 T cells, as well as eosinophils, basophils, and mast cells. IL-4 regulates the differentiation of naive helper T cells (Th0 cells) into Th2 T cells. Steinke and Borish, Respir. Res. 2001, 2, 66-70. Upon activation by IL-4, Th2 T cells then produce additional IL-4 in a positive feedback loop. IL-4 also stimulates B cell proliferation and class II MHC expression, and promotes the production of IgE and IgG from B cells. 1 Recombinant human IL-4 suitable for use in the present invention is commercially available from several sources, including ProSpec-Tany TechnoGene Ltd., East Brunswick, NJ, USA (catalog number CYT-211) and ThermoFisher Scientific, Inc., Waltham, MA, USA (human IL-15 recombinant protein, catalog number Gibco CTP0043). The amino acid sequence of recombinant human IL-4 suitable for use in the present invention is shown in Table 2 (SEQ ID NO: 9).

[0498] 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 sources, 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).

[0499] 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 sources, including ProSpec-Tany TechnoGene Ltd., East Brunswick, NJ, USA (catalog number CYT-230-b) and ThermoFisher Scientific, Inc., Waltham, MA, USA (human IL-15 recombinant protein, catalog number 34-8159-82). The amino acid sequence of recombinant human IL-15 suitable for use in the present invention is shown in Table 2 (SEQ ID NO:11).

[0500] 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 is produced primarily by natural killer T cells and activated human CD4+ T cells. Recombinant human IL-21 is a single, non-glycosylated polypeptide chain containing 132 amino acids with a molecular weight of 15.4 kDa. Recombinant human IL-21 is commercially available from several sources, including ProSpec-Tany TechnoGene Ltd., East Brunswick, NJ, USA (catalog number CYT-408-b) and ThermoFisher Scientific, Inc., Waltham, MA, USA (human IL-21 recombinant protein, catalog number 14-8219-80). The amino acid sequence of recombinant human IL-21 suitable for use in the present invention is shown in Table 2 (SEQ ID NO:21).

[0501] The term "IL-15R agonist" (also referred to herein as "IL-15 agonist") refers to a molecule that activates the IL-15 signaling pathway by binding to the IL-15 receptor (IL-15R) beta and common gamma (γC) subunits. IL-15 is presented in a complex with the membrane-bound α subunit of the IL-15 receptor (IL-15Rα) on the surface of dendritic cells or other cells, and this complex functions through a transpresentation mechanism that interacts with IL-15R beta and gamma subunits expressed on NK, NKT or T cells. See Stonier and Schluns, Immunol Lett. 2010, 127, 85-92, the disclosure of which is incorporated herein by reference. IL-15 agonists are described, for example, in Wu, J Mol Genet Med. 2013, 7, 85, the disclosure of which is incorporated herein by reference. In some embodiments, the IL-15R agonist can be a recombinant IL-15 molecule. In some embodiments, the IL-15R agonist can be a mimic of the IL-15 / IL-15Rα complex presented on the cell surface, e.g., a heterodimeric complex or fusion protein comprising an IL-15 wild-type or mutant (e.g., N72D, D30N, E64Q, N65D) molecule and a partial or extracellular domain of IL-15Rα, e.g., soluble IL-15Rα, sushi domain of IL-15Rα, etc., optionally linked to one or more Fc domains. In some embodiments, the IL-15R agonist can be a modified IL-15 molecule, e.g., an IL-15 mutant molecule (e.g., N72D, D30N, E64Q, N65D), IL-15 with site-specific glycosolation(s), etc., with improved characteristics, e.g., extended half-life, increased affinity for IL-15R, etc.

[0502] 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 concentration 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 10 It may be stated that the TIL (optionally including genetically modified cytotoxic lymphocytes) compositions may be administered in doses of 100-200 mg / kg / day (including all integer values ​​within those ranges). TIL (optionally including genetically engineered TIL) compositions may also be administered multiple times at these doses. TIL (optionally including genetically engineered TIL) may 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 regimens for a particular patient may be readily determined by one skilled in the medical arts by monitoring the patient for signs of disease and adjusting treatment accordingly.

[0503] The term "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.

[0504] 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.

[0505] 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, 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 successful metastasis," as described in Swartz, et al., Cancer Res., 2012, 72, 2473. Although tumors express antigens that should be recognized by T cells, tumor clearance by the immune system is rare due to immunosuppression by the microenvironment.

[0506] In some embodiments, the invention includes a method of treating cancer with a TIL population, where 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, where 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 prior to TIL infusion) and fludarabine 25 mg / m2 / day for 5 days (27-23 days prior to 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 until physiological tolerance.

[0507] 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"). Thus, 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.

[0508] The term "effective amount" or "therapeutically effective amount" refers to an amount of a compound or combination of compounds described herein that is sufficient to achieve the intended use, including but not limited to disease treatment. Therapeutically effective amounts may vary depending on the intended use (in vitro or in vivo), or 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 particular response in target cells (e.g., reduced platelet adhesion and / or cell migration). The particular dose will vary depending on the particular compound selected, the dosing regimen to be followed, whether the compound is administered in combination with other compounds, the timing of administration, the tissue to which it is administered, and the physical delivery system in which the compound is delivered.

[0509] The terms "treatment", "treating", "treating" and the like refer to obtaining a desired pharmacological and / or physiological effect. The effect may be prophylactic 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. As used herein, "treatment" 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 as having the disease, (b) inhibiting the disease, i.e., arresting its onset or progression, and (c) relieving the disease, i.e., causing regression of the disease and / or relieving one or more disease symptoms. "Treatment" is also 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.

[0510] As used herein, the term "immune checkpoint inhibitor (ICI)" has its general meaning in the art and refers to any compound that inhibits the function of immune inhibitory checkpoint proteins. As used herein, the term "immune checkpoint protein" has its general meaning in the art and refers to a molecule expressed by T cells that either increases (stimulatory checkpoint molecules) or decreases (inhibitory checkpoint molecules) signals. Immune checkpoint molecules are recognized in the art as constituting elements of immune checkpoint pathways similar to the CTLA-4 and PD-1 dependent pathways (see, e.g., Pardoll, 2012. Nature Rev Cancer 12:252-264; Mellman et al., 2011. Nature 480:480- 489). Examples of inhibitory checkpoint molecules include A2AR, B7-H3, B7-H4, CD277, IDO, KIR, VISTA, PD-1, CTLA-4, LAG-3, HAVCR2 (TIM-3), Cish, TGFβ, PKA, CBL-B, PPP2CA, PPP2CB, PTPN6, PTPN22, PDCD1, BTLA, CD160, TIGIT, TET2, BAFF (BR3), CD96, CRTAM, LAIR1, SIGLEC7, SIGLEC9, CD244, TNFRSF10B, These include TNFRSF10A, CASP8, CASP10, CASP3, CASP6, CASP7, FADD, FAS, SMAD2, SMAD3, SMAD4, SMAD10, SKI, SKIL, TGIF1, IL10RA, IL10RB, HMOX2, IL6R, IL6ST, EIF2AK4, CSK, PAG1, SIT1, FOXP3, PRDM1, BATF, GUCY1A2, GUCY1A3, GUCY1B2, GUCY1B3, TOX, SOCS1, ANKRD11, and BCOR. For example, immune checkpoint genes that may be silenced or inhibited in the TILs of the present invention may be selected from the group including PD-1, CTLA-4, LAG-3, TIM-3, Cish, CBL-B, TIGIT, TET2, TGFβ, and PKA.BAFF(BR3) is described in Bloom, et al., J. Immunother., 2018, in press. According to another embodiment, the immune checkpoint genes that may be silenced or inhibited in the TILs of the present invention may be selected from the group including PD-1, LAG-3, TIM-3, CTLA-4, TIGIT, TET2, CISH, TGFβR2, PRA, CBLB, BAFF(BR3), and combinations thereof.

[0511] Inhibition includes reduction and complete blockage of function. Preferred immune checkpoint inhibitors are antibodies that specifically recognize immune checkpoint proteins. Several immune checkpoint inhibitors are known, and as well as these known immune checkpoint protein inhibitors, alternative immune checkpoint inhibitors may be developed in the (near) future. Immune checkpoint inhibitors include peptides, antibodies, nucleic acid molecules, and small molecules.

[0512] "Non-myeloablative chemotherapy", "non-myeloablative lymphodepletion", "NMALD", "NMA LD", "NMA-LD", and any variants of the foregoing, are used interchangeably to refer to chemotherapy regimens designed to avoid depletion of a patient's myeloid immune cells while depleting the patient's lymphoid immune cells. Typically, a patient undergoes a course of non-myeloablative chemotherapy prior to administering tumor-infiltrating lymphocytes to the patient, as described herein.

[0513] The term "heterologous" when used in reference to a portion 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, a nucleic acid is typically produced recombinantly and has two or more sequences from unrelated genes arranged to create a new functional nucleic acid, such as a promoter from one source and a coding region from another source, or a coding region from a different source. 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).

[0514] 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 certain percentage of the same nucleotides 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. Programs suitable 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, available from DNASTAR, ALIGN-2 (Genentech, South San Francisco, California), or MegAlign are additional publicly available software programs that can be used to align sequences. Those skilled in the art can determine the appropriate parameters for maximum alignment according to the particular alignment software. In certain embodiments, the default parameters of the alignment software are used.

[0515] As used herein, the term "variant" includes, but is not limited to, an antibody or fusion protein that comprises 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 include 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 the antigen. The term variant also includes pegylated antibodies or proteins.

[0516] 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 TIL cell populations 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, a second expanded TIL or a second additional expanded TIL (e.g., such as those described in step D of FIG. 8, including TILs referred to as reREP TILs).

[0517] TILs can generally be either biochemically defined using cell surface markers or functionally defined 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, e.g., TILs can be considered potent if 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, greater than about 1000 pg / mL.

[0518] 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.

[0519] The term "RNA" defines a molecule that contains at least one ribonucleotide residue. The term "ribonucleotide" defines a nucleotide that has a hydroxyl group at the 2' position of the bD-ribofuranose moiety. The term RNA includes double-stranded RNA, single-stranded RNA, isolated RNA such as partially purified RNA, essentially pure RNA, synthetic RNA, recombinantly produced RNA, and altered RNA that differs from naturally occurring RNA by the addition, deletion, substitution, and / or alteration of one or more nucleotides. The nucleotides of the RNA molecules described herein may also include non-naturally occurring nucleotides or non-standard nucleotides such as chemically synthesized nucleotides or deoxynucleotides. These altered RNAs may be referred to as analogs or analogs of naturally occurring RNA.

[0520] The term "pharmaceutically acceptable carrier" or "pharmaceutically acceptable excipient" is intended to include any and all solvents, dispersion media, coatings, antibacterial and antifungal agents, isotonic and absorption delaying agents, and inactive ingredients. The use of such pharmaceutically acceptable carriers or pharmaceutically acceptable excipients for active pharmaceutical ingredients is well known in the art. Except insofar as any conventional pharmaceutically acceptable carrier or pharmaceutically acceptable excipient is incompatible with the active pharmaceutical ingredient, its use in the therapeutic compositions of the present invention is contemplated. Additional active pharmaceutical ingredients, such as other drugs, can also be incorporated into the compositions and methods described.

[0521] The terms "about" and "approximately" mean within a statistically significant range of values. Such ranges may be within an order of magnitude, preferably within 50%, more preferably within 20%, even more preferably within 10%, and even more preferably within 5% of a given value or range. The allowable deviation encompassed by the terms "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 features are not, and need not be, precise, but may be approximate and / or may be greater or smaller, as appropriate, reflecting tolerances, conversion factors, rounding, measurement errors, and the like, and other factors known to those of ordinary skill in the art. In general, a dimension, size, formulation, parameter, shape, or other quantity or feature is "about" or "approximately" whether or not it is expressly stated as such. It should be noted that embodiments of very different sizes, shapes, and dimensions may employ the described arrangements.

[0522] When used in the appended claims, the transitional terms "comprising," "consisting essentially of," and "consisting of" define the claim in its original and amended form with respect to whether additional unrecited claim elements or steps, if any, are excluded from the claim. 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 be more specifically defined in alternative embodiments by any of the transitional terms "comprising," "consisting essentially of," and "consisting of."

[0523] The terms "antibody" and its plural "antibodies" refer to whole immunoglobulins and to 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. Each light chain is composed of a light chain variable region (abbreviated herein as V L The light chain constant region is composed of one domain, C L The V of the antibody H and V L The regions can be further subdivided into regions of hypervariability, termed complementarity determining regions (CDRs) or hypervariable regions (HVRs), which may be interspersed with more conserved regions, termed framework regions (FRs). H and V Lis composed of three CDRs and four FRs arranged from amino-terminus to carboxy-terminus in the following order: FR1, CDR1, FR2, CDR2, FR3, CDR3, FR4. The variable regions of the heavy and light chains contain 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.

[0524] 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. The term "antigen" as used herein 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 contains or is bound to 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 a large number of other antibodies or TCRs that may be induced by other antigens.

[0525] 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 made 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 antibodies 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 proteins, to obtain the synthesis of the monoclonal antibody in the recombinant host cells. Recombinant production of antibodies is described in more detail below.

[0526] 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 F(ab′)2 fragment, which is a bivalent fragment comprising two Fab fragments linked by disulfide bridges at the hinge region; and (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 (v) an Fv fragment consisting of a V H or VL The Fv fragment includes two domains, the V domain and the V domain. L and V H Although the V L and V H The regions may be joined by synthetic linkers that allow them to be made into a single protein chain that pairs 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 of skill in the art, and the fragments are screened for utility in the same manner as intact antibodies. In some embodiments, the scFv protein domain is a V H Part and V L The scFv molecule comprises a V L If the domain is the N-terminal portion of an scFv molecule, then V L -LV H , or V H If the domain is the N-terminal portion of an scFv molecule, then V H -LV L 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.

[0527] The term "human antibody" as used herein is intended to include antibodies having variable regions in which both the framework and CDR regions are derived from human germline immunoglobulin sequences. Furthermore, if the antibody contains a constant region, the constant region is also derived from human germline immunoglobulin sequences. The human antibodies of the invention may include amino acid residues not encoded by human germline immunoglobulin sequences (e.g., mutations introduced by random or site-specific mutagenesis in vitro or by somatic mutation in vivo). The term "human antibody" as used herein is not intended to include antibodies in which CDR sequences derived from the germline of another mammalian species, such as a mouse, have been grafted onto human framework sequences.

[0528] The term "human monoclonal antibody" refers to antibodies displaying a single binding specificity having variable regions in which both the framework and CDR regions are derived from human germline immunoglobulin sequences. In some embodiments, human monoclonal antibodies are produced by a hybridoma 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.

[0529] The term "recombinant human antibody" as used herein includes all human antibodies that are prepared, expressed, created, or isolated by recombinant means, such as (a) antibodies isolated from 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 improve the V and 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 exist naturally within the human antibody germline repertoire in vivo.

[0530] As used herein, "isotype" refers to the antibody class (e.g., IgM or IgG1) that is encoded by heavy chain constant region genes.

[0531] The phrases "an antibody that recognizes an antigen" and "an antibody specific for an antigen" are used interchangeably herein with the term "an antibody that specifically binds to an antigen."

[0532] 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 term "conjugate," "antibody drug conjugate," "ADC," or "immunoconjugate" refers 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.

[0533] The terms "humanized antibody," "humanized antibodies," and "humanization" are intended to refer to an antibody 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 antibody) in which residues from a hypervariable region of the recipient are replaced by residues from a hypervariable region of a non-human species, such as mouse, rat, rabbit, or non-human primate (donor antibody) having the desired specificity, affinity, and capacity. In some cases, Fv framework (FR) residues of the human immunoglobulin are replaced by corresponding non-human residues. Furthermore, humanized antibodies may include residues that are not found in the recipient antibody or donor antibody. These modifications are made to further improve antibody performance. Generally, a humanized antibody will comprise substantially all of at least one, and typically two, variable domains, in which all or substantially all of the hypervariable loops correspond to those of a non-human immunoglobulin and all or substantially all of the FR regions are from 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 may also be modified to use any Fc variant known to confer improved (e.g., reduced) effector function and / or FcR binding.Fc variants are described 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, WO2002 / 4 4215A2, WO2002 / 060919A2, WO2003 / 074569A2, WO2004 / 016750A2, WO2004 / 029207A2, WO2004 / 03 5752A2, WO2004 / 063351A2, WO2004 / 074455A2, WO2004 / 099249A2, WO2005 / 040217A2, WO2005 / 07 Nos. WO2005 / 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).

[0534] 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., the variable region sequences are derived from a murine antibody and the constant region sequences are derived from a human antibody.

[0535] 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 a heavy chain variable domain (V H ). If a linker that is too short to pair the two domains on the same chain is used, the domains are forced to pair with the complementary domains on another chain and create two antigen-binding sites. Bispecific antibodies are more fully described in, for example, 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.

[0536] The term "glycosylation" refers to modified derivatives of antibodies. An aglycosylated antibody lacks glycosylation. Glycosylation can be altered, for example, to increase the affinity of the antibody for an antigen. Such carbohydrate modifications can be achieved, for example, by altering one or more glycosylation sites within the antibody sequence. For example, one or more amino acid substitutions can be made that result in the elimination of one or more variable region framework glycosylation sites, thereby eliminating glycosylation at that site. As described in U.S. Pat. Nos. 5,714,350 and 6,350,861, aglycosylation can increase the affinity of the antibody for the antigen. Additionally or alternatively, antibodies can be made with altered types of glycosylation, for example, hypofucosylated antibodies with reduced amounts of fucosyl residues or antibodies with increased bisecting GlcNac structures. Such altered glycosylation patterns have been demonstrated to increase the potency of the antibody. 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, cell lines Ms704, Ms705, and Ms709 lack the fucosyltransferase gene FUT8 (alpha(1,6) fucosyltransferase), such that antibodies expressed in Ms704, Ms705, and Ms709 cell lines lack fucose on their carbohydrates. Ms704, Ms705, and Ms709 FUT8- / - cell lines were created by targeted disruption of the FUT8 gene in CHO / DG44 cells using two replacement vectors (see, for example, 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, such that antibodies expressed in such cell lines exhibit hypofucosylation by reducing or eliminating alpha 1,6 linkage-related enzymes, and also describes cell lines with reduced or no enzymatic activity for adding fucose to N-acetylglucosamine attached 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, Lec 13 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, fucose residues of antibodies can be cleaved using a fucosidase enzyme. For example, the fucosidase alpha-L-fucosidase removes fucosyl residues from antibodies, as described in Tarentino, et al., Biochem. 1975, 14, 5516-5523.

[0537] "PEGylation" refers to a modified antibody or fragment thereof that is reacted with polyethylene glycol (PEG), such as a reactive ester or aldehyde derivative of PEG, under conditions in which one or more PEG groups are attached to the antibody or antibody fragment. PEGylation can, for example, increase the biological (e.g., serum) half-life of an antibody. Preferably, PEGylation is carried out via an acylation or 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(C 1 -C 10 It is intended to encompass any of the forms of PEG that have been used to derivatize other proteins, such as alkoxy- or aryloxy-polyethylene glycols, 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 present invention, for example as described in European Patent Nos. EP0154316 and EP0401384, and U.S. Patent No. 5,824,778, the disclosures of each of which are incorporated herein by reference.

[0538] 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. In Europe, a similar bio or "biosimilar" drug is a biological product that is similar to another biological product already approved for use by the European Medicines Agency. The term "biosimilar" is also used synonymously by regulatory agencies in other countries and regions. Biological products or biopharmaceuticals 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), then the protein approved by the drug regulatory agency for aldesleukin is a "biosimilar" of aldesleukin or is 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 relevant 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, and therefore in Europe, a biosimilar may be authorised or approved for the subject of an authorisation or authorisation application under Article 6 of Regulation (EC) No 726 / 2004 and Article 10(4) of Directive 2001 / 83 / EC. The original biological product already authorised may be 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. In addition, product-specific guidelines, including those relevant for monoclonal antibody biosimilars, are provided by the EMA on a product-by-product basis and are published on its website.The biosimilars described herein may be similar to the reference medicinal product in terms of quality characteristics, biological activity, mechanism of action, safety profile, and / or efficacy. Furthermore, the biosimilars may be used or intended to be used to treat the same condition as the reference medicinal product. Thus, the biosimilars described herein may be considered to have similar or very similar quality characteristics as the reference medicinal product. Alternatively, or in addition, the biosimilars described herein may be considered to have similar or very similar biological activity as the reference medicinal product. Alternatively, or in addition, the biosimilars described herein may be considered to have a similar or very similar safety profile as the reference medicinal product. Alternatively, or in addition, the biosimilars described herein may be considered to have similar or very similar efficacy as the reference medicinal product. As described herein, biosimilars in Europe are compared to a reference medicinal product authorized by the EMA. However, in some cases, the biosimilars may be compared to a biopharmaceutical product authorized outside the European Economic Area (a non-EEA authorized "comparator") in certain studies. Such studies include, for example, certain clinical studies and in vivo non-clinical studies. As used herein, the term "biosimilar" also relates to a biopharmaceutical that has been or can be compared to a non-EEA 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 in the amino acid structure (including, e.g., amino acid deletions, additions, and / or substitutions) that do not significantly affect the function of the polypeptide. Biosimilars may include amino acid sequences with 97% or more sequence identity, e.g., 97%, 98%, 99%, or 100%, to the amino acid sequence of its reference pharmaceutical. Biosimilars may include one or more post-translational modifications, e.g., but not limited to, glycosylation, oxidation, deamidation, and / or cleavage, that are different from the post-translational modifications of the reference pharmaceutical, provided that the difference does not result in a change in the safety and / or efficacy of the pharmaceutical. Biosimilars may have the same or different glycosylation pattern as the reference pharmaceutical.In particular, but not exclusively, a biosimilar may have a different glycosylation pattern if the difference addresses or is intended to address a safety concern associated with the reference drug. In addition, a biosimilar may deviate from the reference drug, for example, in its strength, dosage form, formulation, excipients, and / or presentation, provided that the safety and efficacy of the drug is not compromised. A biosimilar may include differences, for example, in its pharmacokinetic (PK) and / or pharmacodynamic (PD) profile compared to the reference drug, but is still considered sufficiently similar to the reference drug to be approved or considered suitable for approval. In certain circumstances, a biosimilar exhibits different binding characteristics compared to the reference drug, and the different binding characteristics are considered by regulatory authorities, such as the EMA, to not 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.

[0539] II. Generation of cryopreserved TIL preparations from tumor harvest Some embodiments of the invention provided herein relate to methods of generating cryopreserved TIL preparations from tumor samples taken from cancer patients before the patients receive immune checkpoint inhibitors (ICIs) and / or standard of care treatments.

[0540] In some embodiments, patients undergo ICI treatment (such as an anti-immune checkpoint antibody described herein) following tumor harvest and cryopreservation.

[0541] In some embodiments, the patient receives standard care treatment for cancer after tumor harvest and cryopreservation. Some of the treatment methods described herein include administering standard care treatment to the patient. As used herein, "standard care treatment" is a treatment process that includes a drug or combination of drugs, radiation therapy, surgery, or other medical interventions that are recognized by physicians as appropriate, accepted, and / or widely used for a particular type of patient, disease, or clinical situation. Standard care treatments for treating different types of cancer are well known by those skilled in the art. For example, the National Comprehensive Cancer Network (NCCN), an alliance of 21 major cancer centers in the United States, publishes the NCCN Clinical Practice Guidelines in Oncology (NCCN GUIDELINES®), which provides detailed, up-to-date information on standard care treatments for a wide variety of cancers (see NCCN GUIDELINES®, 2013). In some embodiments, the standard care treatment is chemotherapy, radiation therapy, surgery, targeted therapy, or any combination thereof.

[0542] In some embodiments, cryopreserved tumor harvest from a patient or subject is used to generate a TIL population after the patient has been treated with an ICI and / or standard of care therapy, and is indicative of cancer progression. In some embodiments, after cryopreservation of the tumor harvest, the patient or subject receives an ICI and / or standard of care therapy and is monitored for cancer progression. In some embodiments, cancer progression indicates the need for autologous TIL therapy.

[0543] In other embodiments, cryopreservation of tumor specimens from patients or subjects is completed prior to the progression of cancer.

[0544] In some embodiments, the methods disclosed herein provide a pharmaceutical economic advantage in the form of avoiding the costs of TIL production in cases where a patient does not develop progressive disease or is otherwise indicated for TIL therapy in the future.

[0545] 15. The method of any one of claims 1 to 14, wherein the patient exhibits progressive disease at least about 1 week, 2 weeks, 3 weeks, 1 month, 2 months, 3 months, 4 months, 5 months, 6 months, 7 months, 8 months, 9 months, 10 months, 11 months, 12 months, 13 months, 14 months, 15 months, 16 months, 17 months, 18 months, 19 months, 20 months, 21 months, 22 months, 23 months, 24 months, 25 months, 26 months, 27 months, 28 months, 29 months, 30 months, 31 months, 32 months, 33 months, 34 months, 35 months, 36 months after the cryopreservation step.

[0546] In some embodiments, the cryopreserved TIL preparations can be made from tumor samples containing TIL populations obtained and / or received from a patient or subject. The patient's tumor sample 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, multilesion 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 multilesion sampling (i.e., obtaining samples from one or more tumor sites and / or locations of a patient, as well as one or more tumors at the same location or in close proximity). In general, the tumor sample can be from any solid tumor, including primary tumors, invasive tumors, or metastatic tumors. The tumor sample can be a liquid tumor, such as a tumor obtained from a hematological malignancy. The solid tumor can be of lung tissue. In some embodiments, useful TILs are obtained from non-small cell lung cancer (NSCLC). The solid tumor can be of skin tissue. In some embodiments, useful TILs are obtained from melanoma.

[0547] Once obtained, tumor samples were generally cut into pieces using sharp dissection ranging from 1 to approximately 8 mm 3 Fragmented into small pieces of about 2-3 mm 3are particularly useful. In some embodiments, TILs are cultured from these fragments using enzymatic tumor digests. Such tumor digests can be generated by incubation in enzymatic medium (e.g., Roswell Park Memorial Institute (RPMI) 1640 buffer, 2 mM glutamate, 10 mcg / mL gentamicin, 30 units / mL DNase, and 1.0 mg / mL collagenase) followed by mechanical dissociation (e.g., using a tissue dissociator). Tumor digests can be generated by placing the tumor in enzymatic medium, mechanically dissociating the tumor for approximately 1 minute, followed by incubation in 5% CO 2 The cells can be generated by incubating in a 50 mL hood at 37° C. for 30 minutes, followed by repeated cycles of mechanical dissociation and incubation under the aforementioned conditions until only small tissue fragments are present. If at the end of this process 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 aforementioned methods can be used in any of the embodiments described herein for the method of expanding TILs or the method of treating cancer.

[0548] In some embodiments, the cryopreserved TIL preparation is stored for future use. In some embodiments, cryopreservation of the tumor harvest is completed prior to progression. In some embodiments, after generation of the cryopreserved TIL preparation, the patient is monitored for the development of progressive disease on or after ICI and / or standard of care treatment. In some embodiments, the patient shows progressive cancer during or after ICI and / or standard of care treatment and is adapted for autologous TIL therapy. In some embodiments, after progression, the cryopreserved TIL preparation is thawed and expanded according to the expansion method described in the following section.

[0549] In some embodiments, the patient is a NSCLC cancer patient. In some embodiments, the patient is suffering from, suffering from, or susceptible to NSCLC. In some embodiments, the patient has metastatic NSCLC. In some embodiments, the patient has metastatic stage IV NSCLC.

[0550] In some embodiments, the subject or patient: i. Predefined tumor proportion score (TPS) of PD-L1 < 1%; ii. PD-L1 TPS score of 1%–49%, or iii. have at least one of the predetermined absence of one or more driver mutations.

[0551] In some embodiments, the NSCLC patient does not have one or more actionable driver mutations. In some embodiments, the actionable driver mutations disclosed herein include EGFR mutations, EGFR insertions, EGFR exon 20, KRAS mutations, BRAF mutations, BRAF V600E mutations, BRAF V600K mutations, BRAF These include, but are not limited to, V600 mutations, ALK mutations, c-ROS mutations (ROS1 mutations), ROS1 fusions, RET mutations, RET fusions, ERBB2 mutations, ERBB2 amplifications, BRCA mutations, MAP2K1 mutations, PIK3CA, CDKN2A, PTEN mutations, UMD mutations, NRAS mutations, KRAS mutations, NF1 mutations, MET mutations, MET splices, and / or altered MET signaling, TP53 mutations, CREBBP mutations, KMT2C mutations, KMT2D mutations, ARID1A mutations, RB1 mutations, ATM mutations, SETD2 mutations, FLT3 mutations, PTPN11 mutations, FGFR1 mutations, EP300 mutations, MYC mutations, EZH2 mutations, JAK2 mutations, FBXW7 mutations, CCND3 mutations, and GNA11 mutations. In some embodiments, the NSCLC exhibits a TPS of <1% with the absence of one or more driver mutations.

[0552] In some embodiments, the patient is naïve to all cancer treatments when the tumor is harvested. In some embodiments, the patient is naïve to targeted therapy. In some embodiments, the patient is naïve to ICI treatment when the tumor is harvested. In some embodiments, the patient is naïve to anti-VEGF (e.g., Avastin / Bevacizumab) treatment when the tumor is harvested. In some embodiments, the patient is naïve to chemotherapy treatment when the tumor is harvested. In some embodiments, the patient is naïve to a combination of two or more of the aforementioned treatments when the tumor is harvested.

[0553] In some embodiments, the patient is naïve to all cancer treatments when the cryopreserved TIL preparation is made. In some embodiments, the patient is naïve to targeted therapy. In some embodiments, the patient is naïve to ICI treatment when the cryopreserved TIL preparation is made. In some embodiments, the patient is naïve to anti-VEGF (e.g., Avastin / Bevacizumab) treatment when the cryopreserved TIL preparation is made. In some embodiments, the patient is naïve to chemotherapy treatment when the cryopreserved TIL preparation is made. In some embodiments, the patient is naïve to a combination of two or more of the aforementioned treatments when the cryopreserved TIL preparation is made.

[0554] In some embodiments, the patient is undergoing maintenance therapy when the tumor is harvested. In some embodiments, the patient's maintenance therapy is discontinued when the tumor is harvested. In some embodiments, the patient is on a drug holiday after discontinuation of maintenance therapy when the tumor is harvested, followed by resumption of maintenance therapy or a different therapy. In some embodiments, maintenance therapy is resumed after a tumor sample is harvested from the patient. In some embodiments, the cancer (such as NSCLC) is treated during or after maintenance therapy.

[0555] In some embodiments, at the time the tumor is harvested, the patient will receive first-line (1L) ICI and / or standard therapy for the cancer.

[0556] In some embodiments, at the time the tumor is harvested, the patient will receive second line (2L) ICI and / or standard therapy for the cancer.

[0557] In some embodiments, the patient's collected tumor sample is cryopreserved using a quick-freezing method or a controlled-rate freezing method. Exemplary quick-freezing and controlled-rate freezing methods can be found in International Patent Publication No. WO / 2020 / 061429, which is incorporated by reference in its entirety for all purposes.

[0558] In some embodiments, the quick-freezing method of the present invention comprises: (i) fragmenting the tumor tissue; (ii) incubating the fragments in a cryopreservation medium; (iii) freezing the fragments, wherein the freezing is flash freezing using the vapor phase of liquid nitrogen.

[0559] In one embodiment, the tumor tissue is fragmented into approximately spherical fragments having a diameter of about 1.5 mm to about 6 mm. In a preferred embodiment, the approximately spherical fragments have a diameter of about 6 mm. In one embodiment, the approximately spherical fragments have a diameter of about 3 mm.

[0560] In some embodiments, the tumor tissue is fragmented into generally rectangular pieces having a shortest edge length of at least 1.5 mm and a longest edge length of about 6 mm. In embodiments, the tumor tissue is fragmented into generally cubic pieces having an edge length of about 1.5 mm to 6 mm. In some embodiments, the generally cubic pieces have an edge length of about 6 mm. In some embodiments, the generally cubic pieces have an edge length of about 3 mm.

[0561] In some embodiments, the tissue sample is trimmed to separate non-tumor tissue from tumor tissue.

[0562] In some embodiments, the tumor tissue is from a dissected tumor. In embodiments, the tumor tissue is from a tumor biopsy. In some embodiments, the tumor tissue is from an incisional biopsy. In some embodiments, the tumor tissue is from an excision biopsy. In some embodiments, the tumor tissue may be from one or more core needle biopsies.

[0563] In some embodiments, fresh tumor tissue is trimmed into pieces having a cross-section of about 1.5 mm x 1.5 mm, about 2 mm x 2 mm, about 2.5 mm x 2.5 mm, about 3 mm x 3 mm, about 3.5 mm x 3.5 mm, about 4 mm x 4 mm, about 4.5 mm x 4.5 mm, about 5 mm x 5 mm, about 5.5 mm x 5.5 mm, or about 6 mm x about 6 mm.

[0564] In some embodiments, the tumor tissue is less than 12 hours old. In some embodiments, the tumor tissue is less than 8 hours old. In some embodiments, the tumor tissue is less than 3 hours old, less than 2 hours old, or less than 1 hour old.

[0565] In view of the present disclosure, any suitable cryopreservation medium known to one of skill in the art may be used in the methods described herein. Examples of suitable cryopreservation media include, but are not limited to, CryoStor® CS10, HypoThermosol®, or combinations thereof. In some embodiments, the cryopreservation medium comprises about 2% v / v DMSO to about 15% v / v DMSO. In some embodiments, the cryopreservation medium comprises about 2% v / v DMSO. In some embodiments, the cryopreservation medium comprises about 2% v / v DMSO. In some embodiments, the cryopreservation medium comprises about 3% v / v DMSO. In some embodiments, the cryopreservation medium comprises about 4% v / v DMSO. In some embodiments, the cryopreservation medium comprises about 5% v / v DMSO. In some embodiments, the cryopreservation medium comprises about 6% v / v DMSO. In some embodiments, the cryopreservation medium comprises about 7% v / v DMSO. In some embodiments, the cryopreservation medium comprises about 8% v / v DMSO. In some embodiments, the cryopreservation medium comprises about 9% v / v DMSO. In some embodiments, the cryopreservation medium comprises about 10% v / v DMSO. In some embodiments, the cryopreservation medium comprises about 11% v / v DMSO. In some embodiments, the cryopreservation medium comprises about 12% v / v DMSO. In some embodiments, the cryopreservation medium comprises about 13% v / v DMSO. In some embodiments, the cryopreservation medium comprises about 14% v / v DMSO. In some embodiments, the cryopreservation medium comprises about 15% v / v DMSO. In some embodiments, the cryopreservation medium comprises at least one antimicrobial agent. Any suitable antimicrobial agent known to one of skill in the art in view of the present disclosure can be used in the methods described herein. In some embodiments, the cryopreservation medium comprises gentamicin. In some embodiments, the cryopreservation medium comprises gentamicin at a concentration of at least 50 μg / mL. In some embodiments, the cryopreservation medium comprises gentamicin at a concentration of at least 40 μg / mL. In some embodiments, the cryopreservation medium comprises gentamicin at a concentration of at least 30 μg / mL.In some embodiments, the cryopreservation medium comprises gentamicin at a concentration of at least 20 μg / mL.

[0566] In some embodiments, the tumor fragments are incubated in the cryopreservation medium for about 20 minutes to about 70 minutes. In some embodiments, the tumor fragments are incubated in the cryopreservation medium for about 30 minutes to about 60 minutes. In some embodiments, the incubation is at least 10 minutes, at least 20 minutes, at least 25 minutes, at least 30 minutes, at least 35 minutes, at least 40 minutes, at least 45 minutes, at least 50 minutes, at least 55 minutes, at least 60 minutes, or at least 70 minutes. In some embodiments, the incubation is about 10 minutes, about 20 minutes, about 25 minutes, about 30 minutes, about 35 minutes, about 40 minutes, about 45 minutes, about 50 minutes, about 55 minutes, about 60 minutes, or about 70 minutes. In some embodiments, the incubation is less than 10 minutes, less than 20 minutes, less than 25 minutes, less than 30 minutes, less than 35 minutes, less than 40 minutes, less than 45 minutes, less than 50 minutes, less than 55 minutes, less than 60 minutes, or less than 70 minutes. In some embodiments, the incubation time is proportional to the tumor fragment density, hi some embodiments, the incubation time is proportional to the surface-to-volume ratio of the tumor fragment.

[0567] In some embodiments, the tumor fragments are incubated in cryopreservation medium at a temperature of about 2°C to about 8°C.

[0568] In some embodiments, the tumor tissue is washed with a physiologically buffered isotonic saline solution. In some embodiments, the washing comprises three successive washes of at least 3 minutes each, with the physiologically buffered isotonic saline being replaced after each successive wash. In some embodiments, the physiologically buffered isotonic saline solution comprises Hanks' Balanced Salt Solution (HBSS). In some embodiments, the physiologically buffered isotonic saline solution comprises Tris-buffered saline (TBS). In some embodiments, the physiologically buffered isotonic saline solution comprises phosphate-buffered saline (PBS). In some embodiments, the physiologically buffered isotonic saline solution comprises Dulbecco's Phosphate-buffered Saline (DPBS). In some embodiments, the physiologically buffered isotonic saline solution in one successive wash can be a different physiologically buffered isotonic saline solution than that used in one or more of the other successive washes.

[0569] In certain embodiments, freezing occurs at a temperature ranging from about -125°C to about -196°C. In certain embodiments, freezing occurs at a temperature ranging from about -140°C to about -185°C. In certain embodiments, freezing occurs at a temperature ranging from about -140°C to about -175°C. In certain embodiments, freezing occurs at a temperature of about -145°C. In some embodiments, freezing occurs in the vapor phase of liquid nitrogen.

[0570] A problem known in the art is cryopreserving cells or tissues without damaging them during the freezing process. Without being bound by theory, one source of damage during freezing is intracellular ice nucleation, resulting in cell rupture. Muldrew and McGann, in "The osmotic rupture hypothesis of intracellular freezing injury", Biophysical Journal, 66:532-41 (1994), outline a quantitative theory of this known and widely recognized difficulty with cell, and particularly whole tissue, cryopreservation. Acker and McGann further develop the basic mechanisms of intracellular ice formation and cell damage in a later article, "Membrane damage occurs during the formation of intracellular ice," Cryo Letter, 22:241-54 (2001).

[0571] Upon thawing, damage during freezing is detected, where the tissue has substantially lost physiological structure or the cells that make up the tissue have substantially lost viability. Viability can be determined by the percentage of cells introduced into the culture medium compared to the number of cells that are allowed to grow or that exhibit markers of normal cell function. Numerous methods for identifying the percentage of viable cells are known in the art, including but not limited to dye exclusion tests such as trypan blue dye exclusion. See, for example, Strober, Curr. Protoc. Immunol., 2001, Appendix 3B, available at https: / / dx.doi.org / 10.1002 / 0471142735.ima03bs21. Viability may also be determined by metabolic activity assays, including but not limited to the MTT assay, where MTT, 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide, is metabolized by cellular enzymes to formazan. This enzymatic reaction converts the yellow MTT to purple formazan. See, e.g., Berridge et al., Tetrazolium dyes as tools in cell biology: new insights into their cellular reduction. Biotechnology Annual Review, 11:127-152 (2005); Mosmann, "Rapid colorimetric assay for cellular growth and survival: application to proliferation and cytotoxicity assays," J. Immunol. Methods 65(1-2):55-63 (1983).

[0572] Without being bound by theory, it is hypothesized that slow cooling produces harmless intracellular ice, see Acker and McGann, “Protective effect of intracellular ice during freezing?” Cryobiology, 46(2):197-202 (2003). The traditional approach to achieve freezing without undue cell damage or without significantly reducing cell viability has been to employ slow freezing rates. Watson et al., U.S. Pat. No. 5,891,617, emphasizes this approach, for example, step (c) of claim 1 teaches a very slow cooling rate of “about −0.3° C. / min or less.” Similarly, Comhaire et al., U.S. Pat. No. 9,938,495, teaches that “high post-thaw cell viability was obtained by slow freezing using DMSO-free cryopreservation medium” for stem cells.

[0573] Based on these exemplary teachings, the methods and products of the present disclosure are surprising and unexpected. Moreover, the present disclosure, including the examples and data therein, demonstrates a technical solution to the problem of rapidly and efficiently cryoprotecting tumor tissue, tumor fragments, or tumor specimens for use in the production of therapeutic tumor infiltrating lymphocytes.

[0574] In some embodiments, the method of cryopreserving tumor tissue for production of tumor infiltrating lymphocytes (TILs) further comprises the step (iv) of storing the frozen fragments at a temperature at least below −130° C. In some embodiments, the frozen fragments are stored in the vapor phase of liquid nitrogen. In some embodiments, the frozen fragments are stored immersed in liquid nitrogen. In some embodiments, the cryopreserved fragments are stored for later production of TILs for autotherapeutic use.

[0575] In some embodiments, the present disclosure provides herein methods for cryopreserving tumor tissue using controlled rate freezing / slow freezing methods.

[0576] In some embodiments, the present invention provides a method for cryopreserving tumor tissue, as well as a cryopreserved tumor tissue, comprising: (i) adding a cryopreservation medium to a closable container; (ii) pre-cooling the closeable container in a controlled rate freezer; (iii) fragmenting the tumor tissue to obtain tumor fragments; (iv) placing the tumor fragments in a closable container containing a cryopreservation medium and closing the container; (v) optionally incubating the sealed container containing the tumor fragments and the cryopreservation medium; (vi) slow freezing the container in a controlled rate freezer; (vii) transferring the container to a liquid nitrogen freezer.

[0577] In some embodiments, the present invention provides a method for cryopreserving tumor tissue, as well as a cryopreserved tumor tissue, comprising: (i) placing tumor fragments obtained from fragmenting tumor tissue in a pre-chilled closable container containing a cryopreservation medium and closing the container; (ii) optionally incubating the sealed container containing the tumor fragments and the cryopreservation medium; (iii) slow freezing the container in a controlled rate freezer; (iv) transferring the container to a liquid nitrogen freezer.

[0578] In some embodiments, the present invention provides a method for cryopreserving tumor tissue, as well as a cryopreserved tumor tissue, comprising: (i) placing a tumor digest obtained from digesting tumor tissue in an enzymatic medium or tumor fragments produced from fragmenting tumor tissue in a pre-chilled closable container containing a cryopreservation medium and closing the container; (ii) optionally incubating the sealed container containing the tumor digest and the cryopreservation medium; (iii) slow freezing the container in a controlled rate freezer; (iv) transferring the container to a liquid nitrogen freezer.

[0579] In some embodiments, the present invention provides a method for cryopreserving tumor tissue, as well as a cryopreserved tumor tissue, comprising: (i) adding a cryopreservation medium to a closable container; (ii) pre-cooling the closeable container in a controlled rate freezer; (iii) digesting the tumor tissue in an enzymatic medium to obtain a tumor digest; (iv) placing the tumor digest in a cryopreservation medium in a closable container and closing the container; (v) optionally incubating the sealed container containing the tumor digest and the cryopreservation medium; (vi) slow freezing the container in a controlled rate freezer; (vii) transferring the container to a liquid nitrogen freezer.

[0580] In view of the present disclosure, any suitable cryopreservation medium known to one of skill in the art may be used in the methods described herein. Examples of suitable cryopreservation media include, but are not limited to, CryoStor® CS10, HypoThermosol®, or combinations thereof. In some embodiments, the cryopreservation medium comprises about 2% v / v DMSO to about 15% v / v DMSO. In some embodiments, the cryopreservation medium comprises about 2% v / v DMSO. In some embodiments, the cryopreservation medium comprises about 2% v / v DMSO. In some embodiments, the cryopreservation medium comprises about 3% v / v DMSO. In some embodiments, the cryopreservation medium comprises about 4% v / v DMSO. In some embodiments, the cryopreservation medium comprises about 5% v / v DMSO. In some embodiments, the cryopreservation medium comprises about 6% v / v DMSO. In some embodiments, the cryopreservation medium comprises about 7% v / v DMSO. In some embodiments, the cryopreservation medium comprises about 8% v / v DMSO. In some embodiments, the cryopreservation medium comprises about 9% v / v DMSO. In some embodiments, the cryopreservation medium comprises about 10% v / v DMSO. In some embodiments, the cryopreservation medium comprises about 11% v / v DMSO. In some embodiments, the cryopreservation medium comprises about 12% v / v DMSO. In some embodiments, the cryopreservation medium comprises about 13% v / v DMSO. In some embodiments, the cryopreservation medium comprises about 14% v / v DMSO. In some embodiments, the cryopreservation medium comprises about 15% v / v DMSO. In some embodiments, the cryopreservation medium comprises at least one antimicrobial agent. Any suitable antimicrobial agent known to one of skill in the art in view of the present disclosure can be used in the methods described herein. In some embodiments, the cryopreservation medium comprises gentamicin. In some embodiments, the cryopreservation medium comprises gentamicin at a concentration of at least 50 μg / mL. In some embodiments, the cryopreservation medium comprises gentamicin at a concentration of at least 40 μg / mL. In some embodiments, the cryopreservation medium comprises gentamicin at a concentration of at least 30 μg / mL.In some embodiments, the cryopreservation medium comprises gentamicin at a concentration of at least 20 μg / mL.

[0581] In view of the present disclosure, any suitable closable container known to one of skill in the art can be used in the methods described herein. Examples of suitable closable containers include, but are not limited to, capped microcentrifuge tubes, lidded microcentrifuge tubes, and cryogenic specimen storage vials. The term "cryogenic sample storage vial" is meant to include terms such as cryogenic sample storage vials, cryocontainers, cryotubes, etc., including closed, sealed, or reclosable containers (e.g., with screw caps or friction-sealing snap caps) that allow the container to be safely and securely stored at low temperatures (meaning temperatures below -80°C, and optionally immersed in liquid nitrogen or suspended in the gas phase above liquid nitrogen at a temperature of about -196°C). Capped or lidded microcentrifuge tubes and cryovials, typically manufactured from polyethylene or polypropylene, are often used as cryogenic sample storage vials.

[0582] In some embodiments, the closable container is filled with cryopreservation medium at about 50% to about 85% volume. In some embodiments, the closable container is filled with cryopreservation medium at about 50% to about 85% volume. In some embodiments, the closable container is filled with cryopreservation medium at about 50% to about 75% volume. In some embodiments, the closable container is filled with cryopreservation medium at about 50% to about 65% volume. In some embodiments, the closable container is filled with cryopreservation medium at about 50% to about 55% volume. In some embodiments, the closable container is filled with cryopreservation medium at about 60% to about 85% volume. In some embodiments, the closable container is filled with cryopreservation medium at about 60% to about 75% volume. In some embodiments, the closable container is filled with cryopreservation medium at about 60% to about 65% volume. In some embodiments, the closable container is filled with cryopreservation medium at about 70% to about 85% volume. In some embodiments, the closable container is filled with cryopreservation medium to about 70% to about 75% volume. In some embodiments, the closable container is filled with cryopreservation medium to about 80% to about 85% volume.

[0583] In some embodiments, the pre-chilling step comprises placing the closeable container in a controlled rate freezer for a period of at least about 5 minutes to about 8 hours at a temperature of about −80° C. to about 8° C. ..., about −79° C., about −78° C., about −77° C., about −76° C., about −75° C., about −70° C., about −65° C., about −60° C., about −55° C., about −50° C., about −45° C., about −40° C., about −35° C., about −30° C., about −25° C., about −20° C., about −15° C., about −10° C., about −5° C., about 0° C., about 1° C., about 2° C., about 3° C., about 4° C., about 5° C., about 6° C., about 7° C., about 8° C., or at least about 5 minutes, at least about 10 minutes, at least about This includes placing in a controlled rate freezer for a period of 15 minutes, at least about 20 minutes, at least about 25 minutes, at least about 30 minutes, at least about 35 minutes, at least about 40 minutes, at least about 45 minutes, at least about 50 minutes, at least about 55 minutes, at least about 1 hour, at least about 1.5 hours, at least about 2 hours, at least about 3 hours, at least about 4 hours, about 5 hours, at least about 6 hours, at least about 7 hours, at least about 8 hours, or more.

[0584] In some embodiments, the sealed container containing the tumor fragments and cryopreservation medium is incubated at a temperature of about 2-8° C. for about 30-60 minutes before slow freezing the container in a controlled rate freezer. In some embodiments, the container containing the tumor fragments and cryopreservation medium is incubated at about 2° C., about 3° C., about 4° C., about 5° C., about 6° C., about 7° C., about 8° C., or any temperature therebetween for about 5 minutes, about 10 minutes, about 15 minutes, about 20 minutes, about 25 minutes, about 30 minutes, about 35 minutes, about 40 minutes, about 45 minutes, about 50 minutes, about 55 minutes, about 60 minutes, or more, before slow freezing the container in a controlled rate freezer.

[0585] In view of the present disclosure, any suitable controlled rate freezer known to one of skill in the art may be used in the methods described herein. Examples of suitable controlled rate freezers include, but are not limited to, a Corning CoolCell™ device or a Nalgene Mr.Frosty™ device. In some embodiments, the controlled rate freezer is an IPA-free controlled rate freezer that cools at a rate of about -0.1°C / min to about -10°C / min. In some embodiments, the controlled rate freezer is an IPA-free controlled rate freezer that cools at a rate of about -0.1°C / min to about -10°C / min, about -0.2°C / min to about -5°C / min, about -0.5°C / min to about -2.5°C / min, about -1°C / min to about -2°C / min. In some embodiments, the controlled rate freezer is an IPA-free controlled rate freezer that cools at a rate of about -1°C / min.

[0586] In some embodiments, all of the controlled rate freezer locations are filled with closable containers containing cryopreservation medium. In some embodiments, 90% or more of the controlled rate freezer locations are filled with closable containers containing cryopreservation medium. In some embodiments, 80% or more of the controlled rate freezer locations are filled with closable containers containing cryopreservation medium. In some embodiments, 70% or more of the controlled rate freezer locations are filled with closable containers containing cryopreservation medium. In some embodiments, 60% or more of the controlled rate freezer locations are filled with closable containers containing cryopreservation medium. In some embodiments, 50% or more of the controlled rate freezer locations are filled with closable containers containing cryopreservation medium. In some embodiments, 40% or more of the controlled rate freezer locations are filled with closable containers containing cryopreservation medium.

[0587] As used herein, the term "slow freezing method" refers to a process in which a sample is cooled at a controlled rate in a refrigerated environment prior to final cryopreservation, such as in liquid nitrogen. In some embodiments, the cooling rate is about -0.1°C / min to about -10°C / min, about -0.2°C / min to about -5°C / min, about -0.5°C / min to about -2.5°C / min, about -1°C / min to about -2°C / min. In some embodiments, the cooling rate is about -1°C / min. In some embodiments, the cooling environment is a -80°C freezer set at about -90°C to about -70°C, e.g., about -90°C, about -89°C, about -88°C, about -87°C, about -86°C, about -85°C, about -84°C, about -83°C, about -82°C, about -81°C, about -80°C, about -79°C, about -78°C, about -77°C, about -76°C, about -75°C, about -74°C, about -73°C, about -72°C, about -71°C, about -70°C, or any temperature in between, or dry ice, etc.

[0588] In some embodiments, the slow freezing comprises incubating the controlled rate freezer at a temperature of about -70°C to about -90°C. In some embodiments, the slow freezing comprises incubating the controlled rate freezer at a temperature of about -75°C to about -85°C. In some embodiments, the slow freezing comprises incubating the controlled rate freezer at a temperature of about -78°C to about -80°C. In some embodiments, the slow freezing comprises incubating the controlled rate freezer with dry ice. In some embodiments, the slow freezing comprises incubating the controlled rate freezer in a -80°C freezer. In some embodiments, the slow freezing comprises incubating the controlled rate freezer in dry ice.

[0589] In some embodiments, the slow freezing comprises incubating the controlled rate freezer at a temperature of about -80°C for about 3-5 hours. In some embodiments, the slow freezing comprises incubating the controlled rate freezer at a temperature of about -80°C for about 3 hours. In some embodiments, the slow freezing comprises incubating the controlled rate freezer at a temperature of about -80°C for about 4 hours. In some embodiments, the slow freezing comprises incubating the controlled rate freezer at a temperature of about -80°C for about 5 hours.

[0590] In some embodiments, after recovery from freezing, the cells have a post-thaw viability of at least about 80%. In some embodiments, after recovery from freezing, the cells have a post-thaw viability of at least about 75%. In some embodiments, after recovery from freezing, the cells have a post-thaw viability of at least about 70%. In some embodiments, after recovery from freezing, the cells have a post-thaw viability of at least about 65%. In some embodiments, after recovery from freezing, the cells have a post-thaw viability of at least about 60%. In some embodiments, after recovery from freezing, the cells have a post-thaw viability of at least about 55%. In some embodiments, after recovery from freezing, the cells have a post-thaw viability of at least about 50%. In some embodiments, after recovery from freezing, the cells have a post-thaw viability of at least about 45%. In some embodiments, after recovery from freezing, the cells have a post-thaw viability of at least about 40%. In some embodiments, after recovery from freezing, the cells have a post-thaw viability of at least about 35%. In some embodiments, after recovery from freezing, the cells have a post-thaw viability of at least about 30%. In some embodiments, after recovery from freezing, the cells have a post-thaw viability of at least about 25%. In some embodiments, after recovery from freezing, the cells have a post-thaw viability of at least about 20%. In view of the present disclosure, any suitable method for measuring or determining post-thaw viability known in the art can be used in the methods described herein.

[0591] In some embodiments, tumor digests are generated by incubating tumors in enzyme media, 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, Calif.). In some embodiments, tumors are placed into a tumor dissociation enzyme mixture, including 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, protease type XIV (pronase), deoxyribonuclease I (DNase), trypsin inhibitor, any other dissociation enzyme or proteolytic enzyme, and any combination thereof. In other embodiments, the tumor is placed in a tumor dissociation enzyme cocktail that includes collagenase (including any blend or type of collagenase), neutral protease (dispase), and deoxyribonuclease I (DNase).

[0592] III. Gene Editing Process A. Overview: TIL expansion + gene editing In an embodiment of the present invention, the present invention relates to a method for expanding a TIL population, the method comprising one or more steps of gene editing at least a portion of the TILs to enhance their therapeutic efficacy. As used herein, "gene editing", "gene editing" and "genome editing" refer to a type of gene modification in which DNA is permanently modified in the genome of a cell, e.g., DNA is inserted, deleted, modified or replaced in the genome of a cell. In some embodiments, gene editing silences (sometimes referred to as gene knockout) or inhibits / reduces (sometimes referred to as gene knockdown) the expression of a DNA sequence. According to an embodiment of the present invention, gene editing technology is used to enhance the efficacy of a therapeutic population of TILs. Exemplary gene editing processes / methods of the present invention, as well as gene edited TIL products, can also be found in International Patent Application No. PCT / US22 / 14425, U.S. Provisional Application Nos. 63 / 304,498 and 63 / 242,373, all of which are incorporated herein by reference in their entirety for all relevant purposes.

[0593] The method for expanding tumor infiltrating lymphocytes (TILs) into a therapeutic TIL population can be carried out according to any embodiment of the method described herein, and the method further comprises gene editing at least a portion of the TILs. According to a further embodiment, the method for expanding TILs into a therapeutic population of TILs is carried out according to any embodiment of the method described in U.S. Pat. No. 10,517,894, U.S. Patent Application Publication No. 2020 / 0121719 A1, or U.S. Pat. No. 10,894,063 (which are incorporated herein by reference in their entirety), and the method further comprises gene editing at least a portion of the TILs. Thus, some embodiments of the present invention provide a therapeutic TIL population expanded according to any embodiment described herein, and at least a portion of the therapeutic population is gene edited, for example, at least a portion of the therapeutic TIL population transferred to an infusion bag is permanently gene edited.

[0594] In some embodiments of the present invention directed to methods for expanding TIL populations, the methods include one or more steps of introducing into at least a portion of a TIL nucleic acid, e.g., an mRNA, for transient expression of an immunomodulatory protein, e.g., an immunomodulatory fusion protein comprising an immunomodulatory protein fused to a membrane anchor, to produce modified TILs with (i) reduced dependency on cytokines when expanded in culture and / or (ii) enhanced therapeutic efficacy. As used herein, the terms "transient gene-editing", "transient gene editing", "transient phenotypic alteration", "transient phenotypic modification", "temporary phenotypic alteration", "temporary phenotypic modification", "transient cellular change", "transient cellular modification", "temporary cellular alteration", "temporary cellular modification", "transient expression", "transient alteration of expression", "transient alteration of protein expression", "transient modification", "transitory phenotypic alteration", "non-permanent phenotypic "transiently modified", "temporarily modified", "non-permanently modified""transiently altered," "temporarily altered," "temporarily altered," grammatical variations of any of the foregoing, and any expressions of similar import, refer to a type of cellular modification or phenotypic change in which a nucleic acid (e.g., mRNA) is introduced into a cell, e.g., by electroporation, calcium phosphate delivery, viral transduction, etc., transferred into the cell, expressed within the cell (e.g., expressing an immune protein, such as an immune modulating fusion protein comprising an immune modulating protein fused to a membrane anchor), to effect a transient or non-permanent phenotypic change within the cell, such as the transient display of a membrane-anchored immune modulating fusion protein on the cell surface. According to embodiments of the invention, transient phenotypic modification techniques are used to reduce dependency on cytokines in the expansion of TILs in culture and / or to enhance the efficacy of therapeutic TIL populations.

[0595] In some embodiments, the microfluidic platform is used for intracellular delivery of nucleic acid encoding the immunomodulatory fusion protein provided herein. In some embodiments, the microfluidic platform is a microfluidic platform that does not contain an SQZ vector. The SQZ platform can deliver nucleic acid and protein to various primary human cells, including T cells (Sharei et al. PNAS 2013, and Sharei et al. PLOS ONE 2015 and Greisbeck et al. J. Immunology vol.195,2015). In the SQZ platform, the cell membrane of the cell for modification (e.g., TIL) is temporarily disrupted by microfluidic contraction, thereby allowing the nucleic acid encoding the immunomodulatory fusion protein to be delivered into the cell. Methods such as those described in International Patent Application Publication Nos. WO2013 / 059343A1, WO2017 / 008063A1, or WO2017 / 123663A1, or US Patent Application Publication Nos. US2014 / 0287509A1, 2018 / 0201889A1, or 2018 / 0245089A1 (incorporated herein by reference in their entirety) can be used with the present invention to deliver nucleic acids encoding the subject immune-modulating fusion proteins to a TIL population. In some embodiments, the delivered nucleic acid allows for transient protein expression of the immune-modulating fusion protein in the modified TIL. In some embodiments, the SQZ platform is used to stably integrate the delivered nucleic acid encoding the immune-modulating fusion protein into the TIL cell genome. Additional exemplary disclosure regarding the SQZ platform and its uses can be found in International Patent Application Publication No. WO / 2019 / 136456, which is incorporated by reference in its entirety for all purposes.

[0596] A. Timing of gene editing / transient phenotypic modification during TIL expansion According to some embodiments, a method for expanding tumor infiltrating lymphocytes (TILs) into a therapeutic TIL population comprises: (a) obtaining a first population of TILs from a tumor excised from a patient by processing a tumor sample obtained from the patient into a plurality of tumor fragments; (b) adding tumor fragments to the closed system; (c) performing a first expansion by culturing the first TIL population in a cell culture medium comprising IL-2 and optionally OKT-3 (e.g., OKT-3 can be present in the cell culture medium starting on the start date of the expansion process) to produce a second TIL population, wherein the first expansion is performed in a closed vessel providing a first gas permeable surface area, and the first expansion is performed for about 3-14 days to obtain a second TIL population, and the transition from step (b) to step (c) occurs without opening the system. (d) performing a second expansion by supplementing the cell culture medium of the second TIL population with additional IL-2, optionally 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 a third TIL population, the third TIL population being a therapeutic TIL population, and wherein the second expansion is performed in a closed vessel providing a second gas permeable surface area, and the transition from step (c) to step (d) occurs without opening the system. (e) harvesting the therapeutic TIL population obtained from step (d), wherein the transition from step (d) to step (e) occurs without opening the line; (f) transferring the harvested TIL population from step (e) into an infusion bag, wherein the transition from step (e) to (f) occurs without opening the system; (g) at any point during the method prior to transfer to the infusion bag in step (f), genetically editing at least a portion of the TIL cells to express an immunomodulatory composition comprising an immunomodulatory agent (e.g., a membrane-anchored immunomodulatory fusion protein described herein) on the surface of the TIL cells. In some embodiments, the immunomodulatory agent is selected from the group consisting of IL-2, IL-7, IL-10, IL-12, IL-15, IL-18, IL-21, and a CD40 agonist (e.g., CD40L or an agonist CD40 binding domain). In some embodiments, the immunomodulatory agent is selected from the group consisting of IL-2, IL-12, IL-15, IL-18, IL-21, and a CD40 agonist. In some embodiments, the immunomodulatory agent is selected from the group consisting of IL-12, IL-15, IL-18, IL-21, and a CD40 agonist.

[0597] As described in step (g) of the above embodiment, the gene editing process may be performed at any point during the TIL expansion method before the transfer to the infusion bag in step (f), which means that gene editing may be performed on the TILs before, during, or after any of the steps of the expansion method, for example, during any of steps (a)-(f) outlined in the above method, or before or after any of steps (a)-(e) outlined in the above method. According to certain embodiments, the TILs are collected during the expansion method (e.g., the expansion method is "paused" for at least a portion of the TILs), the collected TILs are subjected to a gene editing process, and in some cases, are then reintroduced into the expansion method to continue the expansion process (e.g., returned to the culture medium), thereby permanently gene editing at least a portion of the therapeutic TIL population that is eventually transferred to the infusion bag. In some embodiments, the gene editing process may be performed prior to expansion by activating the TILs, performing a gene editing step on the activated TILs, and expanding the gene-edited TILs according to the process described herein. In some embodiments, the nucleic acid for gene editing is delivered to TILs using a microfluidic platform. In some embodiments, the microfluidic platform is a microfluidic platform that does not contain an SQZ vector.

[0598] In some embodiments, the gene editing process is performed after the first TIL expansion step. In some embodiments, the gene editing process is performed after the first TIL expansion step and before the second expansion step. In some embodiments, the gene editing process is performed after the TILs are activated. In some embodiments, the gene editing process is performed after the first expansion step and after the TILs are activated but before the second expansion step. In some embodiments, the gene editing process is performed after the first expansion step and after the TILs are activated, and the TILs are allowed to rest after the gene editing and before the second expansion step. In some embodiments, the TILs are allowed to rest for about 1-2 days after the gene editing and before the second expansion step. In some embodiments, the TILs are activated by exposure to an anti-CD3 agonist and an anti-CD28 agonist. In some embodiments, the anti-CD3 agonist is an anti-CD3 agonist antibody and the anti-CD28 agonist is an anti-CD28 agonist antibody. In some embodiments, the anti-CD3 antibody is OKT-3. In some embodiments, the TILs are activated by exposure to anti-CD3 agonist antibody and anti-CD28 agonist antibody conjugated beads. In some embodiments, the anti-CD3 agonist antibody and anti-CD28 agonist antibody conjugated beads are TransAct™ products from Miltenyi. In some embodiments, the gene editing process is performed by viral transduction. In some embodiments, the gene editing process is performed by retroviral transduction. In some embodiments, the gene editing process is performed by lentiviral transduction. In some embodiments, the immunomodulatory composition is a membrane-anchored immunomodulatory fusion protein. In some embodiments, the immunomodulatory fusion protein comprises IL-15. In some embodiments, the immunomodulatory fusion protein comprises IL-21. In some embodiments, the immunomodulatory composition comprises two or more different membrane-bound fusion proteins. In some embodiments, the immunomodulatory composition comprises a first immunomodulatory protein comprising IL-15 and a second immunomodulatory fusion protein comprising IL-21.In some embodiments, the TILs are gene edited to express an immunomodulatory composition under the control of the NFAT promoter. In some embodiments, the TILs are gene edited to express an immunomodulatory fusion protein comprising IL-15 under the control of the NFAT promoter. In some embodiments, the TILs are gene edited to express an immunomodulatory fusion protein comprising IL-21 under the control of the NFAT promoter. In some embodiments, the TILs are gene edited to express a first immunomodulatory fusion protein comprising IL-15 and a second immunomodulatory fusion protein comprising IL-21 under the control of the NFAT promoter.

[0599] In some embodiments, the gene editing process is carried out by viral transduction. In some embodiments, the gene editing process is carried out by retroviral transduction. In some embodiments, the gene editing process is carried out by lentiviral transduction.

[0600] According to some embodiments, a method for expanding tumor infiltrating lymphocytes (TILs) into a therapeutic TIL population comprises: (a) obtaining a first population of TILs from a tumor excised from a patient by processing a tumor sample obtained from the patient into a plurality of tumor fragments; (b) adding tumor fragments to the closed system; (c) performing a first expansion by culturing the first TIL population in a cell culture medium comprising IL-2 and optionally OKT-3 (e.g., OKT-3 can be present in the cell culture medium starting on the start date of the expansion process) to produce a second TIL population, wherein the first expansion is performed in a closed vessel providing a first gas permeable surface area, and the first expansion is performed for about 3-14 days to obtain a second TIL population, and the transition from step (b) to step (c) occurs without opening the system. (d) genetically editing at least a portion of the TIL cells in the second TIL population to express an immunomodulatory composition comprising an immunomodulatory agent (e.g., a membrane-anchored immunomodulatory fusion protein described herein) on the surface of the TIL cells; (e) performing a second expansion by supplementing the cell culture medium of the second TIL population with additional IL-2, optionally 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 a third TIL population, the third TIL population being a therapeutic TIL population, wherein the second expansion is performed in a closed vessel providing a second gas permeable surface area, and wherein the transition from step (c) to step (d) occurs without opening the system. (f) harvesting the therapeutic TIL population obtained from step (d), wherein the transition from step (d) to step (e) occurs without opening the line;

[0601] (g) transferring the harvested TIL population from step (e) to an infusion bag, where the transition from step (e) to (f) occurs without opening the system. In some embodiments, the immunomodulatory agent is selected from the group consisting of IL-2, IL-7, IL-10, IL-12, IL-15, IL-18, IL-21, and a CD40 agonist (e.g., CD40L or an agonist CD40 binding domain). In some embodiments, the immunomodulatory agent is selected from the group consisting of IL-2, IL-12, IL-15, IL-18, IL-21, and a CD40 agonist. In some embodiments, the immunomodulatory agent is selected from the group consisting of IL-12, IL-15, IL-18, IL-21, and a CD40 agonist. In some embodiments, the TILs are allowed to rest after the gene editing step and before the second expansion step. In some embodiments, the TILs are allowed to rest for about 1-2 days after the gene editing step and before the second expansion step. In some embodiments, the TILs are activated by exposure to an anti-CD3 agonist and an anti-CD28 agonist. In some embodiments, the anti-CD3 agonist is an anti-CD3 agonist antibody and the anti-CD28 agonist is an anti-CD28 agonist antibody. In some embodiments, the anti-CD3 antibody is OKT-3. In some embodiments, the TILs are activated by exposure to an anti-CD3 agonist antibody and an anti-CD28 agonist antibody conjugated beads. In some embodiments, the anti-CD3 agonist antibody and the anti-CD28 agonist antibody conjugated beads are Miltenyi's TransAct™ product. In some embodiments, the gene editing process is performed by viral transduction. In some embodiments, the gene editing process is performed by retroviral transduction of the TILs, optionally for about 2 days. In some embodiments, the gene editing process is performed by lentiviral transduction of the TILs, optionally for about 2 days. In some embodiments, the immunomodulatory composition is a membrane-anchored immunomodulatory fusion protein. In some embodiments, the immunomodulatory fusion protein comprises IL-15. In some embodiments, the immunomodulatory fusion protein comprises IL-21.In some embodiments, the immunomodulatory composition comprises two or more different membrane-bound fusion proteins. In some embodiments, the immunomodulatory composition comprises a first immunomodulatory protein comprising IL-15 and a second immunomodulatory fusion protein comprising IL-21. In some embodiments, the TILs are gene edited to express the immunomodulatory composition under the control of the NFAT promoter. In some embodiments, the TILs are gene edited to express an immunomodulatory fusion protein comprising IL-15 under the control of the NFAT promoter. In some embodiments, the TILs are gene edited to express an immunomodulatory fusion protein comprising IL-21 under the control of the NFAT promoter. In some embodiments, the TILs are gene edited to express a first immunomodulatory fusion protein comprising IL-15 and a second ...

[0602] It should be noted that alternative embodiments of the expansion process may differ from the method shown above, for example, alternative embodiments may not have the same steps (a)-(g) or may have a different number of steps. Regardless of the specific embodiment, the gene editing process may be performed at any point during the TIL expansion method. For example, alternative embodiments may include more than two expansions, gene editing may be performed on the TIL during the third or fourth expansion, etc.

[0603] According to another embodiment, a method for expanding tumor infiltrating lymphocytes (TILs) into a therapeutic TIL population comprises: (a) obtaining a first population of TILs from a tumor excised from a patient by processing a tumor sample obtained from the patient into a plurality of tumor fragments; (b) adding tumor fragments to the closed system; (c) performing a first expansion by culturing the first TIL population in a cell culture medium comprising IL-2 and optionally OKT-3 (e.g., OKT-3 can be present in the cell culture medium starting on the start date of the expansion process) to produce a second TIL population, wherein the first expansion is performed in a closed vessel providing a first gas permeable surface area, and the first expansion is performed for about 3-14 days to obtain a second TIL population, and the transition from step (b) to step (c) occurs without opening the system. (d) performing a second expansion by supplementing the cell culture medium of the second TIL population with additional IL-2, optionally 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 a third TIL population, the third TIL population being a therapeutic TIL population, and wherein the second expansion is performed in a closed vessel providing a second gas permeable surface area, and the transition from step (c) to step (d) occurs without opening the system. (e) harvesting the therapeutic TIL population obtained from step (d), wherein the transition from step (d) to step (e) occurs without opening the line; (f) transferring the harvested TIL population from step (e) into an infusion bag, wherein the transition from step (e) to (f) occurs without opening the system; (g) at any point during the method prior to transfer to the infusion bag in step (f), introducing a transient phenotypic modification to at least a portion of the TIL cells to express an immunomodulatory composition comprising an immunomodulatory agent (e.g., a membrane-anchored immunomodulatory fusion protein described herein) on the surface of the TIL cells. In some embodiments, the immunomodulatory agent is selected from the group consisting of IL-2, IL-7, IL-10, IL-12, IL-15, IL-18, IL-21, and a CD40 agonist (e.g., CD40L or an agonist CD40 binding domain). In some embodiments, the immunomodulatory agent is selected from the group consisting of IL-2, IL-12, IL-15, IL-18, IL-21, and a CD40 agonist. In some embodiments, the immunomodulatory agent is selected from the group consisting of IL-12, IL-15, IL-18, IL-21, and a CD40 agonist. In some embodiments, the immunomodulatory agent is selected from the group consisting of IL-12, IL-15, IL-18, IL-21, and a CD40 agonist. In some embodiments, the nucleic acid for the transient phenotypic modification is delivered to the TILs using a microfluidic platform. In some embodiments, the microfluidic platform is a microfluidic platform that does not contain an SQZ vector.

[0604] As described in step (g) of the above embodiment, the transient phenotypic modification process may be performed at any point during the TIL expansion method prior to transfer to the infusion bag in step (f), meaning that the transient phenotypic modification may be performed on the TILs before, during or after any of the steps of the expansion method, for example, during any of steps (a)-(f) outlined in the above method, or before or after any of steps (a)-(e) outlined in the above method. According to certain embodiments, the TILs are collected during the expansion method (e.g., the expansion method is "paused" for at least a portion of the TILs), the collected TILs are subjected to a transient modification process, and optionally subsequently reintroduced into the expansion method (e.g., returned to culture medium) to continue the expansion process, whereby at least a portion of the therapeutic TIL population that is ultimately transferred to the infusion bag is transiently modified to express the immunomodulatory composition on the surface of the TIL cells. In some embodiments, the transient cell modification process may be carried out prior to expansion by activating the TILs, performing a transient phenotypic modification step on the activated TILs, and expanding the modified TILs according to the processes described herein.

[0605] It should be noted that alternative embodiments of the expansion process may differ from the methods shown above, for example, alternative embodiments may not have the same steps (a)-(g) or may have a different number of steps. Regardless of the particular embodiment, the transient cell modification process may be performed at any point during the TIL expansion method. For example, alternative embodiments may include more than two expansions, transient cell modification may be performed on the TILs during the third or fourth expansion, etc.

[0606] According to some embodiments, the gene editing process is performed on the TILs from one or more of the first population, the second population, and the third population. For example, the gene editing may be performed on the first TIL population, or on a portion of the TILs collected from the first population, and following the gene editing process, the TILs may then be returned to the expansion process (e.g., may be returned to the culture medium). Alternatively, the gene editing may be performed on the TILs from the second or third population, or on a portion of the TILs collected from the second or third population, and following the gene editing process, the TILs may then be returned to the expansion process (e.g., may be returned to the culture medium). According to other embodiments, the gene editing is performed while the TILs are still in the culture medium and while the expansion is being performed, i.e., they are not necessarily "removed" from the expansion to perform the gene editing.

[0607] According to some embodiments, the transient cell modification process is carried out on TILs from one or more of the first population, the second population, and the third population. For example, the transient cell modification may be carried out on the first TIL population, or on a portion of the TILs collected from the first population, and following the gene editing process, the transiently modified TILs may then be returned to the expansion process (e.g., may be returned to the culture medium). Alternatively, the transient cell modification may be carried out on TILs from the second or third population, or on a portion of the TILs collected from the second or third population, and following the gene editing process, the modified TILs may then be returned to the expansion process (e.g., may be returned to the culture medium). According to other embodiments, the transient gene modification is carried out while the TILs are still in the culture medium and while the expansion is being carried out, i.e., they are not necessarily "removed" from the expansion to produce the transient cell modification.

[0608] According to other embodiments, the gene editing process is carried out on the TILs from the first expansion, or the TILs from the second expansion, or both. For example, during the first expansion or the second expansion, gene editing can be carried out on the TILs collected from the culture medium, and after the gene editing process, those TILs can then be returned to the expansion method, for example, by reintroducing them into the culture medium.

[0609] According to other embodiments, the transient cell modification process is performed on the TILs from the first expansion, or the TILs from the second expansion, or both. For example, during the first expansion or the second expansion, the transient cell modification can be performed on the TILs harvested from the culture medium, and after the transient cell modification process, those modified TILs can then be returned to the expansion method, for example, by reintroducing them into the culture medium.

[0610] According to other embodiments, a gene editing process is performed on at least a portion of the TILs after the first expansion and before the second expansion. For example, after the first expansion, gene editing can be performed on the TILs collected from the culture medium, and after the gene editing process, those TILs can then be returned to the expansion method, for example, by reintroducing them into the culture medium for the second expansion.

[0611] According to other embodiments, a transient cell modification process is performed on at least a portion of the TILs after the first expansion and before the second expansion. For example, after the first expansion, a transient cell modification can be performed on the TILs harvested from the culture medium, and after the transient cell modification process, those modified TILs can then be returned to the expansion method, for example, by reintroducing them into the culture medium for the second expansion.

[0612] According to alternative embodiments, the gene editing process is performed before step (c) (e.g., before, during, or after any of steps (a)-(b)), before step (d) (e.g., before, during, or after any of steps (a)-(c)), before step (e) (e.g., before, during, or after any of steps (a)-(d)), or before step (f) (e.g., before, during, or after any of steps (a)-(e)).

[0613] According to alternative embodiments, the transient cell modification process is carried out before step (c) (e.g., before, during, or after any of steps (a)-(b)), before step (d) (e.g., before, during, or after any of steps (a)-(c)), before step (e) (e.g., before, during, or after any of steps (a)-(d)), or before step (f) (e.g., before, during, or after any of steps (a)-(e)).

[0614] With regard to OKT-3, according to certain embodiments, the cell culture medium may include OKT-3 starting on the initiation day (day 0) or day 1 of the first expansion, such that gene editing or transient cell modification is performed on the TILs after exposure to OKT-3 in the cell culture medium on day 0 and / or day 1. According to other embodiments, the cell culture medium includes OKT-3 during the first expansion and / or the second expansion, and gene editing or transient cell modification is performed before OKT-3 is introduced into the cell culture medium. Alternatively, the cell culture medium includes OKT-3 during the first expansion and / or the second expansion, and gene editing or transient cell modification is performed after OKT-3 is introduced into the cell culture medium.

[0615] With regard to the 4-1BB agonist, it should also be noted that according to certain embodiments, the cell culture medium may contain 4-1BB starting on the initiation day (day 0) or on day 1 of the first expansion, so that gene editing or transient cell modification is performed on the TILs after exposure to 4-1BB in the cell culture medium on day 0 and / or day 1. Alternatively, the cell culture medium contains the 4-1BB agonist during the first expansion and / or the second expansion, and gene editing or transient cell modification is performed before the 4-1BB agonist is introduced into the cell culture medium. Alternatively, the cell culture medium contains the 4-1BB agonist during the first expansion and / or the second expansion, and gene editing or transient cell modification is performed after the 4-1BB agonist is introduced into the cell culture medium.

[0616] With regard to IL-2, it should also be noted that according to certain embodiments, the cell culture medium may include IL-2 starting on the initiation day (day 0) or day 1 of the first expansion, such that gene editing or transient cell modification is performed on the TILs after exposure to IL-2 in the cell culture medium on day 0 and / or day 1. According to other embodiments, the cell culture medium includes IL-2 during the first expansion and / or the second expansion, and gene editing or transient cell modification is performed before IL-2 is introduced into the cell culture medium. Alternatively, the cell culture medium includes IL-2 during the first expansion and / or the second expansion, and gene editing or transient cell modification is performed after IL-2 is introduced into the cell culture medium.

[0617] As mentioned above, one or more of OKT-3, 4-1BB agonist and IL-2 may be included in the cell culture medium starting on day 0 or day 1 of the first expansion. According to some embodiments, OKT-3 is included in the cell culture medium starting on day 0 or day 1 of the first expansion, and / or the 4-1BB agonist is included in the cell culture medium starting on day 0 or day 1 of the first expansion, and / or IL-2 is included in the cell culture medium starting on day 0 or day 1 of the first expansion. According to other examples, the cell culture medium includes OKT-3 and 4-1BB agonist starting on day 0 or day 1 of the first expansion. According to other examples, the cell culture medium includes OKT-3, 4-1BB agonist and IL-2 starting on day 0 or day 1 of the first expansion. Of course, as described in various embodiments herein, one or more of OKT-3, 4-1BB agonist and IL-2 may be added to the cell culture medium at one or more additional time points during the expansion process.

[0618] According to some embodiments, a method for expanding tumor infiltrating lymphocytes (TILs) into a therapeutic TIL population comprises: (a) obtaining a first population of TILs from a tumor excised from a patient by processing a tumor sample obtained from the patient into a plurality of tumor fragments; (b) adding tumor fragments to the closed system; (c) performing a first expansion by culturing the first TIL population in a cell culture medium containing IL-2 for about 3-11 days to produce a second TIL population, wherein the first expansion is performed in a sealed container providing a first gas permeable surface area; (d) activating the second TIL population by adding OKT-3 and culturing for about 1-2 days, wherein the transition from step (c) to step (d) occurs without opening the system; and (e) genetically editing at least a portion of the TIL cells in the second TIL population to express an immunomodulatory composition comprising an immunomodulatory agent (e.g., a membrane-anchored immunomodulatory fusion protein described herein) on the surface of the TIL cells; (f) optionally allowing the second population of TILs to rest for about 1 day; (g) performing a second expansion by supplementing the cell culture medium of the second TIL population with additional IL-2, optionally OKT-3 antibody, optionally OX40 antibody, and antigen presenting cells (APCs) to produce a third TIL population, wherein the second expansion is performed for about 7-11 days to obtain a third TIL population, and wherein the second expansion is performed in a closed vessel providing a second gas permeable surface area, and the transition from step (f) to step (g) occurs without opening the system. (h) harvesting the therapeutic TIL population obtained from step (g) to provide a harvested TIL population, wherein the transition from step (g) to step (h) occurs without opening the system, and wherein the harvested TIL population is a therapeutic TIL population; (i) transferring the harvested TIL population to an infusion bag, where the transition from step (h) to (i) occurs without opening the system. In some embodiments, the immunomodulatory agent is selected from the group consisting of IL-2, IL-7, IL-10, IL-12, IL-15, IL-18, IL-21, and a CD40 agonist (e.g., CD40L or an agonist CD40 binding domain). In some embodiments, the immunomodulatory agent is selected from the group consisting of IL-2, IL-12, IL-15, IL-18, IL-21, and a CD40 agonist. In some embodiments, the immunomodulatory agent is selected from the group consisting of IL-12, IL-15, IL-18, IL-21, and a CD40 agonist. In some embodiments, the TILs are allowed to rest after the gene editing step and before the second expansion step. In some embodiments, the TILs are allowed to rest for about 1-2 days after the gene editing step and before the second expansion step. In some embodiments, the TILs are activated by exposure to an anti-CD3 agonist and an anti-CD28 agonist for about 2 days. In some embodiments, the anti-CD3 agonist is an anti-CD3 agonist antibody and the anti-CD28 agonist is an anti-CD28 agonist antibody. In some embodiments, the anti-CD3 antibody is OKT-3. In some embodiments, the TILs are activated by exposure to an anti-CD3 agonist antibody and an anti-CD28 agonist antibody conjugated beads. In some embodiments, the anti-CD3 agonist antibody and the anti-CD28 agonist antibody conjugated beads are Miltenyi's TransAct™ product. In some embodiments, the gene editing process is performed by viral transduction. In some embodiments, the gene editing process is optionally performed by retroviral transduction of the TILs for about 2 days. In some embodiments, the gene editing process is optionally performed by lentiviral transduction of the TILs for about 2 days. In some embodiments, the immunomodulatory composition is a membrane-anchored immunomodulatory fusion protein. In some embodiments, the immunomodulatory fusion protein comprises IL-15. In some embodiments, the immunomodulatory fusion protein comprises IL-21.In some embodiments, the immunomodulatory composition comprises two or more different membrane-bound fusion proteins. In some embodiments, the immunomodulatory composition comprises a first immunomodulatory protein comprising IL-15 and a second immunomodulatory fusion protein comprising IL-21. In some embodiments, the TILs are gene edited to express the immunomodulatory composition under the control of the NFAT promoter. In some embodiments, the TILs are gene edited to express an immunomodulatory fusion protein comprising IL-15 under the control of the NFAT promoter. In some embodiments, the TILs are gene edited to express an immunomodulatory fusion protein comprising IL-21 under the control of the NFAT promoter. In some embodiments, the TILs are gene edited to express a first immunomodulatory fusion protein comprising IL-15 and a second ...

[0619] According to some embodiments, a method for expanding tumor infiltrating lymphocytes (TILs) into a therapeutic TIL population comprises: (a) obtaining a first population of TILs from a tumor excised from a patient by processing a tumor sample obtained from the patient into a plurality of tumor fragments; (b) adding tumor fragments to the closed system; (c) performing a first expansion by culturing the first TIL population in a cell culture medium containing IL-2 and optionally an OKT-3 and / or a 4-1BB agonist antibody for about 3-11 days to produce a second TIL population, wherein the first expansion is performed in a sealed vessel providing a first gas permeable surface area; (d) stimulating a second TIL population by adding OKT-3 and culturing for about 1-3 days, wherein the transition from step (c) to step (d) occurs without opening the system; (e) sterile electroporating the second TIL population to transfer at least one gene editor into a portion of the cells of the second TIL population; (f) allowing the second population of TILs to rest for about 1 day; (g) performing a second expansion by supplementing the cell culture medium of the second TIL population with additional IL-2, optionally OKT-3 antibody, optionally OX40 antibody, and antigen presenting cells (APCs) to produce a third TIL population, wherein the second expansion is performed for about 7-11 days to obtain a third TIL population, and wherein the second expansion is performed in a closed vessel providing a second gas permeable surface area, and the transition from step (f) to step (g) occurs without opening the system. (h) harvesting the therapeutic TIL population obtained from step (g) to provide a harvested TIL population, wherein the transition from step (g) to step (h) occurs without opening the system, and wherein the harvested TIL population is a therapeutic TIL population; (i) transferring the harvested TIL population to an infusion bag, wherein the transition from step (h) to (i) occurs without opening the system; Sterile electroporation of at least one gene editor into a portion of the cells of the second TIL population modifies a plurality of cells in the portion to express an immunomodulatory composition on the surface of the cells. In some embodiments, the immunomodulatory composition comprises an immunomodulatory agent fused to a membrane anchor (e.g., a membrane-anchored immunomodulatory fusion protein described herein). In some embodiments, the immunomodulatory agent is selected from the group consisting of IL-2, IL-7, IL-10, IL-12, IL-15, IL-18, IL-21, and a CD40 agonist (e.g., CD40L or an agonist CD40 binding domain). In some embodiments, the immunomodulatory agent is selected from the group consisting of IL-2, IL-12, IL-15, IL-18, IL-21, and a CD40 agonist. In some embodiments, the immunomodulatory agent is selected from the group consisting of IL-12, IL-15, IL-18, IL-21, and a CD40 agonist.

[0620] According to another embodiment, a method for expanding tumor infiltrating lymphocytes (TILs) into a therapeutic TIL population comprises: (a) obtaining a first population of TILs from a tumor excised from a patient by processing a tumor sample obtained from the patient into a plurality of tumor fragments; (b) adding tumor fragments to the closed system; (c) performing a first expansion by culturing the first TIL population in a cell culture medium containing IL-2 and optionally an OKT-3 and / or a 4-1BB agonist antibody for about 3-11 days to produce a second TIL population, wherein the first expansion is performed in a sealed vessel providing a first gas permeable surface area; (d) stimulating a second TIL population by adding OKT-3 and culturing for about 1-3 days, wherein the transition from step (c) to step (d) occurs without opening the system; (e) sterile electroporating the second TIL population to transfer at least one nucleic acid molecule into a portion of the cells of the second TIL population; (f) allowing the second population of TILs to rest for about 1 day; (g) performing a second expansion by supplementing the cell culture medium of the second TIL population with additional IL-2, optionally OKT-3 antibody, optionally OX40 antibody, and antigen presenting cells (APCs) to produce a third TIL population, wherein the second expansion is performed for about 7-11 days to obtain a third TIL population, and wherein the second expansion is performed in a closed vessel providing a second gas permeable surface area, and the transition from step (f) to step (g) occurs without opening the system. (h) harvesting the therapeutic TIL population obtained from step (g) to provide a harvested TIL population, wherein the transition from step (g) to step (h) occurs without opening the system, and wherein the harvested TIL population is a therapeutic TIL population; (i) transferring the harvested TIL population to an infusion bag, wherein the transition from step (h) to (i) occurs without opening the system; Sterile electroporation of at least one nucleic acid molecule into a portion of the cells of the second TIL population modifies a plurality of cells in the portion to transiently express an immunomodulatory composition on the surface of the cells. In some embodiments, the immunomodulatory composition comprises an immunomodulatory agent fused to a membrane anchor (e.g., a membrane-anchored immunomodulatory fusion protein described herein). In some embodiments, the immunomodulatory agent is selected from the group consisting of IL-2, IL-7, IL-10, IL-12, IL-15, IL-18, IL-21, and a CD40 agonist (e.g., CD40L or an agonist CD40 binding domain). In some embodiments, the immunomodulatory agent is selected from the group consisting of IL-2, IL-12, IL-15, IL-18, IL-21, and a CD40 agonist. In some embodiments, the immunomodulatory agent is selected from the group consisting of IL-12, IL-15, IL-18, IL-21, and a CD40 agonist.

[0621] According to some embodiments, a method for expanding tumor infiltrating lymphocytes (TILs) into a therapeutic TIL population comprises: (a) obtaining a first population of TILs from a tumor excised from a patient by processing a tumor sample obtained from the patient into a plurality of tumor fragments; (b) adding tumor fragments to the closed system; (c) performing a first expansion by culturing the first TIL population in a cell culture medium containing IL-2 and optionally an OKT-3 and / or a 4-1BB agonist antibody for about 3-11 days to produce a second TIL population, wherein the first expansion is performed in a sealed vessel providing a first gas permeable surface area; (d) stimulating a second TIL population by adding OKT-3 and culturing for about 1-3 days, wherein the transition from step (c) to step (d) occurs without opening the system; (e) sterile electroporating the second TIL population to transfer at least one gene editor into a portion of the cells of the second TIL population; (f) allowing the second population of TILs to rest for about 1 day; (g) performing a second expansion by supplementing the cell culture medium of the second TIL population with additional IL-2, optionally OKT-3 antibody, optionally OX40 antibody, and antigen presenting cells (APCs) to produce a third TIL population, wherein the second expansion is performed for about 7-11 days to obtain a third TIL population, and wherein the second expansion is performed in a closed vessel providing a second gas permeable surface area, and the transition from step (f) to step (g) occurs without opening the system. (h) harvesting the therapeutic TIL population obtained from step (g) to provide a harvested TIL population, wherein the transition from step (g) to step (h) occurs without opening the system, and wherein the harvested TIL population is a therapeutic TIL population; (i) transferring the harvested TIL population to an infusion bag, wherein the transition from step (h) to (i) occurs without opening the system; Sterile electroporation of at least one gene editor into a portion of the cells of the second TIL population modifies a plurality of cells in the portion to express an immunomodulatory composition on the surface of the cells. In some embodiments, the immunomodulatory composition comprises an immunomodulatory agent fused to a membrane anchor (e.g., a membrane-anchored immunomodulatory fusion protein described herein). In some embodiments, the immunomodulatory agent is selected from the group consisting of IL-2, IL-7, IL-10, IL-12, IL-15, IL-18, IL-21, and a CD40 agonist (e.g., CD40L or an agonist CD40 binding domain). In some embodiments, the immunomodulatory agent is selected from the group consisting of IL-2, IL-12, IL-15, IL-18, IL-21, and a CD40 agonist. In some embodiments, the immunomodulatory agent is selected from the group consisting of IL-12, IL-15, IL-18, IL-21, and a CD40 agonist.

[0622] According to some embodiments, a method for expanding tumor infiltrating lymphocytes (TILs) into a therapeutic TIL population comprises: (a) obtaining a first population of TILs from a tumor excised from a patient by processing a tumor sample obtained from the patient into a plurality of tumor fragments; (b) adding tumor fragments to the closed system; (c) performing a first expansion by culturing the first TIL population in a cell culture medium containing IL-2 and optionally an OKT-3 and / or a 4-1BB agonist antibody for about 3-11 days to produce a second TIL population, wherein the first expansion is performed in a sealed vessel providing a first gas permeable surface area; (d) stimulating a second TIL population by adding OKT-3 and culturing for about 1-3 days, wherein the transition from step (c) to step (d) occurs without opening the system; (e) transiently disrupting the cell membrane of the second TIL population to transfer at least one gene editor into a portion of the cells of the second TIL population; (f) allowing the second population of TILs to rest for about 1 day; (g) performing a second expansion by supplementing the cell culture medium of the second TIL population with additional IL-2, optionally OKT-3 antibody, optionally OX40 antibody, and antigen presenting cells (APCs) to produce a third TIL population, wherein the second expansion is performed for about 7-11 days to obtain a third TIL population, and wherein the second expansion is performed in a closed vessel providing a second gas permeable surface area, and the transition from step (f) to step (g) occurs without opening the system. (h) harvesting the therapeutic TIL population obtained from step (g) to provide a harvested TIL population, wherein the transition from step (g) to step (h) occurs without opening the system, and wherein the harvested TIL population is a therapeutic TIL population; (i) transferring the harvested TIL population to an infusion bag, wherein the transition from step (h) to (i) occurs without opening the system; At least one gene editor delivered to a portion of the cells of the second TIL population modifies a plurality of cells in the portion to express an immunomodulatory composition on the surface of the cells. In some embodiments, the immunomodulatory composition comprises an immunomodulatory agent fused to a membrane anchor (e.g., a membrane-anchored immunomodulatory fusion protein described herein). In some embodiments, the immunomodulatory agent is selected from the group consisting of IL-2, IL-7, IL-10, IL-12, IL-15, IL-18, IL-21, and a CD40 agonist (e.g., CD40L or an agonist CD40 binding domain). In some embodiments, the immunomodulatory agent is selected from the group consisting of IL-2, IL-12, IL-15, IL-18, IL-21, and a CD40 agonist. In some embodiments, the immunomodulatory agent is selected from the group consisting of IL-12, IL-15, IL-18, IL-21, and a CD40 agonist. In some embodiments, the immunomodulatory agent is selected from the group consisting of IL-12, IL-15, IL-18, IL-21, and a CD40 agonist. In some embodiments, a microfluidic platform is used to transiently disrupt the cell membranes of the second TIL population. In some embodiments, the microfluidic platform is a microfluidic platform that does not contain an SQZ vector.

[0623] According to another embodiment, a method for expanding tumor infiltrating lymphocytes (TILs) into a therapeutic TIL population comprises: (a) obtaining a first population of TILs from a tumor excised from a patient by processing a tumor sample obtained from the patient into a plurality of tumor fragments; (b) adding tumor fragments to the closed system; (c) performing a first expansion by culturing the first TIL population in a cell culture medium containing IL-2 and optionally an OKT-3 and / or a 4-1BB agonist antibody for about 3-11 days to produce a second TIL population, wherein the first expansion is performed in a sealed vessel providing a first gas permeable surface area; (d) stimulating a second TIL population by adding OKT-3 and culturing for about 1-3 days, wherein the transition from step (c) to step (d) occurs without opening the system; (e) transiently disrupting the cell membrane of the second TIL population to transfer at least one nucleic acid molecule into a portion of the cells of the second TIL population; (f) allowing the second population of TILs to rest for about 1 day; (g) performing a second expansion by supplementing the cell culture medium of the second TIL population with additional IL-2, optionally OKT-3 antibody, optionally OX40 antibody, and antigen presenting cells (APCs) to produce a third TIL population, wherein the second expansion is performed for about 7-11 days to obtain a third TIL population, and wherein the second expansion is performed in a closed vessel providing a second gas permeable surface area, and the transition from step (f) to step (g) occurs without opening the system. (h) harvesting the therapeutic TIL population obtained from step (g) to provide a harvested TIL population, wherein the transition from step (g) to step (h) occurs without opening the system, and wherein the harvested TIL population is a therapeutic TIL population; (i) transferring the harvested TIL population to an infusion bag, wherein the transition from step (h) to (i) occurs without opening the system; At least one nucleic acid molecule delivered to a portion of the cells of the second TIL population modifies a plurality of cells in the portion to transiently express an immunomodulatory composition on the surface of the cells. In some embodiments, the immunomodulatory composition comprises an immunomodulatory agent fused to a membrane anchor (e.g., a membrane-anchored immunomodulatory fusion protein described herein). In some embodiments, the immunomodulatory agent is selected from the group consisting of IL-2, IL-7, IL-10, IL-12, IL-15, IL-18, IL-21, and a CD40 agonist (e.g., CD40L or an agonist CD40 binding domain). In some embodiments, the immunomodulatory agent is selected from the group consisting of IL-2, IL-12, IL-15, IL-18, IL-21, and a CD40 agonist. In some embodiments, the immunomodulatory agent is selected from the group consisting of IL-12, IL-15, IL-18, IL-21, and a CD40 agonist. In some embodiments, the immunomodulatory agent is selected from the group consisting of IL-12, IL-15, IL-18, IL-21, and a CD40 agonist. In some embodiments, a microfluidic platform is used to transiently disrupt the cell membranes of the second TIL population. In some embodiments, the microfluidic platform is a microfluidic platform that does not contain an SQZ vector.

[0624] According to some embodiments, a method for expanding tumor infiltrating lymphocytes (TILs) into a therapeutic TIL population comprises: (a) obtaining a first population of TILs from a tumor excised from a patient by processing a tumor sample obtained from the patient into a plurality of tumor fragments; (b) adding tumor fragments to the closed system; (c) performing a first expansion by culturing the first TIL population in a cell culture medium containing IL-2 and optionally an OKT-3 and / or a 4-1BB agonist antibody for about 3-11 days to produce a second TIL population, wherein the first expansion is performed in a sealed vessel providing a first gas permeable surface area; (d) stimulating a second TIL population by adding OKT-3 and culturing for about 1-3 days, wherein the transition from step (c) to step (d) occurs without opening the system; (e) transiently disrupting the cell membrane of the second TIL population to transfer at least one gene editor into a portion of the cells of the second TIL population; (f) allowing the second population of TILs to rest for about 1 day; (g) performing a second expansion by supplementing the cell culture medium of the second TIL population with additional IL-2, optionally OKT-3 antibody, optionally OX40 antibody, and antigen presenting cells (APCs) to produce a third TIL population, wherein the second expansion is performed for about 7-11 days to obtain a third TIL population, and wherein the second expansion is performed in a closed vessel providing a second gas permeable surface area, and the transition from step (f) to step (g) occurs without opening the system. (h) harvesting the therapeutic TIL population obtained from step (g) to provide a harvested TIL population, wherein the transition from step (g) to step (h) occurs without opening the system, and wherein the harvested TIL population is a therapeutic TIL population; (i) transferring the harvested TIL population to an infusion bag, wherein the transition from step (h) to (i) occurs without opening the system; At least one gene editor delivered to a portion of the cells of the second TIL population modifies a plurality of cells in the portion to express an immunomodulatory composition on the surface of the cells. In some embodiments, the immunomodulatory composition comprises an immunomodulatory agent fused to a membrane anchor (e.g., a membrane-anchored immunomodulatory fusion protein described herein). In some embodiments, the immunomodulatory agent is selected from the group consisting of IL-2, IL-7, IL-10, IL-12, IL-15, IL-18, IL-21, and a CD40 agonist (e.g., CD40L or an agonist CD40 binding domain). In some embodiments, the immunomodulatory agent is selected from the group consisting of IL-2, IL-12, IL-15, IL-18, IL-21, and a CD40 agonist. In some embodiments, the immunomodulatory agent is selected from the group consisting of IL-12, IL-15, IL-18, IL-21, and a CD40 agonist. In some embodiments, the immunomodulatory agent is selected from the group consisting of IL-12, IL-15, IL-18, IL-21, and a CD40 agonist. In some embodiments, a microfluidic platform is used to transiently disrupt the cell membranes of the second TIL population. In some embodiments, the microfluidic platform is a microfluidic platform that does not contain an SQZ vector.

[0625] In some embodiments, provided herein is a method for preparing expanded tumor infiltrating lymphocytes (TILs), comprising: (a) obtaining and / or receiving a first population of TILs from tumor tissue resected from a subject or patient; (b) culturing the first TIL population in a first cell culture medium containing IL-2 for about 3-9 days to produce a second TIL population; (c) activating the second TIL population using anti-CD3 and anti-CD28 beads or antibodies for 1-7 days to produce a third TIL population; (d) sterile electroporating the third TIL population to transfer at least one gene editor into a portion of the cells of the third TIL population to produce a fourth TIL population; (e) culturing the fourth population of TILs in a second cell culture medium comprising antigen presenting cells (APCs), OKT-3, and IL-2 for about 5-15 days to produce an expanded number of TILs; Sterile electroporation of at least one gene editor into a portion of the cells of the third TIL population modifies a plurality of cells in the portion to express an immunomodulatory composition on the surface of the cells. In some embodiments, the immunomodulatory composition comprises an immunomodulatory agent fused to a membrane anchor (e.g., a membrane-anchored immunomodulatory fusion protein described herein). In some embodiments, the immunomodulatory agent is selected from the group consisting of IL-2, IL-7, IL-10, IL-12, IL-15, IL-18, IL-21, and a CD40 agonist (e.g., CD40L or an agonist CD40 binding domain). In some embodiments, the immunomodulatory agent is selected from the group consisting of IL-2, IL-12, IL-15, IL-18, IL-21, and a CD40 agonist. In some embodiments, the immunomodulatory agent is selected from the group consisting of IL-12, IL-15, IL-18, IL-21, and a CD40 agonist.

[0626] In some embodiments, provided herein is a method for preparing expanded tumor infiltrating lymphocytes (TILs), comprising: (a) obtaining and / or receiving a first population of TILs from tumor tissue resected from a subject or patient; (b) culturing the first TIL population in a first cell culture medium containing IL-2 for about 3-9 days to produce a second TIL population; (c) activating the second TIL population using anti-CD3 and anti-CD28 beads or antibodies for 1-7 days to produce a third TIL population; (d) genetically editing at least a portion of the TIL cells in the second TIL population to express an immunomodulatory composition comprising an immunomodulatory agent (e.g., a membrane-anchored immunomodulatory fusion protein described herein) on the surface of the TIL cells; (e) culturing the fourth TIL population in a second cell culture medium comprising antigen presenting cells (APCs), OKT-3, and IL-2 for about 5 to 15 days to produce an expanded number of TILs. In some embodiments, the immunomodulatory agent is selected from the group consisting of IL-2, IL-7, IL-10, IL-12, IL-15, IL-18, IL-21, and a CD40 agonist (e.g., CD40L or an agonist CD40 binding domain). In some embodiments, the immunomodulatory agent is selected from the group consisting of IL-2, IL-12, IL-15, IL-18, IL-21, and a CD40 agonist. In some embodiments, the immunomodulatory agent is selected from the group consisting of IL-12, IL-15, IL-18, IL-21, and a CD40 agonist. In some embodiments, the TILs are allowed to rest after the gene editing step and before the second expansion step. In some embodiments, the TILs are allowed to rest for about 1-2 days after the gene editing step and before the second expansion step. In some embodiments, the TILs are activated by exposure to an anti-CD3 agonist and an anti-CD28 agonist for about 2 days. In some embodiments, the anti-CD3 agonist is an anti-CD3 agonist antibody and the anti-CD28 agonist is an anti-CD28 agonist antibody. In some embodiments, the anti-CD3 antibody is OKT-3. In some embodiments, the TILs are activated by exposure to an anti-CD3 agonist antibody and an anti-CD28 agonist antibody conjugated beads. In some embodiments, the anti-CD3 agonist antibody and the anti-CD28 agonist antibody conjugated beads are Miltenyi's TransAct™ products. In some embodiments, the gene editing process is performed by viral transduction. In some embodiments, the gene editing process is performed by retroviral transduction of the TILs, optionally for about 2 days. In some embodiments, the gene editing process is performed by lentiviral transduction of the TILs, optionally for about 2 days. In some embodiments, the immunomodulatory composition is a membrane-anchored immunomodulatory fusion protein. In some embodiments, the immunomodulatory fusion protein comprises IL-15. In some embodiments, the immunomodulatory fusion protein comprises IL-21. In some embodiments, an immunomodulatory composition comprises two or more different membrane-bound fusion proteins.In some embodiments, the immunomodulatory composition comprises a first immunomodulatory protein comprising IL-15 and a second immunomodulatory fusion protein comprising IL-21. In some embodiments, the TILs are gene edited to express the immunomodulatory composition under the control of an NFAT promoter. In some embodiments, the TILs are gene edited to express an immunomodulatory fusion protein comprising IL-15 under the control of an NFAT promoter. In some embodiments, the TILs are gene edited to express an immunomodulatory fusion protein comprising IL-21 under the control of an NFAT promoter. In some embodiments, the TILs are gene edited to express a first immunomodulatory fusion protein comprising IL-15 and a second immunomodulatory fusion protein comprising IL-21 under the control of an NFAT promoter.

[0627] In some embodiments, provided herein is a method for preparing expanded tumor infiltrating lymphocytes (TILs), comprising: (a) obtaining and / or receiving a first population of TILs from tumor tissue resected from a subject or patient; (b) culturing the first TIL population in a first cell culture medium containing IL-2 for about 3-9 days to produce a second TIL population; (c) activating the second TIL population using anti-CD3 and anti-CD28 beads or antibodies for 1-7 days to produce a third TIL population; (d) sterile electroporating the third TIL population to transfer at least one nucleic acid molecule into a portion of the cells of the third TIL population to produce a fourth TIL population; (e) culturing the fourth population of TILs in a second cell culture medium comprising antigen presenting cells (APCs), OKT-3, and IL-2 for about 5-15 days to produce an expanded number of TILs; At least one nucleic acid molecule delivered to a portion of the cells of the third TIL population modifies a plurality of cells in the portion to transiently express an immunomodulatory composition on the surface of the cells. In some embodiments, the immunomodulatory composition comprises an immunomodulatory agent fused to a membrane anchor (e.g., a membrane-anchored immunomodulatory fusion protein described herein). In some embodiments, the immunomodulatory agent is selected from the group consisting of IL-2, IL-7, IL-10, IL-12, IL-15, IL-18, IL-21, and a CD40 agonist (e.g., CD40L or an agonist CD40 binding domain). In some embodiments, the immunomodulatory agent is selected from the group consisting of IL-2, IL-12, IL-15, IL-18, IL-21, and a CD40 agonist. In some embodiments, the immunomodulatory agent is selected from the group consisting of IL-12, IL-15, IL-18, IL-21, and a CD40 agonist.

[0628] In some embodiments, provided herein is a method for preparing expanded tumor infiltrating lymphocytes (TILs), comprising: (a) obtaining and / or receiving a first population of TILs from tumor tissue resected from a subject or patient; (b) digesting the tumor tissue in an enzymatic medium to produce a tumor digest; (c) culturing the first TIL population in a first cell culture medium containing IL-2 for about 3-9 days to produce a second TIL population; (d) activating the second TIL population for 1-7 days using anti-CD3 and anti-CD28 beads or antibodies to produce a third TIL population; (e) genetically editing at least a portion of the TIL cells in the second TIL population to express an immunomodulatory composition comprising an immunomodulatory agent (e.g., a membrane-anchored immunomodulatory fusion protein described herein) on the surface of the TIL cells; (f) culturing the fourth TIL population in a second cell culture medium comprising antigen presenting cells (APCs), OKT-3, and IL-2 for about 5-15 days to produce an expanded number of TILs. In some embodiments, the immunomodulatory agent is selected from the group consisting of IL-2, IL-7, IL-10, IL-12, IL-15, IL-18, IL-21, and a CD40 agonist (e.g., CD40L or an agonist CD40 binding domain). In some embodiments, the immunomodulatory agent is selected from the group consisting of IL-2, IL-12, IL-15, IL-18, IL-21, and a CD40 agonist. In some embodiments, the immunomodulatory agent is selected from the group consisting of IL-12, IL-15, IL-18, IL-21, and a CD40 agonist. In some embodiments, the TILs are allowed to rest after the gene editing step and before the second expansion step. In some embodiments, the TILs are allowed to rest for about 1-2 days after the gene editing step and before the second expansion step. In some embodiments, the TILs are activated by exposure to an anti-CD3 agonist and an anti-CD28 agonist for about 2 days. In some embodiments, the anti-CD3 agonist is an anti-CD3 agonist antibody and the anti-CD28 agonist is an anti-CD28 agonist antibody. In some embodiments, the anti-CD3 antibody is OKT-3. In some embodiments, the TILs are activated by exposure to an anti-CD3 agonist antibody and an anti-CD28 agonist antibody conjugated beads. In some embodiments, the anti-CD3 agonist antibody and the anti-CD28 agonist antibody conjugated beads are Miltenyi's TransAct™ product. In some embodiments, the gene editing process is performed by viral transduction. In some embodiments, the gene editing process is optionally performed by retroviral transduction of the TILs for about 2 days. In some embodiments, the gene editing process is optionally carried out by lentiviral transduction of TILs for about 2 days. In some embodiments, the immunomodulatory composition is a membrane-anchored immunomodulatory fusion protein. In some embodiments, the immunomodulatory fusion protein comprises IL-15. In some embodiments, the immunomodulatory fusion protein comprises IL-21.In some embodiments, the immunomodulatory composition comprises two or more different membrane-bound fusion proteins. In some embodiments, the immunomodulatory composition comprises a first immunomodulatory protein comprising IL-15 and a second immunomodulatory fusion protein comprising IL-21. In some embodiments, the TILs are gene edited to express the immunomodulatory composition under the control of the NFAT promoter. In some embodiments, the TILs are gene edited to express an immunomodulatory fusion protein comprising IL-15 under the control of the NFAT promoter. In some embodiments, the TILs are gene edited to express an immunomodulatory fusion protein comprising IL-21 under the control of the NFAT promoter. In some embodiments, the TILs are gene edited to express a first immunomodulatory fusion protein comprising IL-15 and a second ...

[0629] In some embodiments, provided herein is a method for preparing expanded tumor infiltrating lymphocytes (TILs), comprising: (a) obtaining and / or receiving a first population of TILs from tumor tissue resected from a subject or patient; (b) digesting the tumor tissue in an enzymatic medium to produce a tumor digest; (c) culturing the first TIL population in a first cell culture medium containing IL-2 for about 3-9 days to produce a second TIL population; (d) activating the second TIL population using anti-CD3 and anti-CD28 beads or antibodies for 1-7 days to produce a third TIL population; (e) sterile electroporating the third TIL population to transfer at least one gene editor into a portion of the cells of the third TIL population to produce a fourth TIL population; (f) culturing the fourth population of TILs in a second cell culture medium comprising antigen presenting cells (APCs), OKT-3, and IL-2 for about 5-15 days to produce an expanded number of TILs; Sterile electroporation of at least one gene editor into a portion of the cells of the third TIL population modifies a plurality of cells in the portion to express an immunomodulatory composition on the surface of the cells. In some embodiments, the immunomodulatory composition comprises an immunomodulatory agent fused to a membrane anchor (e.g., a membrane-anchored immunomodulatory fusion protein described herein). In some embodiments, the immunomodulatory agent is selected from the group consisting of IL-2, IL-7, IL-10, IL-12, IL-15, IL-18, IL-21, and a CD40 agonist (e.g., CD40L or an agonist CD40 binding domain). In some embodiments, the immunomodulatory agent is selected from the group consisting of IL-2, IL-12, IL-15, IL-18, IL-21, and a CD40 agonist. In some embodiments, the immunomodulatory agent is selected from the group consisting of IL-12, IL-15, IL-18, IL-21, and a CD40 agonist.

[0630] In some embodiments, provided herein is a method for preparing expanded tumor infiltrating lymphocytes (TILs), comprising: (a) obtaining and / or receiving a first population of TILs from tumor tissue resected from a subject or patient; (b) digesting the tumor tissue in an enzymatic medium to produce a tumor digest; (c) culturing the first TIL population in a first cell culture medium containing IL-2 for about 3-9 days to produce a second TIL population; (d) activating the second TIL population using anti-CD3 and anti-CD28 beads or antibodies for 1-7 days to produce a third TIL population; (e) sterile electroporating the third TIL population to transfer at least one nucleic acid molecule into a portion of the cells of the third TIL population to produce a fourth TIL population; (f) culturing the fourth population of TILs in a second cell culture medium comprising antigen presenting cells (APCs), OKT-3, and IL-2 for about 5-15 days to produce an expanded number of TILs; At least one nucleic acid molecule delivered to a portion of the cells of the third TIL population modifies a plurality of cells in the portion to transiently express an immunomodulatory composition on the surface of the cells. In some embodiments, the immunomodulatory composition comprises an immunomodulatory agent fused to a membrane anchor (e.g., a membrane-anchored immunomodulatory fusion protein described herein). In some embodiments, the immunomodulatory agent is selected from the group consisting of IL-2, IL-7, IL-10, IL-12, IL-15, IL-18, IL-21, and a CD40 agonist (e.g., CD40L or an agonist CD40 binding domain). In some embodiments, the immunomodulatory agent is selected from the group consisting of IL-2, IL-12, IL-15, IL-18, IL-21, and a CD40 agonist. In some embodiments, the immunomodulatory agent is selected from the group consisting of IL-12, IL-15, IL-18, IL-21, and a CD40 agonist.

[0631] In some embodiments, provided herein is a method for preparing expanded tumor infiltrating lymphocytes (TILs), comprising: (a) obtaining and / or receiving a first population of TILs from tumor tissue resected from a subject or patient; (b) culturing the first TIL population in a first cell culture medium containing IL-2 for about 3-9 days to produce a second TIL population; (c) activating the second TIL population using anti-CD3 and anti-CD28 beads or antibodies for 1-7 days to produce a third TIL population; (d) transiently disrupting the cell membrane of the third TIL population and transferring at least one gene editor into a portion of the cells of the third TIL population to produce a fourth TIL population; (e) culturing the fourth population of TILs in a second cell culture medium comprising antigen presenting cells (APCs), OKT-3, and IL-2 for about 5-15 days to produce an expanded number of TILs; Transfer of at least one gene editor into a portion of the cells of the third TIL population modifies a plurality of cells in the portion to express an immunomodulatory composition on the surface of the cells. In some embodiments, the immunomodulatory composition comprises an immunomodulatory agent fused to a membrane anchor (e.g., a membrane-anchored immunomodulatory fusion protein described herein). In some embodiments, the immunomodulatory agent is selected from the group consisting of IL-2, IL-7, IL-10, IL-12, IL-15, IL-18, IL-21, and a CD40 agonist (e.g., CD40L or an agonist CD40 binding domain). In some embodiments, the immunomodulatory agent is selected from the group consisting of IL-2, IL-12, IL-15, IL-18, IL-21, and a CD40 agonist. In some embodiments, the immunomodulatory agent is selected from the group consisting of IL-12, IL-15, IL-18, IL-21, and a CD40 agonist. In some embodiments, the immunomodulatory agent is selected from the group consisting of IL-12, IL-15, IL-18, IL-21, and a CD40 agonist. In some embodiments, a microfluidic platform is used to transiently disrupt the cell membranes of the second TIL population. In some embodiments, the microfluidic platform is a microfluidic platform that does not contain an SQZ vector.

[0632] In some embodiments, provided herein is a method for preparing expanded tumor infiltrating lymphocytes (TILs), comprising: (a) obtaining and / or receiving a first population of TILs from tumor tissue resected from a subject or patient; (b) culturing the first TIL population in a first cell culture medium containing IL-2 for about 3-9 days to produce a second TIL population; (c) activating the second TIL population using anti-CD3 and anti-CD28 beads or antibodies for 1-7 days to produce a third TIL population; (d) transiently disrupting the cell membrane of the third TIL population to transfer at least one nucleic acid molecule into a portion of the cells of the third TIL population to produce a fourth TIL population; (e) culturing the fourth population of TILs in a second cell culture medium comprising antigen presenting cells (APCs), OKT-3, and IL-2 for about 5-15 days to produce an expanded number of TILs; Transfer of at least one nucleic acid molecule into a portion of the cells of the third TIL population modifies a plurality of cells in the portion to transiently express an immunomodulatory composition on the surface of the cells. In some embodiments, the immunomodulatory composition comprises an immunomodulatory agent fused to a membrane anchor (e.g., a membrane-anchored immunomodulatory fusion protein described herein). In some embodiments, the immunomodulatory agent is selected from the group consisting of IL-2, IL-7, IL-10, IL-12, IL-15, IL-18, IL-21, and a CD40 agonist (e.g., CD40L or an agonist CD40 binding domain). In some embodiments, the immunomodulatory agent is selected from the group consisting of IL-2, IL-12, IL-15, IL-18, IL-21, and a CD40 agonist. In some embodiments, the immunomodulatory agent is selected from the group consisting of IL-12, IL-15, IL-18, IL-21, and a CD40 agonist. In some embodiments, the immunomodulatory agent is selected from the group consisting of IL-12, IL-15, IL-18, IL-21, and a CD40 agonist. In some embodiments, a microfluidic platform is used to transiently disrupt the cell membranes of the second TIL population. In some embodiments, the microfluidic platform is a microfluidic platform that does not contain an SQZ vector.

[0633] In some embodiments, provided herein is a method for preparing expanded tumor infiltrating lymphocytes (TILs), comprising: (a) obtaining and / or receiving a first population of TILs from tumor tissue resected from a subject or patient; (b) digesting the tumor tissue in an enzymatic medium to produce a tumor digest; (c) culturing the first TIL population in a first cell culture medium containing IL-2 for about 3-9 days to produce a second TIL population; (d) activating the second TIL population for 1-7 days using anti-CD3 and anti-CD28 beads or antibodies to produce a third TIL population; (e) transiently disrupting the cell membrane of the third TIL population and transferring at least one gene editor into a portion of the cells of the third TIL population to produce a fourth TIL population; (f) culturing the fourth population of TILs in a second cell culture medium comprising antigen presenting cells (APCs), OKT-3, and IL-2 for about 5-15 days to produce an expanded number of TILs; Transfer of at least one gene editor into a portion of the cells of the third TIL population modifies a plurality of cells in the portion to express an immunomodulatory composition on the surface of the cells. In some embodiments, the immunomodulatory composition comprises an immunomodulatory agent fused to a membrane anchor (e.g., a membrane-anchored immunomodulatory fusion protein described herein). In some embodiments, the immunomodulatory agent In some embodiments, the immunomodulatory agent is selected from the group consisting of IL-2, IL-7, IL-10, IL-12, IL-15, IL-18, IL-21, and a CD40 agonist (e.g., CD40L or an agonist CD40 binding domain). In some embodiments, the immunomodulatory agent is selected from the group consisting of IL-2, IL-12, IL-15, IL-18, IL-21, and a CD40 agonist. In some embodiments, the immunomodulatory agent is selected from the group consisting of IL-12, IL-15, IL-18, IL-21, and a CD40 agonist. In some embodiments, a microfluidic platform is used to temporarily disrupt the cell membrane of the second TIL population. In some embodiments, the microfluidic platform is a microfluidic platform that does not contain an SQZ vector.

[0634] In some embodiments, provided herein is a method for preparing expanded tumor infiltrating lymphocytes (TILs), comprising: (a) obtaining and / or receiving a first population of TILs from tumor tissue resected from a subject or patient; (b) digesting the tumor tissue in an enzymatic medium to produce a tumor digest; (c) culturing the first TIL population in a first cell culture medium containing IL-2 for about 3-9 days to produce a second TIL population; (d) activating the second TIL population for 1-7 days using anti-CD3 and anti-CD28 beads or antibodies to produce a third TIL population; (e) transiently disrupting the cell membrane of the third TIL population to transfer at least one nucleic acid molecule into a portion of the cells of the third TIL population to produce a fourth TIL population; (f) culturing the fourth population of TILs in a second cell culture medium comprising antigen presenting cells (APCs), OKT-3, and IL-2 for about 5-15 days to produce an expanded number of TILs; Transfer of at least one nucleic acid molecule into a portion of the cells of the third TIL population modifies a plurality of cells in the portion to transiently express an immunomodulatory composition on the surface of the cells. In some embodiments, the immunomodulatory composition comprises an immunomodulatory agent fused to a membrane anchor (e.g., a membrane-anchored immunomodulatory fusion protein described herein). In some embodiments, the immunomodulatory agent is selected from the group consisting of IL-2, IL-7, IL-10, IL-12, IL-15, IL-18, IL-21, and a CD40 agonist (e.g., CD40L or an agonist CD40 binding domain). In some embodiments, the immunomodulatory agent is selected from the group consisting of IL-2, IL-12, IL-15, IL-18, IL-21, and a CD40 agonist. In some embodiments, the immunomodulatory agent is selected from the group consisting of IL-12, IL-15, IL-18, IL-21, and a CD40 agonist.

[0635] In some embodiments, any of the aforementioned methods further comprise culturing the fourth population of TILs by: (f) culturing the fourth TIL population in a second cell culture medium comprising antigen presenting cells (APCs), OKT-3, and IL-2 for about 1-7 days to produce a culture of a fifth TIL population; (g) splitting the culture of the fifth TIL population into multiple subcultures, culturing each of the multiple subcultures for about 3-7 days in a third cell culture medium containing IL-2, and combining the multiple subcultures to provide an expanded number of TILs.

[0636] In some embodiments, the invention provides a method as set forth in any of the relevant preceding paragraphs above, modified such that the step of activating the second TIL population is performed for about 1 day, 2 days, 3 days, 4 days, 5 days, 6 days, or 7 days.

[0637] In some embodiments, the invention provides a method as described in any of the relevant preceding paragraphs above, modified such that the step of activating the second TIL population is performed for about 2-7 days.

[0638] In some embodiments, the invention provides a method as described in any of the relevant preceding paragraphs above, modified such that the step of activating the second TIL population is performed for about 3 to 7 days.

[0639] In some embodiments, the invention provides a method as described in any of the relevant preceding paragraphs above, modified such that the step of activating the second TIL population is performed for about 4 to 7 days.

[0640] In some embodiments, the invention provides a method as described in any of the applicable preceding paragraphs above, modified such that the step of activating the second TIL population is performed for about 5 to 7 days.

[0641] In some embodiments, the invention provides a method as described in any of the relevant preceding paragraphs above, modified such that the step of activating the second TIL population is performed for about 6 to 7 days.

[0642] In some embodiments, the invention provides a method as described in any of the relevant preceding paragraphs above, modified such that the step of activating the second TIL population is performed for about 1 to 6 days.

[0643] In some embodiments, the invention provides a method as described in any of the relevant preceding paragraphs above, modified such that the step of activating the second TIL population is performed for about 1 to 5 days.

[0644] In some embodiments, the invention provides a method as described in any of the relevant preceding paragraphs above, modified such that the step of activating the second TIL population is performed for about 1 to 4 days.

[0645] In some embodiments, the invention provides a method as described in any of the relevant preceding paragraphs above, modified such that the step of activating the second TIL population is performed for about 1 to 3 days.

[0646] In some embodiments, the invention provides a method as described in any of the relevant preceding paragraphs above, modified such that the step of activating the second TIL population is performed for about 1 to 2 days.

[0647] In some embodiments, the invention provides a method as described in any of the relevant preceding paragraphs above, modified such that the step of activating the second TIL population is performed for about 2-6 days.

[0648] In some embodiments, the invention provides a method as described in any of the relevant preceding paragraphs above, modified such that the step of activating the second TIL population is performed for about 3-6 days.

[0649] In some embodiments, the invention provides a method as described in any of the relevant preceding paragraphs above, modified such that the step of activating the second TIL population is performed for about 4 to 6 days.

[0650] In some embodiments, the invention provides a method as described in any of the relevant preceding paragraphs above, modified such that the step of activating the second TIL population is performed for about 5-6 days.

[0651] In some embodiments, the invention provides a method as described in any of the relevant preceding paragraphs above, modified such that the step of activating the second TIL population is performed for about 3 to 5 days.

[0652] In some embodiments, the invention provides a method as described in any of the relevant preceding paragraphs above, modified such that the step of activating the second TIL population is performed for about 3 to 4 days.

[0653] In some embodiments, the invention provides a method as described in any of the relevant preceding paragraphs above, modified such that the step of activating the second TIL population is performed for about 2-5 days.

[0654] In some embodiments, the invention provides a method as described in any of the relevant preceding paragraphs above, modified such that the step of activating the second TIL population is performed for about 2-4 days.

[0655] In some embodiments, the invention provides a method as described in any of the relevant preceding paragraphs above, modified such that the step of activating the second TIL population is performed for about 2-3 days.

[0656] In some embodiments, the invention provides a method as described in any of the relevant preceding paragraphs above, modified such that the step of activating the second TIL population is performed for about 4 to 5 days.

[0657] In some embodiments, the invention provides a method as described in any of the relevant preceding paragraphs above, modified such that the step of activating the second population of TILs is performed for about 1 day.

[0658] In some embodiments, the invention provides a method as described in any of the relevant preceding paragraphs above, modified such that the step of activating the second population of TILs is performed for about 2 days.

[0659] In some embodiments, the invention provides a method as described in any of the relevant preceding paragraphs above, modified such that the step of activating the second TIL population is performed for about 3 days.

[0660] In some embodiments, the invention provides a method as described in any of the relevant preceding paragraphs above, modified such that the step of activating the second TIL population is performed for about 4 days.

[0661] In some embodiments, the invention provides a method as described in any of the relevant preceding paragraphs above, modified such that the step of activating the second TIL population is performed for about 5 days.

[0662] In some embodiments, the invention provides a method as described in any of the relevant preceding paragraphs above, modified such that the step of activating the second population of TILs is performed for about 6 days.

[0663] In some embodiments, the invention provides a method as described in any of the relevant preceding paragraphs above, modified such that the step of activating the second TIL population is performed for about 7 days.

[0664] In some embodiments, provided herein is a method for preparing expanded tumor infiltrating lymphocytes (TILs), comprising: (a) obtaining and / or receiving a first population of TILs from tumor tissue resected from a subject or patient; (b) culturing the first TIL population in a first cell culture medium comprising IL-2 and OKT-3 for about 3-9 days to produce a second TIL population; (c) sterile electroporating the second TIL population to transfer at least one gene editor into a portion of the cells of the second TIL population to produce a third TIL population; (d) culturing the third population of TILs in a second cell culture medium comprising antigen presenting cells (APCs), OKT-3, and IL-2 for about 5-15 days to produce an expanded number of TILs; Sterile electroporation of at least one gene editor into a portion of the cells of the third TIL population modifies a plurality of cells in the portion to express an immunomodulatory composition on the surface of the cells. In some embodiments, the immunomodulatory agent is selected from the group consisting of IL-2, IL-7, IL-10, IL-12, IL-15, IL-18, IL-21, and a CD40 agonist (e.g., CD40L or an agonist CD40 binding domain). In some embodiments, the immunomodulatory agent is selected from the group consisting of IL-2, IL-12, IL-15, IL-18, IL-21, and a CD40 agonist. In some embodiments, the immunomodulatory agent is selected from the group consisting of IL-12, IL-15, IL-18, IL-21, and a CD40 agonist.

[0665] In some embodiments, provided herein is a method for preparing expanded tumor infiltrating lymphocytes (TILs), comprising: (a) obtaining and / or receiving a first population of TILs from tumor tissue resected from a subject or patient; (b) culturing the first TIL population in a first cell culture medium comprising IL-2 and OKT-3 for about 3-9 days to produce a second TIL population; (c) sterile electroporating the second TIL population to transfer at least one nucleic acid molecule into a portion of the cells of the second TIL population to produce a third TIL population; (d) culturing the third population of TILs in a second cell culture medium comprising antigen presenting cells (APCs), OKT-3, and IL-2 for about 5-15 days to produce an expanded number of TILs; Sterile electroporation of at least one nucleic acid molecule into a portion of the cells of the third TIL population modifies a plurality of cells in the portion to transiently express an immunomodulatory composition on the surface of the cells. In some embodiments, the immunomodulatory composition comprises an immunomodulatory agent fused to a membrane anchor (e.g., a membrane-anchored immunomodulatory fusion protein described herein). In some embodiments, the immunomodulatory agent is selected from the group consisting of IL-2, IL-7, IL-10, IL-12, IL-15, IL-18, IL-21, and a CD40 agonist (e.g., CD40L or an agonist CD40 binding domain). In some embodiments, the immunomodulatory agent is selected from the group consisting of IL-2, IL-12, IL-15, IL-18, IL-21, and a CD40 agonist. In some embodiments, the immunomodulatory agent is selected from the group consisting of IL-12, IL-15, IL-18, IL-21, and a CD40 agonist.

[0666] In some embodiments, provided herein is a method for preparing expanded tumor infiltrating lymphocytes (TILs), comprising: (a) obtaining and / or receiving a first population of TILs from tumor tissue resected from a subject or patient; (b) digesting the tumor tissue in an enzymatic medium to produce a tumor digest; (c) culturing the first TIL population in a first cell culture medium comprising IL-2 and OKT-3 for about 3-9 days to produce a second TIL population; (d) sterile electroporating the second TIL population to transfer the at least one gene editor into a portion of the cells of the second TIL population to produce a third TIL population; (e) culturing the third population of TILs in a second cell culture medium comprising antigen presenting cells (APCs), OKT-3, and IL-2 for about 5-15 days to produce an expanded number of TILs; Sterile electroporation of at least one gene editor into a portion of the cells of the second TIL population modifies a plurality of cells in the portion to express an immunomodulatory composition on the surface of the cells. In some embodiments, the immunomodulatory composition comprises an immunomodulatory agent fused to a membrane anchor (e.g., a membrane-anchored immunomodulatory fusion protein described herein). In some embodiments, the cytokine is selected from the group consisting of IL-2, IL-7, IL-10, IL-12, IL-15, IL-18, IL-21, and a CD40 agonist. In some embodiments, the cytokine is selected from the group consisting of IL-2, IL-12, IL-15, IL-18, and IL-21. In some embodiments, the cytokine is selected from the group consisting of IL-12, IL-15, IL-18, IL-21, and a CD40 agonist.

[0667] In some embodiments, provided herein is a method for preparing expanded tumor infiltrating lymphocytes (TILs), comprising: (a) obtaining and / or receiving a first population of TILs from tumor tissue resected from a subject or patient; (b) digesting the tumor tissue in an enzymatic medium to produce a tumor digest; (c) culturing the first TIL population in a first cell culture medium comprising IL-2 and OKT-3 for about 3-9 days to produce a second TIL population; (d) sterile electroporating the second TIL population to transfer at least one nucleic acid molecule into a portion of the cells of the second TIL population to produce a third TIL population; (e) culturing the third population of TILs in a second cell culture medium comprising antigen presenting cells (APCs), OKT-3, and IL-2 for about 5-15 days to produce an expanded number of TILs; Sterile electroporation of at least one nucleic acid molecule into a portion of the cells of the second TIL population modifies a plurality of cells in the portion to transiently express an immunomodulatory composition on the surface of the cells. In some embodiments, the immunomodulatory composition comprises an immunomodulatory agent fused to a membrane anchor (e.g., a membrane-anchored immunomodulatory fusion protein described herein). In some embodiments, the immunomodulatory agent is selected from the group consisting of IL-2, IL-7, IL-10, IL-12, IL-15, IL-18, IL-21, and a CD40 agonist (e.g., CD40L or an agonist CD40 binding domain). In some embodiments, the immunomodulatory agent is selected from the group consisting of IL-2, IL-12, IL-15, IL-18, IL-21, and a CD40 agonist. In some embodiments, the immunomodulatory agent is selected from the group consisting of IL-12, IL-15, IL-18, IL-21, and a CD40 agonist.

[0668] In some embodiments, provided herein is a method for preparing expanded tumor infiltrating lymphocytes (TILs), comprising: (a) obtaining and / or receiving a first population of TILs from tumor tissue resected from a subject or patient; (b) culturing the first TIL population in a first cell culture medium comprising IL-2 and OKT-3 for about 3-9 days to produce a second TIL population; (c) transiently disrupting the cell membrane of the second TIL population and transferring at least one gene editor into a portion of the cells of the second TIL population to produce a third TIL population; (d) culturing the third population of TILs in a second cell culture medium comprising antigen presenting cells (APCs), OKT-3, and IL-2 for about 5-15 days to produce an expanded number of TILs; Transfer of at least one gene editor into a portion of the cells of the second TIL population modifies a plurality of cells in the portion to express an immunomodulatory composition on the surface of the cells. In some embodiments, the immunomodulatory composition comprises an immunomodulatory agent fused to a membrane anchor (e.g., a membrane-anchored immunomodulatory fusion protein described herein). In some embodiments, the immunomodulatory agent is selected from the group consisting of IL-2, IL-7, IL-10, IL-12, IL-15, IL-18, IL-21, and a CD40 agonist (e.g., CD40L or an agonist CD40 binding domain). In some embodiments, the immunomodulatory agent is selected from the group consisting of IL-2, IL-12, IL-15, IL-18, IL-21, and a CD40 agonist. In some embodiments, the immunomodulatory agent is selected from the group consisting of IL-12, IL-15, IL-18, IL-21, and a CD40 agonist. In some embodiments, the immunomodulatory agent is selected from the group consisting of IL-12, IL-15, IL-18, IL-21, and a CD40 agonist. In some embodiments, a microfluidic platform is used to transiently disrupt the cell membranes of the second TIL population. In some embodiments, the microfluidic platform is a microfluidic platform that does not contain an SQZ vector.

[0669] In some embodiments, provided herein is a method for preparing expanded tumor infiltrating lymphocytes (TILs), comprising: (a) obtaining and / or receiving a first population of TILs from tumor tissue resected from a subject or patient; (b) culturing the first TIL population in a first cell culture medium comprising IL-2 and OKT-3 for about 3-9 days to produce a second TIL population; (c) transiently disrupting the cell membrane of the second TIL population to transfer at least one nucleic acid molecule into a portion of the cells of the second TIL population to produce a third TIL population; (d) culturing the third population of TILs in a second cell culture medium comprising antigen presenting cells (APCs), OKT-3, and IL-2 for about 5-15 days to produce an expanded number of TILs; Transfer of at least one gene editor into a portion of the cells of the second TIL population modifies a plurality of cells in the portion to transiently express an immunomodulatory composition on the surface of the cells. In some embodiments, the immunomodulatory composition comprises an immunomodulatory agent fused to a membrane anchor (e.g., a membrane-anchored immunomodulatory fusion protein described herein). In some embodiments, the immunomodulatory agent is selected from the group consisting of IL-2, IL-7, IL-10, IL-12, IL-15, IL-18, IL-21, and a CD40 agonist (e.g., CD40L or an agonist CD40 binding domain). In some embodiments, the immunomodulatory agent is selected from the group consisting of IL-2, IL-12, IL-15, IL-18, IL-21, and a CD40 agonist. In some embodiments, the immunomodulatory agent is selected from the group consisting of IL-12, IL-15, IL-18, IL-21, and a CD40 agonist. In some embodiments, the immunomodulatory agent is selected from the group consisting of IL-12, IL-15, IL-18, IL-21, and a CD40 agonist. In some embodiments, a microfluidic platform is used to transiently disrupt the cell membranes of the second TIL population. In some embodiments, the microfluidic platform is a microfluidic platform that does not contain an SQZ vector.

[0670] In some embodiments, provided herein...

Claims

1. A tumor-infiltrating lymphocyte (TIL) population for use in a method of treating cancer in a patient requiring treatment, wherein the method comprises administering the TIL population and an IL-15R agonist, the TIL population comprising human IL-15 or polymer-conjugated IL-15 conjugated with a soluble form of human IL-15Ra.

2. The IL-15R agonist is selected from the group consisting of NIZ985 (recombinant heterodimer of IL-15 / IL-15Rα; Novartis), NKTR-255 (polymer conjugate IL-15; Nektar), N-803 (IL-15 / IL-15Rα-Fc; ImmunityBio), XmAb306 (reduced-potency IL15 / IL15Rα-Fc fusion protein; Xencor), BJ-001 (tumor-targeted IL-15 / IL-15Rα-Fc; BJ Bioscience), CYP0150 (Cytune), and combinations thereof, and optionally, the IL-15R agonist is selected from the group consisting of NIZ985 (recombinant heterodimer of IL-15 / IL-15Rα; Novartis), NKTR-255 (polymer conjugate IL-15; Nektar), N-803 (IL-15 / IL-15Rα-Fc; ImmunityBio), XmAb306 (reduced-potency IL15 / IL15Rα-Fc fusion protein; Xencor), BJ-001 (tumor-targeted IL-15 / IL-15Rα-Fc; BJ Bioscience), CYP0150 (Cytune), and combinations thereof. (a) NIZ985 (recombinant heterodimer of IL-15 / IL-15Rα; Novartis) (b) NKTR-255 (polymer conjugate IL-15; Nektar) (c) N-803 (IL-15 / IL-15Rα-Fc; ImmunityBio) or (d) XmAb306 (a reduced-potency IL15 / IL15Rα-Fc fusion protein; Xencor), a population for use according to claim 1.

3. The aforementioned IL-15R agonist, (a) On the same day that the TIL population is administered, the patient: (b) Approximately 1 to 10 days after administering the TIL population, the patient was given (c) Once a day, once every two days, once every three days, once every four days, once every five days, once a week, once every two weeks, once every three weeks, or once a month. (d) In total doses of approximately 1 to 28 times, (e) at doses of approximately 1 μg / kg to approximately 100 μg / kg, and / or (f) A dose of 20 μg / kg, administered once every 5 days, for a total dose of up to 3 times. A population for use according to claim 1 or 2, to be administered.

4. The procedure further includes treating the patient with a non-myeloablative lymphocyte depletion regimen before administering the TIL to the patient, Optionally, the above non-myeloablative lymphocyte depletion regimen is, (i) Administer cyclophosphamide at a dose of 60 mg / kg / day for 2 days, followed by fludarabine at a dose of 25 mg / m². ２ The procedure involves administering the drug at a daily dose for five days, and optionally administering the cyclophosphamide together with mesna, or (ii) Cyclophosphamide at a dose of 60 mg / kg / day, and fludarabine at a dose of 25 mg / m². ２ Administer at a daily dose for two days, followed by fludarabine at 25 mg / m². ２ The procedure involves administering the drug at a daily dose for three days, and optionally administering the cyclophosphamide together with mesna. A group for use according to claim 1 or 2, including the group for use according to claim 1 or 2.

5. The patient receives a reduced-intensity non-myeloablative lymphocyte depletion regimen, and optionally, the reduced-intensity non-myeloablative lymphocyte depletion regimen includes cyclophosphamide at 750 mg / m². ２ Administer at a daily dose for 4 days, followed by fludarabine at 30 mg / m². ２ A population for use according to claim 4, comprising the step of administering a dose of / day for 4 days, wherein optionally, the cyclophosphamide is administered together with mesna.

6. A population for use according to claim 1 or 2, wherein the patient does not undergo a non-myeloablative lymphocyte depletion regimen.

7. The further step includes treating the patient with an IL-2 regimen initiated the day following the administration of the TIL population to the patient, or Optionally, the procedure further includes the step of treating the patient with an IL-2 regimen initiated on the same day as the administration of the TIL population to the patient, (a) The IL-2 regimen is a high-dose IL-2 regimen containing 600,000 or 720,000 IU / kg of aldesleukin, or its biosimilar or variant, administered as a 15-minute bolus intravenous infusion every 8 hours up to an acceptable level, or (b) The IL-2 regimen is a reduced-dose IL-2 regimen comprising 600,000 or 720,000 IU / kg of aldesleukin or its biosimilar or variant in a reduced number, e.g., 1, 2, 3, 4, or 5 doses, administered as a 15-minute bolus intravenous infusion every 8 hours. A group for use according to claim 1 or 2.

8. A population for use according to claim 1 or 2, wherein the patient does not receive the IL-2 regimen.

9. The aforementioned IL-15R agonist, (a) resulting in increased survival and / or expansion of the TIL population, (b) causing persistence of the TIL on day 14, day 28, and / or day 42 after administration of the TIL, (c) A population for use according to claim 1 or 2, administered in doses of approximately 0.5 μg / kg, approximately 1.0 μg / kg, approximately 1.5 μg / kg, approximately 2.0 μg / kg, approximately 2.5 μg / kg, approximately 3.0 μg / kg, approximately 3.5 μg / kg, approximately 4.0 μg / kg, approximately 4.5 μg / kg, approximately 5.0 μg / kg, approximately 10 μg / kg, approximately 15 μg / kg, approximately 20 μg / kg, approximately 30 μg / kg, approximately 40 μg / kg, approximately 50 μg / kg, or approximately 100 μg / kg.

10. The further includes administering an immune checkpoint inhibitor (ICI) to the patient, optionally, (a) Administering a PD-1 or PD-L1 inhibitor or its biosimilar to the patient, wherein the PD-1 inhibitor is selected from the group consisting of nivolumab, pembrolizumab, and their biosimilars, or the PD-L1 inhibitor is selected from the group consisting of avelumab, atezolizumab, durvalumab, and their biosimilars, and / or (b) Administering a CTLA-4 inhibitor or a biosimilar thereof to the patient, wherein the CTLA-4 inhibitor is optionally selected from the group consisting of ipilumumab, tremelimumab, and their biosimilars, and / or (c) Administering a chemotherapeutic agent to the patient. A group for use according to claim 1 or 2, further comprising:

11. The aforementioned TIL group, (a) Before the patient receives an ICI or chemotherapy agent, the step of obtaining and / or receiving a first TIL population from a tumor resected from the patient by processing a tumor sample obtained from the patient into a plurality of tumor fragments or tumor digests, (b) prepared using a method comprising the step of cryopreserving the tumor fragment or tumor digest containing the first TIL population from step (a) to produce cryopreserved tumor fragment or tumor digest, If the patient exhibits a progressive disease during or after treatment with the ICI or chemotherapy agent, the first TIL population is expanded to include the TIL population. Optionally, the extension of the first TIL group is (c) Thawing the cryopreserved tumor fragments or tumor digests and adding the first TIL population to the closed system, (d) A step of producing a second TIL population by performing a first expansion by culturing the first TIL population in a first cell culture medium containing IL-2, wherein the first expansion is performed in a sealed container providing a first gas-permeable surface area, the first expansion is performed for about 3 to 11 days to obtain the second TIL population, and the transition from step (c) to step (d) occurs without opening the system, (e) A step of producing a third TIL population, wherein the cell culture medium of the second TIL population is supplemented with additional IL-2, OKT-3, and antigen-presenting cells (APCs) to produce a third TIL population, the second expansion being carried out for about 7 to 11 days to obtain the third TIL population, the third TIL population being a therapeutic TIL population, the second expansion being carried out in a sealed container providing a second gas-permeable surface area, and the transition from step (d) to step (e) occurring without opening the system, (f) A step of collecting the therapeutic TIL population obtained from step (e), wherein the transition from step (e) to step (f) occurs without opening the system, (g) A step of transferring the TIL sample collected from step (f) to an injection bag, wherein the transition from step (f) to (g) occurs without opening the system, (h) a group for use according to claim 10, comprising the step of freezing the infusion bag containing the TIL group taken from step (g) using a cryopreservation process.

12. A population for use according to claim 11, wherein the patient exhibits a progressive disease at least about one week, two weeks, three weeks, one month, two months, three months, four months, five months, six months, seven months, eight months, nine months, ten months, eleven months, twelve months, thirteen months, fourteen months, fifteen months, sixteen months, seventeen months, eighteen months, nineteen months, twenty months, twenty months, twenty-one months, twenty-two months, twenty-three months, twenty-four months, twenty-five months, twenty-six months, twenty-seven months, twenty-eight months, twenty-nine months, thirty-one months, thirty-one months, thirty-two months, thirty-three months, thirty-four months, thirty-five months, thirty-five months, thirty-five months, thirty-two months, thirty-three months, thirty-fivesix months, thirty-five months, thirty-six months, thirty-five months, thirty-five months, thirty-five months, thirty-five months, thirty-six months, thirty-five months, thirty-five months, thirty-five months, thirty-five months, thirty-five months, thirty-six months, thirty-five months, thirty-five months, thirty-five months, thirty-five months, thirty-five months, thirty-five months, thirty-five months, thirty-five months, thirty-five months, thirty-five months, thirty-five months, thirty-five months, thirty-five months, thirty-five months, thirty-f

13. Step (b) is, (a) Rapid freezing of the tumor fragment or tumor digest, wherein the rapid freezing is optionally performed i) Incubating the tumor fragment or tumor digest in a cryopreservation medium, optionally incubating for about 30 minutes to about 60 minutes at about 2°C to about 8°C in a cryopreservation medium containing 10% v / v DMSO, ii) Freezing the tumor, wherein the freezing is rapid freezing using the gas phase of liquid nitrogen, and the rapid freezing includes, (b) Freezing of the tumor fragment or tumor digest, wherein the freezing of the controlled rate is: i) Adding the cryopreservation medium to a resealable container, ii) Pre-cooling the closable container in a controlled-speed refrigeration device, iii) Freezing The tumor is placed in the resealable container containing the preservation culture medium, and the container is closed. iv) Incubating the sealed container containing the tumor and the cryopreservation medium at a temperature of approximately 2 to 8°C for approximately 30 to 60 minutes, v) The controlled-speed freezing, which includes slow freezing the container in a controlled-speed freezing device. A group for use according to claim 11, including the group for use described in claim 11.

14. The aforementioned TIL group, (a) Before the patient receives the ICI or chemotherapy agent, the step of obtaining a first TIL population from a tumor resected from the patient by processing a tumor sample obtained from the patient into a plurality of tumor fragments or tumor digests, (b) The step of adding the first TIL group to the closed system, (c) A step of producing a second TIL population by performing a first expansion by culturing the first TIL population in a first cell culture medium containing IL-2, wherein the first expansion is performed in a sealed container providing a first gas-permeable surface area, the first expansion is performed for about 3 to 11 days to obtain the second TIL population, and the transition from step (b) to step (c) occurs without opening the system, (d) A step of producing a third TIL population by performing a second expansion by supplementing the second cell culture medium of the second TIL population with additional IL-2, OKT-3, and antigen-presenting cells (APCs), wherein the second expansion is carried out for about 7 to 11 days to obtain the third TIL population, and the second expansion is carried out in a sealed container providing a second gas-permeable surface area, and the transition from step (c) to step (d) occurs without opening the system, (e) A step of collecting the third TIL group obtained from step (d), wherein the transition from step (d) to step (e) occurs without opening the system, (f) A step of transferring the third TIL group collected from step (e) to an injection bag, wherein the transfer step occurs without opening the system, (g) a group for use according to claim 10, prepared using a method comprising the step of cryopreserving the infusion bag containing the TIL group taken from step (f) using a cryopreservation process.

15. The population for use according to claim 10, wherein the patient is naive to treatment with the ICI and / or chemotherapeutic agent.

16. The aforementioned TIL group, (a) A step of obtaining a first TIL population from tumors excised from a subject by processing a tumor sample obtained from the subject into multiple tumor fragments, (b) The step of adding the tumor fragment to the closed system, (c) A step of producing a second TIL population by performing a first expansion by culturing the first TIL population in a first cell culture medium containing IL-2, wherein the first expansion is performed in a sealed container providing a first gas-permeable surface area, the first expansion is performed for about 3 to 11 days to obtain the second TIL population, and the transition from step (b) to step (c) occurs without opening the system, (d) A step of producing a third TIL population by performing a second expansion by supplementing the second cell culture medium of the second TIL population with additional IL-2, OKT-3, and antigen-presenting cells (APCs), wherein the second expansion is carried out for about 7 to 11 days to obtain the third TIL population, and the second expansion is carried out in a sealed container providing a second gas-permeable surface area, and the transition from step (c) to step (d) occurs without opening the system, (e) A step of collecting the third TIL group obtained from step (d), wherein the transition from step (d) to step (e) occurs without opening the system, (f) A step of transferring the third TIL sample collected from step (e) to an injection bag, wherein the transition from step (e) to (f) occurs without opening the system, (g) Prepared using a method comprising the step of cryopreserving the infusion bag containing the TIL population collected from step (f) using a cryopreservation process, The group for use according to claim 1 or 2, wherein the second TIL group is optionally at least 50 times larger in number than the first TIL group.

17. The first cell culture medium further comprises an IL-15R agonist, or the second cell culture medium further comprises an IL-15R agonist, optionally comprising: (a) Selected from the group consisting of NIZ985, NKTR-255, N-803, XmAb306, BJ-001, CYP0150 (Cytune), and combinations thereof, (b) NIZ985, (c) NKTR-255, (d) N-803, (e) XmAb306, or (f) Replenished at concentrations of approximately 0.1 ng / mL, approximately 0.5 ng / mL, approximately 1 ng / mL, approximately 5 ng / mL, approximately 10 ng / mL, approximately 50 ng / mL, approximately 100 ng / mL, approximately 150 ng / mL, or approximately 200 ng / mL. A group for use according to claim 11.

18. The expression of one or more genes in the TIL population is regulated, and by arbitrary selection, one or more genes are selected from the group consisting of PD-1, CTLA-4, LAG-3, CISH, TIGIT, and CBL-B, and further by arbitrary selection, (a) The expression of PD-1 and CTLA-4 is regulated in the TIL population. (b) The expression of PD-1 and LAG-3 is regulated in the TIL population. (c) The expression of PD-1 and CISH is regulated in the TIL population. (d) The expression of PD-1 and CBL-B is regulated in the TIL population. (e) The expression of PD-1 and TIGIT is regulated in the TIL population. (f) The expression of CTLA-4 and LAG-3 is regulated in the TIL population. (g) The expression of CTLA-4 and CISH is regulated in the TIL population. (h) The expression of CTLA-4 and CBL-B is regulated in the TIL population. (i) The expression of LAG-3 and CISH is regulated in the TIL population. (j) The expression of LAG-3 and CBL-B is regulated in the TIL population. (k) The expression of CISH and CBL-B is regulated in the TIL population. (l) PD-1 expression is regulated in the TIL population, (m) The expression of CTLA-4 is regulated in the TIL population. (n) LAG-3 expression is regulated in the TIL population, (o) CISH expression is regulated in the TIL population, (p) CBL-B expression is regulated in the TIL population, or (q) The expression of TIGIT is regulated in the TIL population. A group for use according to claim 1 or 2.

19. The aforementioned cancer has been previously treated with a PD-1 inhibitor and / or a PD-L1 inhibitor or a biosimilar thereof, and at the discretion of the patient, (a) The cancer has been previously treated with a PD-1 inhibitor or a biosimilar thereof, and the PD-1 inhibitor is further optionally selected from the group consisting of nivolumab, pembrolizumab, and their biosimilars. (b) The patient has been previously treated with a PD-L1 inhibitor or a biosimilar thereof, and the PD-L1 inhibitor is further selected at the discretion of the group consisting of avelumab, atezolizumab, durvalumab, and their biosimilars. (c) The cancer has been previously treated with a CTLA-4 inhibitor or a biosimilar thereof, and the CTLA-4 inhibitor is optionally selected from the group consisting of ipilumumab, tremelimumab, and their biosimilars, or (d) The cancer has been previously treated with a chemotherapy regimen, and the chemotherapy regimen optionally includes dacarbazine or temozolimide. A group for use according to claim 1 or 2.

20. i) In the first expansion, IL-2 is present in the cell culture medium at an initial concentration of 1000 IU / mL to 6000 IU / mL. ii) In the second expansion step, the IL-2 is present at an initial concentration of 1000 IU / mL to 6000 IU / mL, the OKT-3 antibody is present at an initial concentration of about 30 ng / mL, and / or iii) In the rapid expansion step, the IL-2 is present at an initial concentration of 1000 IU / mL to 6000 IU / mL, and the OKT-3 antibody is present at an initial concentration of approximately 30 ng / mL, and optionally, (a) The first expansion is carried out using a gas-permeable container. (b) The second expansion is carried out using a gas-permeable container, or (c) The rapid expansion is carried out using a gas-permeable container. A group for use according to claim 11.

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